Alexandra L. Joyner, Ph.D.

Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei
Blaess, Sandra; Bodea, Gabriela O; Kabanova, Anna; Chanet, Soline; Mugniery, Emilie; Derouiche, Amin; Stephen, Daniel; Joyner, Alexandra L
2011 ;6:29-29, Neural development
BACKGROUND: The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points. RESULTS: We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes. CONCLUSIONS: We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions
— id: 141264, year: 2011, vol: 6, page: 29, stat: Journal Article,

Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells
Brownell, Isaac; Guevara, Elizabeth; Bai, C Brian; Loomis, Cynthia A; Joyner, Alexandra L
2011 May 6;8(5):552-565, Cell Stem Cell
In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. The degree to which all bulge cells have equal regenerative potential is not known. We found that Sonic hedgehog (Shh) from neurons signals to a population of cells in the telogen bulge marked by the Hedgehog response gene Gli1. Gli1-expressing bulge cells function as multipotent stem cells in their native environment and repeatedly regenerate the anagen follicle. Shh-responding perineural bulge cells incorporate into healing skin wounds where, notably, they can change their lineage into epidermal stem cells. The perineural niche (including Shh) is dispensable for follicle contributions to acute wound healing and skin homeostasis, but is necessary to maintain bulge cells capable of becoming epidermal stem cells. Thus, nerves cultivate a microenvironment where Shh creates a molecularly and phenotypically distinct population of hair follicle stem cells
— id: 133414, year: 2011, vol: 8, page: 552, stat: Journal Article,

Ascl1 genetics reveals insights into cerebellum local circuit assembly
Sudarov, Anamaria; Turnbull, Rowena K; Kim, Euiseok J; Lebel-Potter, Melanie; Guillemot, Francois; Joyner, Alexandra L
2011 Jul 27;31(30):11055-11069, Journal of neuroscience
Two recently generated targeted mouse alleles of the neurogenic gene Ascl1 were used to characterize cerebellum circuit formation. First, genetic inducible fate mapping (GIFM) with an Ascl1(CreER) allele was found to specifically mark all glial and neuron cell types that arise from the ventricular zone (vz). Moreover, each cell type has a unique temporal profile of marking with Ascl1(CreER) GIFM. Of great utility, Purkinje cells (Pcs), an early cohort of Bergmann glia, and four classes of GABAergic interneurons can be genetically birth dated during embryogenesis using Ascl1(CreER) GIFM. Astrocytes and oligodendrocytes, in contrast, express Ascl1(CreER) throughout their proliferative phase in the white matter. Interestingly, the final position each neuron type acquires differs depending on when it expresses Ascl1. Interneurons (including candelabrum) attain a more outside position the later they express Ascl1, whereas Pcs have distinct settling patterns each day they express Ascl1. Second, using a conditional Ascl1 allele, we discovered that Ascl1 is differentially required for generation of most vz-derived cells. Mice lacking Ascl1 in the cerebellum have a major decrease in three types of interneurons with a tendency toward a loss of later-born interneurons, as well as an imbalance of oligodendrocytes and astrocytes. Double-mutant analysis indicates that a related helix-loop-helix protein, Ptf1a, functions with Ascl1 in generating interneurons and Pcs. By fate mapping vz-derived cells in Ascl1 mutants, we further discovered that Ascl1 plays a specific role during the time period when Pcs are generated in restricting vz progenitors from becoming rhombic lip progenitors
— id: 141263, year: 2011, vol: 31, page: 11055, stat: Journal Article,

Spatially restricted and developmentally dynamic expression of engrailed genes in multiple cerebellar cell types
Wilson, Sandra L; Kalinovsky, Anna; Orvis, Grant D; Joyner, Alexandra L
2011 Sep;10(3):356-372, Cerebellum
The cerebellum is a highly organized structure partitioned into lobules along the anterior-posterior (A-P) axis and into striped molecular domains along the medial-lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P) 21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCss4 and EPHA4
— id: 141265, year: 2011, vol: 10, page: 356, stat: Journal Article,

Morphological and functional midbrain phenotypes in Fibroblast Growth Factor 17 mutant mice detected by Mn-enhanced MRI
Yu, Xin; Nieman, Brian J; Sudarov, Anamaria; Szulc, Kamila U; Abdollahian, Davood J; Bhatia, Nitin; Lalwani, Anil K; Joyner, Alexandra L; Turnbull, Daniel H
2011 Jun 1;56(3):1251-1258, Neuroimage
With increasing efforts to develop and utilize mouse models of a variety of neuro-developmental diseases, there is an urgent need for sensitive neuroimaging methods that enable in vivo analysis of subtle alterations in brain anatomy and function in mice. Previous studies have shown that the brains of Fibroblast Growth Factor 17 null mutants (Fgf17(-/-)) have anatomical abnormalities in the inferior colliculus (IC)-the auditory midbrain-and minor foliation defects in the cerebellum. In addition, changes in the expression domains of several cortical patterning genes were detected, without overt changes in forebrain morphology. Recently, it has also been reported that Fgf17(-/-) mutants have abnormal vocalization and social behaviors, phenotypes that could reflect molecular changes in the cortex and/or altered auditory processing / perception in these mice. We used manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) to analyze the anatomical phenotype of Fgf17(-/-) mutants in more detail than achieved previously, detecting changes in IC, cerebellum, olfactory bulb, hypothalamus and frontal cortex. We also used MEMRI to characterize sound-evoked activity patterns, demonstrating a significant reduction of the active IC volume in Fgf17(-/-) mice. Furthermore, tone-specific (16- and 40-kHz) activity patterns in the IC of Fgf17(-/-) mice were observed to be largely overlapping, in contrast to the normal pattern, separated along the dorsal-ventral axis. These results demonstrate that Fgf17 plays important roles in both the anatomical and functional development of the auditory midbrain, and show the utility of MEMRI for in vivo analyses of mutant mice with subtle brain defects
— id: 131957, year: 2011, vol: 56, page: 1251, stat: Journal Article,

The Hedgehog response gene Gli1 marks multipotent stem cells in the telogen bulge
Brownell, Isaac; Loomis, Cynthia A.; Joyner, Alexandra L.
2010 SEP ;130(4):S88-S88, Journal of investigative dermatology
— id: 113755, year: 2010, vol: 130, page: S88, stat: Journal Article,

The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum
Cheng, Yulan; Sudarov, Anamaria; Szulc, Kamila U; Sgaier, Sema K; Stephen, Daniel; Turnbull, Daniel H; Joyner, Alexandra L
2010 Feb;137(3):519-529, Development
Little is known about the genetic pathways and cellular processes responsible for regional differences in cerebellum foliation, which interestingly are accompanied by regionally distinct afferent circuitry. We have identified the Engrailed (En) homeobox genes as being crucial to producing the distinct medial vermis and lateral hemisphere foliation patterns in mammalian cerebella. By producing a series of temporal conditional mutants in En1 and/or En2, we demonstrate that both En genes are required to ensure that folia exclusive to the vermis or hemispheres form in the appropriate mediolateral position. Furthermore, En1/En2 continue to regulate foliation after embryonic day 14, at which time Fgf8 isthmic organizer activity is complete and the major output cells of the cerebellar cortex have been specified. Changes in spatially restricted gene expression occur prior to foliation in mutants, and foliation is altered from the onset and is accompanied by changes in the thickness of the layer of proliferating granule cell precursors. In addition, the positioning and timing of fissure formation are altered. Thus, the En genes represent a new class of genes that are fundamental to patterning cerebellum foliation throughout the mediolateral axis and that act late in development
— id: 112032, year: 2010, vol: 137, page: 519, stat: Journal Article,

Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain
Garcia, A Denise R; Petrova, Ralitsa; Eng, Liane; Joyner, Alexandra L
2010 Oct 13;30(41):13597-13608, Journal of neuroscience
Astrocytes are an essential component of the CNS, and recent evidence points to an increasing diversity of their functions. Identifying molecular pathways that mediate distinct astrocyte functions, is key to understanding how the nervous system operates in the intact and pathological states. In this study, we demonstrate that the Hedgehog (Hh) pathway, well known for its roles in the developing CNS, is active in astrocytes of the mature mouse forebrain in vivo. Using multiple genetic approaches, we show that regionally distinct subsets of astrocytes receive Hh signaling, indicating a molecular diversity between specific astrocyte populations. Furthermore, we identified neurons as a source of Sonic hedgehog (Shh) in the adult forebrain, suggesting that Shh signaling is involved in neuron-astrocyte communication. Attenuation of Shh signaling in postnatal astrocytes by targeted removal of Smoothened, an obligate Shh coreceptor, resulted in upregulation of GFAP and cellular hypertrophy specifically in astrocyte populations regulated by Shh signaling. Collectively, our findings demonstrate a role for neuron-derived Shh in regulating specific populations of differentiated astrocytes
— id: 114456, year: 2010, vol: 30, page: 13597, stat: Journal Article,

Genetic fate mapping using site-specific recombinases
Legue, Emilie; Joyner, Alexandra L
2010 ;477:153-181, Methods in enzymology
Understanding how cells are assembled in three dimensions to generate an organ, or a whole organism, is a pivotal question in developmental biology. Similarly, it is critical to understand how adult stem cells integrate into an existing organ during regeneration or in response to injury. Key to discovering the answers to these questions is being able to study the various behaviors of distinct cell types during development or regeneration. Fate mapping techniques are fundamental to studying cell behaviors such as proliferation, movement, and lineage segregation, as the techniques allow precursor cells to be marked and their descendants followed and characterized over time. The generation of transgenic mice, combined with the use of site-specific recombinases (SSR) in the mouse genome, has provided a means to develop powerful genetic fate mapping approaches. A key advantage of genetic fate mapping is that it allows cells to be genetically marked, and therefore the mark is transmitted to all the descendants of the initially marked cells. By making modifications to the SSRs that render their enzymatic activity inducible, and the development of an assortment of reporter alleles for marking cells, increasingly sophisticated genetic fate mapping studies can be performed. In this chapter, we review the four main genetic fate mapping methods that utilize intrachromosomal recombination to mark cells (cumulative, inducible, clonal, and intersectional) and one interchromosomal method, the tools required to carry out each approach, and the practical considerations that have to be taken into account before embarking on each type of genetic fate mapping study
— id: 114457, year: 2010, vol: 477, page: 153, stat: Journal Article,

In vivo MRI of neural cell migration dynamics in the mouse brain
Nieman, Brian J; Shyu, Jeffrey Y; Rodriguez, Joe J; Garcia, A Denise; Joyner, Alexandra L; Turnbull, Daniel H
2010 Apr 1;50(2):456-464, Neuroimage
Multipotent neuroblasts (NBs) are produced throughout life by neural stem cells in the forebrain subventricular zone (SVZ), and are able to travel long distances to the olfactory bulb. On arrival in the bulb, migrating NBs normally replace olfactory neurons, raising interest in their potential for novel cell replacement therapies in various disease conditions. An understanding of the migratory capabilities of NBs is therefore important, but as yet quantitative in vivo measurement of cell migration has not been possible. In this study, targeted intracerebral injections of iron-oxide particles to the mouse SVZ were used to label resident NBs in situ, and their migration was tracked noninvasively over time with magnetic resonance imaging (MRI). Quantitative intensity metrics were employed to identify labeled cells and to show that cells are able to travel at speeds up to 100 microm/h en route to the olfactory bulb, but that distribution through the olfactory bulb occurs at a much slower rate. In addition, comparison of histological and MRI measures of iron-oxide particle distribution were in excellent agreement. Immunohistochemistry analysis 1-3 weeks after labeling revealed that the majority of labeled cells in the olfactory bulb were immature neurons, although iron-oxide particles were also found in astrocytes and microglia. This work indicates that dynamic measurements of endogenous cell migration can be made with MRI and represents the first in vivo measurement of NB migration rates. The use of MRI in future studies tracking endogenous NB cells will permit a more complete evaluation of their role during homeostasis at various developmental stages and during disease progression
— id: 107770, year: 2010, vol: 50, page: 456, stat: Journal Article,

An interview with Alex Joyner and Liz Robertson: development editors at the helm of Developmental Biology Societies
Robertson, Liz; Joyner, Alexandra
2010 Jul;137(13):2075-2077, Development
— id: 114459, year: 2010, vol: 137, page: 2075, stat: Journal Article,

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor
Seidel, Kerstin; Ahn, Christina P; Lyons, David; Nee, Alexander; Ting, Kevin; Brownell, Isaac; Cao, Tim; Carano, Richard A D; Curran, Tom; Schober, Markus; Fuchs, Elaine; Joyner, Alexandra; Martin, Gail R; de Sauvage, Frederic J; Klein, Ophir D
2010 Nov;137(22):3753-3761, Development
In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells
— id: 114455, year: 2010, vol: 137, page: 3753, stat: Journal Article,

Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map
Sillitoe, Roy V; Vogel, Michael W; Joyner, Alexandra L
2010 Jul 28;30(30):10015-10024, Journal of neuroscience
The spatial organization of the cerebellar afferent map has remarkable correspondence to two aspects of intrinsic patterning within the cerebellum embodied by a series of lobules and Purkinje cell (PC)-striped gene expression. Using male and female mice, we tested whether the Engrailed (En) homeobox genes are a common genetic substrate regulating all three systems, since they are expressed in spatially restricted domains within the cerebellum and are critical for patterning PC gene expression and foliation. Indeed, we discovered that En1/2 are necessary for the precise targeting of mossy fibers to distinct lobules, as well as their subsequent resolution into discrete parasagittal bands. Moreover, each En gene coordinately regulates afferent targeting and the striped pattern of PC protein expression (e.g., ZebrinII/AldolaseC) independent of regulating foliation. We further found that En1/2, rather than the presence of a full complement of lobules, are critical for generating PC protein stripes and mossy fiber bands, and that PC striped gene expression is determined before afferent banding. Thus, the En transcription factors not only regulate cerebellum circuit topography, but they also link afferent and efferent neurons precisely enough that alterations in PC protein expression can be used as a read out for underlying defects in circuitry. In summary, our data suggest that En1/2 are master regulators of three-dimensional organization of the cerebellum and coordinately regulate morphology, patterned gene expression, and afferent topography
— id: 114458, year: 2010, vol: 30, page: 10015, stat: Journal Article,

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development (vol 135, pg 889, 2008)
Basson, MA; Echevarria, D; Ahn, CP; Sudarov, A; Joyner, AL; Mason, IJ; Martinez, S; Martin, GR
2009 ;136(11):1962-1962, Development
— id: 104585, year: 2009, vol: 136, page: 1962, stat: Journal Article,

Three-dimensional micro-MRI analysis of cerebral artery development in mouse embryos
Berrios-Otero, Cesar A; Wadghiri, Youssef Zaim; Nieman, Brian J; Joyner, Alexandra L; Turnbull, Daniel H
2009 Dec;62(6):1431-1439, Magnetic resonance in medicine
Vascular system development involves a complex, three-dimensional branching process that is critical for normal embryogenesis. In the brain, the arterial systems appear to develop in a stereotyped fashion, but no detailed quantitative analyses of the mouse embryonic cerebral arteries have been described. In this study, a gadolinium-based contrast perfusion method was developed to selectively enhance the cerebral arteries in fixed mouse embryos. Three-dimensional magnetic resonance micro-imaging (micro-MRI) data were acquired simultaneously from multiple embryos staged between 10 and 17 days of gestation, and a variety of image analysis methods was used to extract and analyze the cerebral arterial patterns. The results show that the primary arterial branches in the mouse brain are very similar between individuals, with the patterns established early and growth occurring by extension of the segments, while maintaining the underlying vascular geometry. To investigate the utility of this method for mutant mouse phenotype analysis, contrast-enhanced micro-MRI data were acquired from Gli2(-/-) mutant embryos and their wild-type littermates, showing several previously unreported vascular phenotypes in Gli2(-/-) embryos, including the complete absence of the basilar artery. These results demonstrate that contrast-enhanced micro-MRI provides a powerful tool for analyzing vascular phenotypes in a variety of genetically engineered mice
— id: 105501, year: 2009, vol: 62, page: 1431, stat: Journal Article,

Deciphering the mechanism of engrailed function during mouse cerebellar foliation
Orvis, GD; Joyner, AL
2009 ;331(2):524-524 484, Developmental biology (Orlando)
— id: 104587, year: 2009, vol: 331, page: 524, stat: Journal Article,

The duration of Fgf8 isthmic organizer expression is key to patterning different tectal-isthmo-cerebellum structures
Sato, Tatsuya; Joyner, Alexandra L
2009 Nov;136(21):3617-3626, Development
The isthmic organizer and its key effector molecule, fibroblast growth factor 8 (Fgf8), have been cornerstones in studies of how organizing centers differentially pattern tissues. Studies have implicated different levels of Fgf8 signaling from the mid/hindbrain boundary (isthmus) as being responsible for induction of different structures within the tectal-isthmo-cerebellum region. However, the role of Fgf8 signaling for different durations in patterning tissues has not been studied. To address this, we conditionally ablated Fgf8 in the isthmus and uncovered that prolonged expression of Fgf8 is required for the structures found progressively closer to the isthmus to form. We found that cell death cannot be the main factor accounting for the loss of brain structures near the isthmus, and instead demonstrate that tissue transformation underlies the observed phenotypes. We suggest that the remaining Fgf8 and Fgf17 signaling in our temporal Fgf8 conditional mutants is sufficient to ensure survival of most midbrain/hindbrain cells near the isthmus. One crucial role for sustained Fgf8 function is in repressing Otx2 in the hindbrain, thereby allowing the isthmus and cerebellum to form. A second requirement for sustained Fgf8 signaling is to induce formation of a posterior tectum. Finally, Fgf8 is also required to maintain the borders of expression of a number of key genes involved in tectal-isthmo-cerebellum development. Thus, the duration as well as the strength of Fgf8 signaling is key to patterning of the mid/hindbrain region. By extrapolation, the length of Fgf8 expression could be crucial to Fgf8 function in other embryonic organizers
— id: 114460, year: 2009, vol: 136, page: 3617, stat: Journal Article,

Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections
Sillitoe, R V; Gopal, N; Joyner, A L
2009 Sep 1;162(3):574-588, Neuroscience
The establishment of neural circuits involves both the precise positioning of cells within brain regions and projection of axons to specific target cells. In the cerebellum (Cb), the medial-lateral (M-L) and anterior-posterior (A-P) position of each Purkinje cell (PC) and the topography of its axon can be defined with respect to two coordinate systems within the Cb; one based on the pattern of lobules and the other on PC gene expression in parasagittal clusters in the embryo (e.g. Pcp2) and stripes in the adult (e.g. ZebrinII). The relationship between the embryonic clusters of molecularly defined PCs and particular adult PC stripes is not clear. Using a mouse genetic inducible fate mapping (GIFM) approach and a Pcp2-CreER-IRES-hAP transgene, we marked three bilateral clusters of PC clusters with myristolated green fluorescent protein (mGfp) on approximately embryonic day (E) 15 and followed their fate into adulthood. We found that these three clusters contributed specifically to ZebrinII-expressing PCs, including nine of the adult stripes. This result suggests that embryonic PCs maintain a particular molecular identity, and that each embryonic cluster can contribute PCs to more than one adult M-L stripe. Each PC projects a primary axon to one of the deep cerebellar nuclei (DCN) or the vestibular nuclei in the brainstem in an organized fashion that relates to the position of the PCs along the M-L axis. We characterized when PC axons from the three M-L clusters acquire topographic projections. Using a combination of GIFM to mark the PC clusters with mGfp and staining for human placental alkaline phosphatase (hAP) in Pcp2-CreER-IRES-hAP transgenic embryos we found that axons from each embryonic PC cluster intermingled with neurons within particular DCN or projected out of the Cb toward the vestibular nuclei by E14.5. These studies show that PC molecular patterning, efferent circuitry, and DCN nucleogenesis occur simultaneously, suggesting a link between these processes
— id: 141266, year: 2009, vol: 162, page: 574, stat: Journal Article,

Characterizing developmental fates of elements of the cerebellar microcircuitry based on Mash1 expression
Sudarov, A; Turnbull, R; Joyner, AL
2009 ;331(2):444-444 191, Developmental biology (Orlando)
— id: 104586, year: 2009, vol: 331, page: 444, stat: Journal Article,

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development
Basson, M Albert; Echevarria, Diego; Ahn, Christina Petersen; Sudarov, Anamaria; Joyner, Alexandra L; Mason, Ivor J; Martinez, Salvador; Martin, Gail R
2008 Mar;135(5):889-898, Development
Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development
— id: 96755, year: 2008, vol: 135, page: 889, stat: Journal Article,

Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh and Fgf8 signaling
Blaess, Sandra; Stephen, Daniel; Joyner, Alexandra L
2008 Jun;135(12):2093-2103, Development
The coordination of anterior-posterior (AP) and dorsal-ventral (DV) patterning of the mesencephalon (mes) and rhombomere 1 (r1) is instrumental for the development of three distinct brain structures: the tectum and cerebellum dorsally and the tegmentum ventrally. Patterning of the mes/r1 is primarily mediated by signaling molecules secreted from two organizers: sonic hedgehog (Shh) from the floor plate (DV) and Fgf8 from the isthmus (AP). Gli3, a zinc-finger transcription factor in the Shh signaling pathway, has been implicated in regulating Fgf8 expression and is therefore a potential candidate for coordinating the action of the two organizers. By inactivating mouse Gli3 at successive embryonic time points in vivo, we uncovered the extent and the underlying mechanism of Gli3 function in the mes/r1. We demonstrate that before E9.0, Gli3 is required for establishing a distinct posterior tectum, isthmus and cerebellum, but does not play a role in the development of the tegmentum. Between E9.0 and E11.0, Gli3 continues to be required for isthmus and cerebellum development, but primarily for defining the cerebellar foliation pattern. We show that Gli3 regulates patterning of the isthmus and cerebellar anlage by confining Fgf8 expression to the isthmus, and attenuates growth of dorsal r1 (before E11.0) and the dorsal mes and isthmus (beyond E11.0) through regulation of cell proliferation and viability. In conclusion, our results show that Gli3 is essential for the coordinated three-dimensional patterning and growth of the dorsal mes/r1
— id: 96754, year: 2008, vol: 135, page: 2093, stat: Journal Article,

Hedgehog-responding stem cells in the bulge expand to regenerate the anagen follicle
Brownell, I; Patel, A; Joyner, AL
2008 ;128(5):896-896, Journal of investigative dermatology
— id: 104588, year: 2008, vol: 128, page: 896, stat: Journal Article,

Hedgehog-responding stem cells regenerate the anagen hair follicle
Brownell, I; Patel, A; Joyner, AL
2008 ;319(2):556-556, Developmental biology (Orlando)
— id: 104589, year: 2008, vol: 319, page: 556, stat: Journal Article,

Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum
Sillitoe, Roy V; Stephen, Daniel; Lao, Zhimin; Joyner, Alexandra L
2008 Nov 19;28(47):12150-12162, Journal of neuroscience
Underlying the seemingly uniform cellular composition of the adult mammalian cerebellum (Cb) are striking parasagittal stripes of gene expression along the medial-lateral (ML) axis that are organized with respect to the lobules that divide the Cb along the anterior-posterior (AP) axis. Although there is a clear correlation between the organization of gene expression stripes and Cb activity patterns, little is known about the genetic pathways that determine the intrinsic stripe molecular code. Here we establish that ML molecular code patterning is highly dependent on two homeobox transcription factors, Engrailed1 (En1) and En2, both of which are also required for patterning the lobules. Gene expression analysis of an allelic series of En1/2 mutant mice that have an intact Purkinje cell layer revealed severe patterning defects using three known components of the ML molecular code and a new marker of Hsp25 negative stripes (Neurofilament heavy chain, Nfh). Importantly, the complementary expression of ZebrinII/PhospholipaseC beta4 and Hsp25/Nfh changes in unison in each mutant. Furthermore, each En gene has unique as well as overlapping functions in patterning the ML molecular code and each En protein has dominant functions in different AP domains (subsets of lobules). Remarkably, in En1/2 mutants with almost normal foliation, ML molecular code patterning is severely disrupted. Thus, independent mechanisms that use En1/2 must pattern foliation and spatial gene expression separately. Our studies reveal that En1/2 are fundamental components of the genetic pathways that pattern the two intersecting coordinate systems of the Cb, morphological divisions and the molecular code
— id: 96753, year: 2008, vol: 28, page: 12150, stat: Journal Article,

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential
Thomas, Natalie A; Koudijs, Marco; van Eeden, Fredericus J M; Joyner, Alexandra L; Yelon, Deborah
2008 Nov;135(22):3789-3799, Development
Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells
— id: 92167, year: 2008, vol: 135, page: 3789, stat: Journal Article,

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential
Yelon, D; Thomas, NA; Koudijs, M; Van Eeden, F; Joyner, AL
2008 JUL 15 ;319(2):603-604, Developmental biology (Orlando)
— id: 86843, year: 2008, vol: 319, page: 603, stat: Journal Article,

Fibroblast growth factor signaling controls development of the cerebellar vermis by inhibiting signals permissive for roofplate formation in anterior rhombomere 1
Basson, MA; Echevarria, D; Peterson, C; Minowada, G; Sudarov, A; Joyner, A; Mason, J; Martinez, S
2007 ;306(1):302-302 40, Developmental biology (Orlando)
— id: 104590, year: 2007, vol: 306, page: 302, stat: Journal Article,

Cerebellum patterning: From folds and stripes to circuits
Joyner, AL; Sillitoe, RV; Sgaier, S; Cheng, YL; Sudarov, A
2007 ;306(1):310-310 54, Developmental biology (Orlando)
— id: 104591, year: 2007, vol: 306, page: 310, stat: Journal Article,

Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins
Sgaier, Sema K; Lao, Zhimin; Villanueva, Melissa P; Berenshteyn, Frada; Stephen, Daniel; Turnbull, Rowena K; Joyner, Alexandra L
2007 Jun;134(12):2325-2335, Development
The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains
— id: 73296, year: 2007, vol: 134, page: 2325, stat: Journal Article,

Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum
Sillitoe, Roy V; Joyner, Alexandra L
2007 ;23:549-577, Annual review of cell & developmental biology
The most noticeable morphological feature of the cerebellum is its folded appearance, whereby fissures separate its anterior-posterior extent into lobules. Each lobule is molecularly coded along the medial-lateral axis by parasagittal stripes of gene expression in one cell type, the Purkinje cells (PCs). Additionally, within each lobule distinct combinations of afferents terminate and supply the cerebellum with synchronized sensory and motor information. Strikingly, afferent terminal fields are organized into parasagittal domains, and this pattern bears a close relationship to PC molecular coding. Thus, cerebellum three-dimensional complexity obeys a basic coordinate system that can be broken down into morphology and molecular coding. In this review, we summarize the sequential stages of cerebellum development that produce its laminar structure, foliation, and molecular organization. We also introduce genes that regulate morphology and molecular coding, and discuss the establishment of topographical circuits within the context of the two coordinate systems. Finally, we discuss how abnormal cerebellar organization may result in neurological disorders like autism
— id: 96757, year: 2007, vol: 23, page: 549, stat: Journal Article,

Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers
Sudarov, Anamaria; Joyner, Alexandra L
2007 ;2:26-26, Neural development
BACKGROUND: The cerebellum has a striking morphology consisting of folia separated by fissures of different lengths. Since folia in mammals likely serve as a broad platform on which the anterior-posterior organization of the sensory-motor circuits of the cerebellum are built, it is important to understand how such complex morphology arises. RESULTS: Using a combination of genetic inducible fate mapping, high-resolution cellular analysis and mutant studies in mouse, we demonstrate that a key event in initiation of foliation is the acquisition of a distinct cytoarchitecture in the regions that will become the base of each fissure. We term these regions 'anchoring centers'. We show that the first manifestation of anchoring centers when the cerebellar outer surface is smooth is an increase in proliferation and inward thickening of the granule cell precursors, which likely causes an associated slight invagination of the Purkinje cell layer. Thereafter, granule cell precursors within anchoring centers become distinctly elongated along the axis of the forming fissure. As the outer cerebellar surface begins to fold inwards, Bergmann glial fibers radiate in towards the base of the immature fissure in a fan shape. Once the anchoring center is formed, outgrowth of folia seems to proceed in a self-sustaining manner driven by granule cell migration along Bergmann glial fibers. Finally, by analyzing a cerebellum foliation mutant (Engrailed 2), we demonstrate that changing the timing of anchoring center formation leads to predictable changes in the shape and size of the surrounding folia. CONCLUSION: We present a new cellular model of the initial formation of cerebellar fissures with granule cells providing the driving physical force. Both the precise timing of the appearance of anchoring centers at the prospective base of each fissure and the subsequent coordinated action of granule cells and Bergmann glial fibers within the anchoring centers dictates the shape of the folia
— id: 96756, year: 2007, vol: 2, page: 26, stat: Journal Article,

Beta-catenin activation is necessary and sufficient to specify the dorsal dermal fate in the mouse
Atit, Radhika; Sgaier, Sema K; Mohamed, Othman A; Taketo, Makoto M; Dufort, Daniel; Joyner, Alexandra L; Niswander, Lee; Conlon, Ronald A
2006 Aug 1;296(1):164-176, Developmental biology (Orlando)
Dorsal dermis and epaxial muscle have been shown to arise from the central dermomyotome in the chick. En1 is a homeobox transcription factor gene expressed in the central dermomyotome. We show by genetic fate mapping in the mouse that En1-expressing cells of the central dermomyotome give rise to dorsal dermis and epaxial muscle and, unexpectedly, to interscapular brown fat. Thus, the En1-expressing central dermomyotome normally gives rise to three distinct fates in mice. Wnt signals are important in early stages of dermomyotome development, but the signal that acts to specify the dermal fate has not been identified. Using a reporter transgene for Wnt signal transduction, we show that the En1-expressing cells directly underneath the surface ectoderm transduce Wnt signals. When the essential Wnt transducer beta-catenin is mutated in En1 cells, it results in the loss of Dermo1-expressing dorsal dermal progenitors and dermis. Conversely, when beta-catenin was activated in En1 cells, it induces Dermo1 expression in all cells of the En1 domain and disrupts muscle gene expression. Our results indicate that the mouse central dermomyotome gives rise to dermis, muscle, and brown fat, and that Wnt signalling normally instructs cells to select the dorsal dermal fate
— id: 96758, year: 2006, vol: 296, page: 164, stat: Journal Article,

Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region
Blaess, Sandra; Corrales, Jomichelle D; Joyner, Alexandra L
2006 May;133(9):1799-1809, Development
The midbrain and anterior hindbrain offer an ideal system in which to study the coordination of tissue growth and patterning in three dimensions. Two organizers that control anteroposterior (AP) and dorsoventral (DV) development are known, and the regulation of AP patterning by Fgf8 has been studied in detail. Much less is known about the mechanisms that control mid/hindbrain development along the DV axis. Using a conditional mutagenesis approach, we have determined how the ventrally expressed morphogen sonic hedgehog (Shh) directs mid/hindbrain development over time and space through positive regulation of the Gli activators (GliA) and inhibition of the Gli3 repressor (Gli3R). We have discovered that Gli2A-mediated Shh signaling sequentially induces ventral neurons along the medial to lateral axis, and only before midgestation. Unlike in the spinal cord, Shh signaling plays a major role in patterning of dorsal structures (tectum and cerebellum). This function of Shh signaling involves inhibition of Gli3R and continues after midgestation. Gli3R levels also regulate overall growth of the mid/hindbrain region, and this largely involves the suppression of cell death. Furthermore, inhibition of Gli3R by Shh signaling is required to sustain expression of the AP organizer gene Fgf8. Thus, the precise spatial and temporal regulation of Gli2A and Gli3R by Shh is instrumental in coordinating mid/hindbrain development in three dimensions
— id: 96760, year: 2006, vol: 133, page: 1799, stat: Journal Article,

The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation
Corrales, JoMichelle D; Blaess, Sandra; Mahoney, Eamonn M; Joyner, Alexandra L
2006 May;133(9):1811-1821, Development
Foliation of the mouse cerebellum occurs primarily during the first 2 weeks after birth and is accompanied by tremendous proliferation of granule cell precursors (GCPs). We have previously shown that sonic hedgehog (Shh) signaling correlates spatially and temporally with fissure formation, and that Gli2 is the main activator driving Shh induced proliferation of embryonic GCPs. Here, we have tested whether the level of Shh signaling regulates the extent of cerebellar foliation. By progressively lowering signaling by removing Gli1 and Gli2 or the Shh receptor smoothened, we found the extent of foliation is gradually reduced, and that this correlates with a decrease in the duration of GCP proliferation. Importantly, the pattern of the remaining fissures in the mutants corresponds to the first fissures that form during normal development. In a complementary manner, an increase in the level and length of Shh signaling results in formation of an extra fissure in a position conserved in rat. The complexity of cerebellar foliation varies greatly between vertebrate species. Our studies have uncovered a mechanism by which the level and length of Shh signaling could be integral to determining the distinct number of fissures in each species
— id: 96761, year: 2006, vol: 133, page: 1811, stat: Journal Article,

Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development (vol 7, pg 772, 2006)
Fuccillo, M; Joyner, AL; Fishell, G
2006 ;7(11):902-902, Nature reviews. Neuroscience
— id: 104592, year: 2006, vol: 7, page: 902, stat: Journal Article,

Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development
Fuccillo, Marc; Joyner, Alexandra L; Fishell, Gord
2006 Oct;7(10):772-783, Nature reviews. Neuroscience
Sonic hedgehog has received an enormous amount of attention since its role as a morphogen that directs ventral patterning in the spinal cord was discovered a decade ago. Since that time, a bewildering array of information has been generated concerning both the components of the hedgehog signalling pathway and the remarkable number of contexts in which it functions. Nowhere is this more evident than in the nervous system, where hedgehog signalling has been implicated in events as disparate as axonal guidance and stem cell maintenance. Here we review our present knowledge of the hedgehog signalling pathway and speculate about areas in which further insights into this versatile pathway might be forthcoming
— id: 69028, year: 2006, vol: 7, page: 772, stat: Journal Article,

Genetic inducible fate mapping in mouse: establishing genetic lineages and defining genetic neuroanatomy in the nervous system
Joyner, Alexandra L; Zervas, Mark
2006 Sep;235(9):2376-2385, Developmental dynamics
A fascinating aspect of developmental biology is how organs are assembled in three dimensions over time. Fundamental to understanding organogenesis is the ability to determine when and where specific cell types are generated, the lineage of each cell, and how cells move to reside in their final position. Numerous methods have been developed to mark and follow the fate of cells in various model organisms used by developmental biologists, but most are not readily applicable to mouse embryos in utero because they involve physical marking of cells through injection of tracers. The advent of sophisticated transgenic and gene targeting techniques, combined with the use of site-specific recombinases, has revolutionized fate mapping studies in mouse. Furthermore, using genetic fate mapping to mark cells has opened up the possibility of addressing fundamental questions that cannot be studied with traditional methods of fate mapping in other organisms. Specifically, genetic fate mapping allows both the relationship between embryonic gene expression and cell fate (genetic lineage) to be determined, as well as the link between gene expression domains and anatomy (genetic anatomy) to be established. In this review, we present the ever-evolving development of genetic fate mapping techniques in mouse, especially the recent advance of Genetic Inducible Fate Mapping. We then review recent studies in the nervous system (focusing on the anterior hindbrain) as well as in the limb and with adult stem cells to highlight fundamental developmental processes that can be discovered using genetic fate mapping approaches. We end with a look toward the future at a powerful new approach that combines genetic fate mapping with cellular phenotyping alleles to study cell morphology, physiology, and function using examples from the nervous system
— id: 69244, year: 2006, vol: 235, page: 2376, stat: Journal Article,

Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain
Olsen, Shaun K; Li, James Y H; Bromleigh, Carrie; Eliseenkova, Anna V; Ibrahimi, Omar A; Lao, Zhimin; Zhang, Fuming; Linhardt, Robert J; Joyner, Alexandra L; Mohammadi, Moosa
2006 Jan 15;20(2):185-198, Genes & development
Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the 'c' splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the 'b' isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b(F32A) mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution
— id: 62746, year: 2006, vol: 20, page: 185, stat: Journal Article,

Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation
Pan, Yong; Bai, Chunyang Brian; Joyner, Alexandra L; Wang, Baolin
2006 May;26(9):3365-3377, Molecular & cellular biology
Gli2 and Gli3 are the primary transcription factors that mediate Sonic hedgehog (Shh) signals in the mouse. Gli3 mainly acts as a transcriptional repressor, because the majority of full-length Gli3 protein is proteolytically processed. Gli2 is mostly regarded as a transcriptional activator, even though it is also suggested to have a weak repressing activity. What the molecular basis for its possible dual function is and how its activity is regulated by Shh signaling are largely unknown. Here we demonstrate that unlike the results seen with Gli3 and Cubitus Interruptus, the fly homolog of Gli, only a minor fraction of Gli2 is proteolytically processed to form a transcriptional repressor in vivo and that in addition to being processed, Gli2 full-length protein is readily degraded. The degradation of Gli2 requires the phosphorylation of a cluster of numerous serine residues in its carboxyl terminus by protein kinase A and subsequently by casein kinase 1 and glycogen synthase kinase 3. The phosphorylated Gli2 interacts directly with betaTrCP in the SCF ubiquitin-ligase complex through two binding sites, which results in Gli2 ubiquitination and subsequent degradation by the proteasome. Both processing and degradation of Gli2 are suppressed by Shh signaling in vivo. Our findings provide the first demonstration of a molecular mechanism by which the Gli2 transcriptional activity is regulated by Shh signaling
— id: 96759, year: 2006, vol: 26, page: 3365, stat: Journal Article,

Recycling to the plasma membrane is delayed in EHD1 knockout mice
Rapaport, Debora; Auerbach, Wojtek; Naslavsky, Naava; Pasmanik-Chor, Metsada; Galperin, Emilia; Fein, Amos; Caplan, Steve; Joyner, Alexandra L; Horowitz, Mia
2006 Jan;7(1):52-60, Traffic
EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model
— id: 96762, year: 2006, vol: 7, page: 52, stat: Journal Article,

Reduced Ptc or Gli3 function enhances tumorigenicity of Shh-induced medulloblastomas in mice
Weiner, HL; Pompeiano, M; Mohan, A; Bakst, R; Piedimonte, L; Stephen, D; Babb, JS; Zagzag, D; Turnbull, DH; Joyner, AL
2006 OCT ;8(4):470-470, Neuro-oncology
— id: 70328, year: 2006, vol: 8, page: 470, stat: Journal Article,

In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog
Ahn, Sohyun; Joyner, Alexandra L
2005 Oct 6;437(7060):894-897, Nature
Sonic hedgehog (Shh) has been implicated in the ongoing neurogenesis in postnatal rodent brains. Here we adopted an in vivo genetic fate-mapping strategy, using Gli1 (GLI-Kruppel family member) as a sensitive readout of Shh activity, to systematically mark and follow the fate of Shh-responding cells in the adult mouse forebrain. We show that initially, only a small population of cells (including both quiescent neural stem cells and transit-amplifying cells) responds to Shh in regions undergoing neurogenesis. This population subsequently expands markedly to continuously provide new neurons in the forebrain. Our study of the behaviour of quiescent neural stem cells provides in vivo evidence that they can self-renew for over a year and generate multiple cell types. Furthermore, we show that the neural stem cell niches in the subventricular zone and dentate gyrus are established sequentially and not until late embryonic stages
— id: 58734, year: 2005, vol: 437, page: 894, stat: Journal Article,

Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh
Briegel, Karoline J; Baldwin, H Scott; Epstein, Jonathan A; Joyner, Alexandra L
2005 Jul;132(14):3305-3316, Development
Partial trisomy 2p syndrome includes a spectrum of congenital heart disease (CHD) that is characterized by complex malformations of the outflow and inflow tracts, defects in cardiac septation, heart position, as well as abnormal ventricular development. Lbh (limb-bud and heart) is a novel, highly conserved putative transcriptional regulatory protein, which displays a unique spatiotemporal gene expression pattern during early mouse heart development. Here we show that human LBH maps to chromosome 2p23, a genomic region related to CHD in partial trisomy 2p syndrome. Remarkably, transgenic overexpression of Lbh in mice throughout the embryonic myocardium from a cardiomyocyte-specific promoter of the cardiac ankyrin repeat protein gene (Carp/Ankrd1) models CHD reported in humans with partial trisomy 2p syndrome. The malformations in Carp-Lbh transgenic mice reflect impaired pulmonary outflow tract valvulogenesis, cardiac septation, inflow tract morphogenesis, as well as abnormalities in ventricular cardiomyocyte growth. Furthermore, we demonstrate that overexpression of Lbh in cultured mammalian cells represses the synergistic activity of key cardiac transcription factors, Nkx2.5 and Tbx5, leading to reduced activation of the common target gene, Anf (Nppa). Strikingly, reduced levels of Anf expression were also observed in embryonic day 9.5 Carp-Lbh transgenic mice. Thus, repression of Nkx2.5 and Tbx5-mediated gene expression by deregulated Lbh may account in part for the cardiac anomalies observed in these mice. Our findings implicate LBH as a candidate gene for CHD associated with partial trisomy 2p syndrome and suggest an important role of Lbh in transcriptional control during normal cardiogenesis
— id: 57869, year: 2005, vol: 132, page: 3305, stat: Journal Article,

Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh (vol 132, pg 3305, 2005)
Briegel, KJ; Baldwin, HS; Epstein, JA; Joyner, AL
2005 ;132(17):4015-4015, Development
— id: 104593, year: 2005, vol: 132, page: 4015, stat: Journal Article,

Genetic dissection of the role of En2 during cerebellum development
Cheng, YL; Sgaier, SK; Rocco, G; Villanueva, M; Berenshteyn, F; Joyner, AL
2005 JUL 15 ;283(2):667-667, Developmental biology (Orlando)
— id: 58648, year: 2005, vol: 283, page: 667, stat: Journal Article,

The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion
Cohen, Tsadok; Auerbach, Wojtek; Ravid, Liat; Bodennec, Jacques; Fein, Amos; Futerman, Anthony H; Joyner, Alexandra L; Horowitz, Mia
2005 Mar;25(6):2431-2440, Molecular & cellular biology
Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids
— id: 114462, year: 2005, vol: 25, page: 2431, stat: Journal Article,

Gli1 is important for medulloblastoma formation in Ptc1+/- mice
Kimura, Hiromichi; Stephen, Daniel; Joyner, Alexandra; Curran, Tom
2005 Jun 9;24(25):4026-4036, Oncogene
Germline mutations in the human homolog of the patched1 (PTCH1) are associated with basal cell nevus carcinoma syndrome (BCNS or Gorlin syndrome), which is characterized by developmental anomalies, radiation hypersensitivity and a predisposition to medulloblastomas and skin tumors. Patched1 (Ptc1) functions as a receptor for Sonic hedgehog (Shh) in a wide range of biological processes. Binding of Shh to Ptc1 results in activation of Smoothened (Smo), which in turn stimulates expression of downstream target genes including Ptc1 and Gli1. Gli1 is a member of a family of DNA-binding zinc-finger proteins, including Gli2 and Gli3, that function in transcription control. Here, we report that inactivation of both Gli1 alleles in Ptc1+/- mice significantly reduces spontaneous medulloblastoma formation. Therefore, Gli1 is not only a marker of pathway activation but also plays a functional role in medulloblastoma formation. Interestingly, Gli2 levels were elevated in medulloblastoma cells but not in normal granule neuron precursors during cerebellar development in mice lacking Gli1. In cultured fibroblasts, Gli1 was more potent than Gli2 at inducing cell transformation. These results demonstrate that Gli1 plays a central role in medulloblastoma formation in Ptc1+/- mice and that Gli2 may also contribute to oncogenesis
— id: 114461, year: 2005, vol: 24, page: 4026, stat: Journal Article,

New regulatory interactions and cellular responses in the isthmic organizer region revealed by altering Gbx2 expression
Li, James Y H; Lao, Zhimin; Joyner, Alexandra L
2005 Apr;132(8):1971-1981, Development
The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2-/-; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2-/-; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival
— id: 56065, year: 2005, vol: 132, page: 1971, stat: Journal Article,

Morphogenetic and cellular movements that shape the mouse cerebellum; insights from genetic fate mapping
Sgaier, Sema K; Millet, Sandrine; Villanueva, Melissa P; Berenshteyn, Frada; Song, Christian; Joyner, Alexandra L
2005 Jan 6;45(1):27-40, Neuron
We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis
— id: 49007, year: 2005, vol: 45, page: 27, stat: Journal Article,

Genetic dissection of midbrain and anterior hindbrain development
Zervas, M; Joyner, A
2005 JUL 15 ;283(2):623-623, Developmental biology (Orlando)
— id: 58646, year: 2005, vol: 283, page: 623, stat: Journal Article,

Classical embryological studies and modern genetic analysis of midbrain and cerebellum development
Zervas, Mark; Blaess, Sandra; Joyner, Alexandra L
2005 ;69:101-138, Current topics in developmental biology
The brain is a remarkably complex anatomical structure that contains a diverse array of subdivisions, cell types, and synaptic connections. It is equally extraordinary in its physiological properties, as it constantly evaluates and integrates external stimuli as well as controls a complicated internal environment. The brain can be divided into three primary broad regions: the forebrain, midbrain (Mb), and hindbrain (Hb), each of which contain further subdivisions. The regions considered in this chapter are the Mb and most-anterior Hb (Mb/aHb), which are derived from the mesencephalon (mes) and rhombomere 1 (r1), respectively. The dorsal Mb consists of the laminated superior colliculus and the globular inferior colliculus (Fig. 1A and B), which modulate visual and auditory stimuli, respectively. The dorsal component of the aHb is the highly foliated cerebellum (Cb), which is primarily attributed to controlling motor skills (Fig. 1A and B). In contrast, the ventral Mb/aHb (Fig. 1B) consists of distinct clusters of neurons that together comprise a network of nuclei and projections-notably, the Mb dopaminergic and Hb serotonergic and Mb/aHb cholinergic neurons (Fig. 1G and H), which modulate a collection of behaviors, including movement, arousal, feeding, wakefulness, and emotion. Historically, the dorsal Mb and Cb have been studied using the chick as a model system because of the ease of performing both cell labeling and tissue transplants in the embryo in ovo; currently DNA electroporation techniques are also used. More recently the mouse has emerged as a powerful genetic system with numerous advantages to study events underpinning Mb/aHb development. There is a diverse array of spontaneous mutants with both Mb- and Cb-related phenotypes. In addition, numerous gene functions have been enumerated in mouse, gene expression is similar across vertebrates, and powerful genetic tools have been developed. Finally, additional insight into Mb/aHb function has been gained from studies of genetic diseases, such as Parkinson's disease, schizophrenia, cancer, and Dandy Walker syndrome, that afflict the Mb/aHb in humans and have genetic counterparts in mouse. Accordingly, this chapter discusses a spectrum of experiments, including classic embryology, in vitro assays, sophisticated genetic methods, and human diseases. We begin with an overview of Mb and aHb anatomy and physiology and mes/r1 gene expression patterns. We then provide a summary of fate-mapping studies that collectively demonstrate the complex cell behaviors that occur while the Mb and aHb primordia are established during embryogenesis and discuss the integration of both anterior-posterior (A-P) and dorsal-ventral (D-V) patterning. Finally, we describe some aspects of postnatal development and some of the insights gained from human diseases
— id: 96763, year: 2005, vol: 69, page: 101, stat: Journal Article,

Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning
Ahn, Sohyun; Joyner, Alexandra L
2004 Aug 20;118(4):505-516, Cell
In the vertebrate limb, the posteriorly located zone of polarizing activity (ZPA) regulates digit identity through the morphogen Sonic Hedgehog (Shh). By genetically marking Shh-responding cells in mice, we have addressed whether the cumulative influence of positive Shh signaling over time and space reflects a linear gradient of Shh responsiveness and whether Shh could play additional roles in limb patterning. Our results show that all posterior limb mesenchyme cells, as well as the ectoderm, respond to Shh from the ZPA and become the bone, muscle, and skin of the posterior limb. Further, the readout of Shh activator function integrated over time and space does not display a stable and linear gradient along the A-P axis, as in a classical morphogen view. Finally, by fate mapping Shh-responding cells in Gli2 and Gli3 mutant limbs, we demonstrate that a specific level of positive Hh signaling is not required to specify digit identities
— id: 44885, year: 2004, vol: 118, page: 505, stat: Journal Article,

The knockout mouse project
Austin, Christopher P; Battey, James F; Bradley, Allan; Bucan, Maja; Capecchi, Mario; Collins, Francis S; Dove, William F; Duyk, Geoffrey; Dymecki, Susan; Eppig, Janan T; Grieder, Franziska B; Heintz, Nathaniel; Hicks, Geoff; Insel, Thomas R; Joyner, Alexandra; Koller, Beverly H; Lloyd, K C Kent; Magnuson, Terry; Moore, Mark W; Nagy, Andras; Pollock, Jonathan D; Roses, Allen D; Sands, Arthur T; Seed, Brian; Skarnes, William C; Snoddy, Jay; Soriano, Philippe; Stewart, David J; Stewart, Francis; Stillman, Bruce; Varmus, Harold; Varticovski, Lyuba; Verma, Inder M; Vogt, Thomas F; von Melchner, Harald; Witkowski, Jan; Woychik, Richard P; Wurst, Wolfgang; Yancopoulos, George D; Young, Stephen G; Zambrowicz, Brian
2004 Sep;36(9):921-924, Nature genetics
Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain
— id: 44884, year: 2004, vol: 36, page: 921, stat: Journal Article,

All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3
Bai, C Brian; Stephen, Daniel; Joyner, Alexandra L
2004 Jan;6(1):103-115, Developmental cell
An important question is how the gradient of Hedgehog is interpreted by cells at the level of the Gli transcription factors. The full range of Gli activity and its dependence on Hh have not been determined, although the Gli2 activator and Gli3 repressor have been implicated. Using the spinal cord as a model system, we demonstrate that Gli3 can transduce Hedgehog signaling as an activator. All expression of the Hh target gene Gli1 is dependent on both Gli2 and Gli3. Unlike Gli2, however, Gli3 requires endogenous Gli1 for induction of floor plate and V3 interneurons. Strikingly, embryos lacking all Gli function develop motor neurons and three ventral interneuron subtypes, similar to embryos lacking Hh signaling and Gli3. Therefore, in the spinal cord all Hh signaling is Gli dependent. Furthermore, a combination of Gli2 and Gli3 is required to regulate motor neuron development and spatial patterning of ventral spinal cord progenitors
— id: 42619, year: 2004, vol: 6, page: 103, stat: Journal Article,

Spatial pattern of sonic hedgehog signaling through Gli genes during cerebellum development
Corrales, JoMichelle D; Rocco, Gina L; Blaess, Sandra; Guo, Qiuxia; Joyner, Alexandra L
2004 Nov;131(22):5581-5590, Development
The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. We studied how Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo, and tested whether they influence foliation. We demonstrate that Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, we show that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth
— id: 47904, year: 2004, vol: 131, page: 5581, stat: Journal Article,

Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality
Hopkins, Kevin M; Auerbach, Wojtek; Wang, Xiang Yuan; Hande, M Prakash; Hang, Haiying; Wolgemuth, Debra J; Joyner, Alexandra L; Lieberman, Howard B
2004 Aug;24(16):7235-7248, Molecular & cellular biology
The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls. Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation. Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development
— id: 44887, year: 2004, vol: 24, page: 7235, stat: Journal Article,

EHD1 regulates recycling in mouse embryonic fibroblasts
Naslavsky, N; Rapaport, D; Auerbach, W; Joyner, AL; Horowitz, M; Caplan, S
2004 NOV ;15(3):113A-114A, Molecular biology of the cell
— id: 50419, year: 2004, vol: 15, page: 113A, stat: Journal Article,

How does Fgf signaling from the isthmic organizer induce midbrain and cerebellum development?
Sato, Tatsuya; Joyner, Alexandra L; Nakamura, Harukazu
2004 Dec;46(6):487-494, Development, growth & differentiation
The mesencephalic/rhombomere 1 border (isthmus) is an organizing center for early development of midbrain and cerebellum. In this review, we summarize recent progress in studies of Fgf signaling in the isthmus and discuss how the isthmus instructs the differentiation of the midbrain versus cerebellum. Fgf8 is shown to play a pivotal role in isthmic organizer activity. Only a strong Fgf signal mediated by Fgf8b activates the Ras-extracellular signal-regulated kinase (ERK) pathway, and this is sufficient to induce cerebellar development. A lower level of signaling transduced by Fgf8a, Fgf17 and Fgf18 induce midbrain development. Numerous feedback loops then maintain appropriate mesencephalon/rhombomere1 and organizer gene expression
— id: 56066, year: 2004, vol: 46, page: 487, stat: Journal Article,

Manganese-enhanced magnetic resonance imaging (MEMRI) of mouse brain development
Wadghiri, Youssef Zaim; Blind, Jeffrey A; Duan, Xiaohong; Moreno, Clement; Yu, Xin; Joyner, Alexandra L; Turnbull, Daniel H
2004 Dec;17(8):613-619, NMR in biomedicine
Given the importance of genetically modified mice in studies of mammalian brain development and human congenital brain diseases, MRI has the potential to provide an efficient in vivo approach for analyzing mutant phenotypes in the early postnatal mouse brain. The combination of reduced tissue contrast at the high magnetic fields required for mice, and the changing cellular composition of the developing mouse brain make it difficult to optimize MRI contrast in neonatal mouse imaging. We have explored an easily implemented approach for contrast-enhanced imaging, using systemically administered manganese (Mn) to reveal fine anatomical detail in T1-weighted MR images of neonatal mouse brains. In particular, we demonstrate the utility of this Mn-enhanced MRI (MEMRI) method for analyzing early postnatal patterning of the mouse cerebellum. Through comparisons with matched histological sections, we further show that MEMRI enhancement correlates qualitatively with granule cell density in the developing cerebellum, suggesting that the cerebellar enhancement is due to uptake of Mn in the granule neurons. Finally, variable cerebellar defects in mice with a conditional mutation in the Gbx2 gene were analyzed with MEMRI to demonstrate the utility of this method for mutant mouse phenotyping. Taken together, our results indicate that MEMRI provides an efficient and powerful in vivo method for analyzing neonatal brain development in normal and genetically engineered mice
— id: 52631, year: 2004, vol: 17, page: 613, stat: Journal Article,

Mutations in the sonic hedgehog pathway: Enhancement of medulloblastoma induction
Weiner, HL; Pompeiano, M; Mohan, A; Turnbull, DH; Joyner, AL
2004 ;100(4):780-780, Journal of neurosurgery
— id: 104594, year: 2004, vol: 100, page: 780, stat: Journal Article,

Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1
Zervas, Mark; Millet, Sandrine; Ahn, Sohyun; Joyner, Alexandra L
2004 Aug 5;43(3):345-357, Neuron
Brain structures derived from the mesencephalon (mes) and rhombomere 1 (r1) modulate distinct motor and sensory modalities. The precise origin and cellular behaviors underpinning the cytoarchitectural organization of the mes and r1, however, are unknown. Using a novel inducible genetic fate mapping approach in mouse, we determined the fate and lineage relationships of mes/r1 cells with fine temporal and spatial resolution. We demonstrate that the mes and r1 are neuromeres that along with the isthmic organizer are partitioned along the anterior-posterior axis by lineage restriction boundaries established sequentially between E8.5 and E9.5. Furthermore, a small group of cells originating from the most posterior mes exhibit anterior intracompartmental expansion and contribute throughout the inferior colliculus. Finally, we also uncovered transient and differential genetic lineages of ventral midbrain dopaminergic and ventral hindbrain serotonergic neuronal precursors with respect to Wnt1 and Gli1 expression
— id: 44886, year: 2004, vol: 43, page: 345, stat: Journal Article,

Strain-dependent differences in the efficiency of transgenic mouse production
Auerbach, Anna B; Norinsky, Rada; Ho, Weilan; Losos, Kasia; Guo, Qiuxia; Chatterjee, Samprit; Joyner, Alexandra L
2003 Feb;12(1):59-69, Transgenic research
Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving approximately 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production
— id: 35028, year: 2003, vol: 12, page: 59, stat: Journal Article,

A gene expression atlas of the central nervous system based on bacterial artificial chromosomes
Gong, Shiaoching; Zheng, Chen; Doughty, Martin L; Losos, Kasia; Didkovsky, Nicholas; Schambra, Uta B; Nowak, Norma J; Joyner, Alexandra; Leblanc, Gabrielle; Hatten, Mary E; Heintz, Nathaniel
2003 Oct 30;425(6961):917-925, Nature
The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community
— id: 44890, year: 2003, vol: 425, page: 917, stat: Journal Article,

Fate map of mouse ventral limb ectoderm and the apical ectodermal ridge
Guo, Qiuxia; Loomis, Cynthia; Joyner, Alexandra L
2003 Dec 1;264(1):166-178, Developmental biology (Orlando)
The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5-E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral border nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb
— id: 44888, year: 2003, vol: 264, page: 166, stat: Journal Article,

FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors
Liu, Aimin; Li, James Y H; Bromleigh, Carrie; Lao, Zhimin; Niswander, Lee A; Joyner, Alexandra L
2003 Dec;130(25):6175-6185, Development
Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We and others have found that FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. In this study we used a chick electroporation assay and in vitro mouse brain explant experiments to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. First, FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. Second, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as Fgf8 is. Third, analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b showed that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. As Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, our studies have broad implications for how these FGFs differentially control development
— id: 44889, year: 2003, vol: 130, page: 6175, stat: Journal Article,

Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway
Bai, C Brian; Auerbach, Wojtek; Lee, Joon S; Stephen, Daniel; Joyner, Alexandra L
2002 Oct;129(20):4753-4761, Development
The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, we generated a Gli1 null allele expressing lacZ. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, we show that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. We show that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Of significance, we found instead that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. Our studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc
— id: 35030, year: 2002, vol: 129, page: 4753, stat: Journal Article,

Formation of a signaling center that patterns the midbrain and cerebellum in mouse
Joyner, A; Li, YJ; Zervas, M
2002 Mar 22;16(5):A1111-A1111, FASEB journal
— id: 27481, year: 2002, vol: 16, page: A1111, stat: Journal Article,

Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer
Li, James Y H; Lao, Zhimin; Joyner, Alexandra L
2002 Sep 26;36(1):31-43, Neuron
We examined whether Gbx2 is required after embryonic day 9 (E9) to repress Otx2 in the cerebellar anlage and position the midbrain/hindbrain organizer. In contrast to Gbx2 null mutants, mice lacking Gbx2 in rhombomere 1 (r1) after E9 (Gbx2-CKO) are viable and develop a cerebellum. A Gbx2-independent pathway can repress Otx2 in r1 after E9. Mid/hindbrain organizer gene expression, however, continues to be dependent on Gbx2. We found that Fgf8 expression normally correlates with the isthmus where cells undergo low proliferation and that in Gbx2-CKO mutants this domain is expanded. We propose that Fgf8 permits lateral cerebellar development through repression of Otx2 and also suppresses medial cerebellar growth in Gbx2-CKO embryos. Our work has uncovered distinct requirements for Gbx2 during cerebellum formation and provided a model for how a transcription factor can play multiple roles during development
— id: 35029, year: 2002, vol: 36, page: 31, stat: Journal Article,

A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development
Nakashima, M; Tanese, N; Ito, M; Auerbach, W; Bai, C; Furukawa, T; Toyono, T; Akamine, A; Joyner, A L
2002 Nov;119(1):21-21, Mechanisms of development
The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development
— id: 32540, year: 2002, vol: 119, page: 21, stat: Journal Article,

Induction of medulloblastomas in mice, in the absence of Gli1
Weiner HL; Joyner A; Turnbull DH
2002 ;96:450-450, Journal of neurosurgery
— id: 34019, year: 2002, vol: 96, page: 450, stat: Journal Article,

Induction of medulloblastomas in mice by sonic hedgehog, independent of Gli1
Weiner, Howard L; Bakst, Richard; Hurlbert, Marc S; Ruggiero, Jason; Ahn, Esther; Lee, Wing Shing; Stephen, Daniel; Zagzag, David; Joyner, Alexandra L; Turnbull, Daniel H
2002 Nov 15;62(22):6385-6389, Cancer research
The Sonic hedgehog (Shh) signaling pathway plays a critical role in normal cerebellar development and has been implicated in medulloblastomas, common malignant childhood tumors of the cerebellum. To test whether Shh mis-expression is sufficient for medulloblastoma formation, we used ultrasound biomicroscopy-guided in utero injection of a Shh-expressing retrovirus into the cerebellum of 13.5-day mouse embryos to show that direct activation of the Shh pathway can lead to tumor formation. Significantly, medulloblastomas were observed in 76% of the mice infected with Shh-expressing retrovirus. Furthermore, contrary to recent suggestions that the Shh transcriptional target Gli1 plays a critical role in Shh-induced tumorigenesis, we found that medulloblastomas form in Gli1 null mutant mice. We have developed an efficient mouse model of medulloblastoma and shown that Gli1 is not required for tumorigenesis when Shh signaling is activated upstream in the pathway
— id: 34734, year: 2002, vol: 62, page: 6385, stat: Journal Article,

Location, development, control, and function of extraadrenal phenylethanolamine N-methyltransferase
Ziegler, MG; Bao, X; Kennedy, BP; Joyner, A; Enns, R
2002 ;971(22):76-82, Annals of the New York Academy of Sciences
Phenylethanolamine N-methyltransferase (PNMT) methylates norepinephrine (NE) to form epinephrine (E). It is present in a high concentration in the adrenal medula but occurs in many other tissues throughout the body. In the brain stem and retina PNNIT is present in specific neurons. Cardiac PNNIT develops early in the fetal heart and is found in relatively high levels in the adult left atrium. Intrinsic cardiac adrenergic cells are distributed throughout the adult myocardium and contain all the enzymes necessary for E synthesis. The PNNIT gene promoter region contains a glucocorticoid response element; however, the initial development of brain and cardiac fetal PNMT is glucocorticoid independent. Rat fetal heart PNMT peaks at embryonic day 11 and becomes sensitive to glucocorticoid induction by day 12. PNMT-containing cells are concentrated in the atrioventricular canal and interventricular septum during cardiac development, areas important in the development of the cardiac conduction system. In the adult rat, cardiac PNMT is inducible by glucocorticoids and synthesizes E. Glucocorticoids are essential for development of the high levels of PNMT in the adrenal, but are less important outside the adrenal. The PNMT gene contains 3 exons and 2 introns. Adrenal PNMT mRNA exists as a single type, but in the heart PNMT mRNA is present as both an intronless and an intron-containing type. In some cardiac tissues, glucocorticoids decrease levels of intron-containing PNMT mRNA and increase intronless PNMT mRNA and PNMT activity. Studies in adrenalectomized animals suggest that extraadrenal PNMT increases blood pressure, blood glucose, and lymphocyte cytokine production. PNMT may also play a role in the regulation of fetal heart rate prior to development of the adrenal medulla. :
— id: 104595, year: 2002, vol: 971, page: 76, stat: Journal Article,

The HD mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin
Auerbach W; Hurlbert MS; Hilditch-Maguire P; Wadghiri YZ; Wheeler VC; Cohen SI; Joyner AL; MacDonald ME; Turnbull DH
2001 Oct 15;10(22):2515-2523, Human molecular genetics
Huntingtin is an essential protein that with mutant polyglutamine tracts initiates dominant striatal neurodegeneration in Huntington's disease (HD). To assess the consequences of mutant protein when huntingtin is limiting, we have studied three lines of compound heterozygous mice in which both copies of the HD gene homolog (Hdh) were altered, resulting in greatly reduced levels of huntingtin with a normal human polyglutamine length (Q20) and/or an expanded disease-associated segment (Q111): Hdh(neoQ20)/Hdh(neoQ20), Hdh(neoQ20)/Hdh(null) and Hdh(neoQ20)/Hdh(neoQ111). All surviving mice in each of the three lines were small from birth, and had variable movement abnormalities. Magnetic resonance micro-imaging and histological evaluation showed enlarged ventricles in approximately 50% of the Hdh(neoQ20)/Hdh(neoQ111) and Hdh(neoQ20)/Hdh(null) mice, revealing a developmental defect that does not worsen with age. Only Hdh(neoQ20)/Hdh(neoQ111) mice exhibited a rapidly progressive movement disorder that, in the absence of striatal pathology, begins with hind-limb clasping during tail suspension and tail stiffness during walking by 3-4 months of age, and then progresses to paralysis of the limbs and tail, hypokinesis and premature death, usually by 12 months of age. Thus, dramatically reduced huntingtin levels fail to support normal development in mice, resulting in reduced body size, movement abnormalities and a variable increase in ventricle volume. On this sensitized background, mutant huntingtin causes a rapid neurological disease, distinct from the HD-pathogenic process. These results raise the possibility that therapeutic elimination of huntingtin in HD patients could lead to unintended neurological, as well as developmental side-effects
— id: 35033, year: 2001, vol: 10, page: 2515, stat: Journal Article,

Gli1 can rescue the in vivo function of Gli2
Bai CB; Joyner AL
2001 Dec;128(24):5161-5172, Development
In mice, three Gli genes are thought to mediate sonic hedgehog (Shh) signaling collectively. Mis-expression studies and analysis of null mutants for each gene have indicated that the Gli proteins have different functions. In particular, Gli1 appears to be a constitutive activator, and Gli2 and Gli3 have repressor functions. To determine the precise functional differences between Gli1 and Gli2, we have expressed Gli1 in place of Gli2 from the endogenous Gli2 locus in mice. Strikingly, a low level of Gli1 can rescue all the Shh signaling defects in Gli2 mutants; however, only in the presence of a wild-type Shh gene. These studies demonstrate that only the activator function of Gli2 is actually required, and indicates that in specific situations, Shh can modulate the ability of Gli1 to activate target genes. Furthermore, expression of both copies of Gli1 in place of Gli2 does not disrupt spinal cord patterning, but does result in new gain-of-function defects that lead to lethality. We show that the defects are enhanced when Gli3 function is reduced, demonstrating that an important difference between Gli1 and Gli2 is the ability of Gli1 to antagonize Gli3 function
— id: 35031, year: 2001, vol: 128, page: 5161, stat: Journal Article,

Identification and characterization of Lbh, a novel conserved nuclear protein expressed during early limb and heart development
Briegel KJ; Joyner AL
2001 May 15;233(2):291-304, Developmental biology (Orlando)
We report the cloning, protein characterization, and expression of a novel vertebrate gene, termed Lbh (Limb-bud-and-heart), with a spatiotemporal expression pattern that marks embryologically significant domains in the developing limbs and heart. Lbh encodes a highly conserved nuclear protein, which in tissue culture cells possesses a transcriptional activator function. During limb development, expression of Lbh initiates in the ectoderm of the presumptive limb territory in the lateral body wall. As the limb buds appear, Lbh expression is restricted primarily to the distal ventral limb ectoderm and the apical ectodermal ridge, and overlaps in these ectodermal compartments with En1 and Fgf8 expression. During heart formation, Lbh is expressed as early as Nkx2.5 and dHand in the bilateral heart primordia, with the highest levels in the anterior promyocardium. After heart tube fusion and looping, Lbh expression is confined to the ventricular myocardium, with the highest intensity in the right ventricle and atrioventricular canal, as well as in the sinus venosus. Based on the molecular characteristics and the domain-specific expression pattern, it is possible that Lbh functions in synergy with other genes known to be required for heart and limb development.
— id: 20688, year: 2001, vol: 233, page: 291, stat: Journal Article,

Unique and conserved pathways across species
Joyner A; Martinez-Arias A
2001 Aug;11(4):363-366, Current opinion in genetics & development
— id: 21129, year: 2001, vol: 11, page: 363, stat: Journal Article,

Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression
Li JY; Joyner AL
2001 Dec;128(24):4979-4991, Development
Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes
— id: 35032, year: 2001, vol: 128, page: 4979, stat: Journal Article,

Early anterior/posterior patterning of the midbrain and cerebellum
Liu A; Joyner AL
2001 ;24(1):869-896, Annual review of neuroscience
Transplantation studies performed in chicken embryos indicated that early anterior/posterior patterning of the vertebrate midbrain and cerebellum might be regulated by an organizing center at the junction between the midbrain and hindbrain. More than a decade of molecular and genetic studies have shown that such an organizer is indeed central to development of the midbrain and anterior hindbrain. Furthermore, a complicated molecular network that includes multiple positive and negative feedback loops underlies the establishment and refinement of a mid/hindbrain organizer, as well as the subsequent function of the organizer. In this review, we first introduce the expression patterns of the genes known to be involved in this patterning process and the quail-chick transplantation experiments that have provided the foundation for understanding the genetic pathways regulating mid/hindbrain patterning. Subsequently, we discuss the molecular genetic studies that have revealed the roles for many genes in normal early patterning of this region. Finally, some of the remaining questions and future directions are discussed
— id: 26695, year: 2001, vol: 24, page: 869, stat: Journal Article,

EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region
Liu A; Joyner AL
2001 Jan;128(2):181-191, Development
Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address through what genetic pathways FGF8 acts, we examined the epistatic relationships of mid/hindbrain genes that respond to FGF8, using a novel mouse brain explant culture system. We found that En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone
— id: 26815, year: 2001, vol: 128, page: 181, stat: Journal Article,

Multiple levels of autoregulation on FGF signaling during mouse mid-/hindbrain early patterning
Liu, AM; Joyner, AL
2001 JUL 1 ;235(1):224-224, Developmental biology (Orlando)
— id: 55004, year: 2001, vol: 235, page: 224, stat: Journal Article,

Formation of the mesencephalic/metencephalic boundary
Zervas, M; Millet, S; Joyner, A
2001 JUL 1 ;235(1):221-221, Developmental biology (Orlando)
— id: 55003, year: 2001, vol: 235, page: 221, stat: Journal Article,

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines
Auerbach W; Dunmore JH; Fairchild-Huntress V; Fang Q; Auerbach AB; Huszar D; Joyner AL
2000 Nov;29(5):1024-8, 1030, 1032, Biotechniques
Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline
— id: 35034, year: 2000, vol: 29, page: 1024, stat: Journal Article,

Progressive neurological disease in mice expressing reduced amounts of huntingtin with 20 and 111 glutamine repeat
Auerbach, W; Hurlbert, MS; Hilditch-Maguire, P; Wadghiri, YZ; Wheeler, VC; Cohen, SI; Joyner, AL; MacDonald, ME; Turnbull, DH
2000 OCT ;67(4):364-364, American journal of human genetics
— id: 54432, year: 2000, vol: 67, page: 364, stat: Journal Article,

Classical genetics and gene targeting
Bultman, Scott; Magnuson, Terry; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5214, year: 2000, vol: , page: 255, stat: Chapter,

Site-specific recombination in cellss and mice
Dymecki, Susan M.; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5215, year: 2000, vol: , page: 37, stat: Chapter,

Members of the bHLH-PAS family regulate Shh transcription in forebrain regions of the mouse CNS
Epstein DJ; Martinu L; Michaud JL; Losos KM; Fan C; Joyner AL
2000 Nov;127(21):4701-4709, Development
The secreted protein sonic hedgehog (Shh) is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified which direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. We show here that the activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2(-)(/)(-) embryos, it was determined to be absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon
— id: 35036, year: 2000, vol: 127, page: 4701, stat: Journal Article,

Gene targeting, principles, and practice in mammalian cells
Hasty, Paul; Abuin, Alejandro; Bradley, Allan; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5216, year: 2000, vol: , page: 1, stat: Chapter,

Progressive neurological disease in mice expressing reduced amounts of Huntingtin with 18 and 109 glutamine repeats
Hurlbert, MS; Auerbach, W; Wadghiri, YZ; Wheeler, V; MacDonald, ME; Joyner, AL; Tumbull, DH
2000 FEB ;74(2):S16-S16, Journal of neurochemistry
— id: 54743, year: 2000, vol: 74, page: S16, stat: Journal Article,

LSP1 modulates leukocyte populations in resting and inflamed peritoneum
Jongstra-Bilen J; Misener VL; Wang C; Ginzberg H; Auerbach A; Joyner AL; Downey GP; Jongstra J
2000 Sep 1;96(5):1827-1835, Blood
Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1(-/-)) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen. Lsp1(-/-) mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5(-) macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in Lsp1(-/-) mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1(-/-) mice are similar to those of wt mice and Lsp1(-/-) mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the Lsp1(-/-) mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1(-) neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis. (Blood. 2000;96:1827-1835)
— id: 35037, year: 2000, vol: 96, page: 1827, stat: Journal Article,

Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer
Joyner AL; Liu A; Millet S
2000 Dec;12(6):736-741, Current opinion in cell biology
A decade ago, chick-quail transplantation studies demonstrated that the junction between the midbrain and hindbrain has the properties of an organizing center capable of patterning the midbrain and cerebellum. Many of the genes that function to pattern these tissues have been identified and extensively studied. Recent experiments have shown that Otx2, Gbx2 and Fgf8 genes play a major role in the positioning and functioning of this organizing center
— id: 35035, year: 2000, vol: 12, page: 736, stat: Journal Article,

Gene Targeting
Joyner, Alexandra L
New York : Oxford University Press, 2000,
It is now possible to make any mutation in the germline of mice by utilizing recombination and embryonic stem (ES) cells. Homologous recombination when applied to altering endogenous genes is called gene targeting. This laboratory manual contains 7 separately authored chapters which describe laboratory methods for studying gene targeitng. The manual updates previously described techniques and also describes new techniques that have expanded the types of mutations that can be made using recombination in ES cells. Experimental protocols are provided. Diagrams, graphs, photographs, and tables illustrate the text. An index and a list of suppliers are included
— id: 627, year: 2000, vol: , page: , stat: ,

Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation
Kimmel RA; Turnbull DH; Blanquet V; Wurst W; Loomis CA; Joyner AL
2000 Jun 1;14(11):1377-1389, Genes & development
Proximal-distal outgrowth of the vertebrate limb bud is regulated by the apical ectodermal ridge (AER), which forms at an invariant position along the dorsal-ventral (D/V) axis of the embryo. We have studied the genetic and cellular events that regulate AER formation in the mouse. In contrast to implications from previous studies in chick, we identified two distinct lineage boundaries in mouse ectoderm prior to limb bud outgrowth using a Cre/loxP-based fate-mapping approach and a novel retroviral cell-labeling technique. One border is transient and at the limit of expression of the ventral gene En1, which corresponds to the D/V midline of the AER, and the second border corresponds to the dorsal AER margin. Labeling of AER precursors using an inducible Cre showed that not all cells that initially express AER genes form the AER, indicating that signaling is required to maintain an AER phenotype. Misexpression of En1 at moderate levels specifically in the dorsal AER of transgenic mice was found to produce dorsally shifted AER fragments, whereas high levels of En1 abolished AER formation. In both cases, the dorsal gene Wnt7a was repressed in cells adjacent to the En1-expressing cells, demonstrating that signaling regulated by EN1 occurs across the D/V border. Finally, fate mapping of AER domains in these mutants showed that En1 plays a part in positioning and maintaining the two lineage borders
— id: 11671, year: 2000, vol: 14, page: 1377, stat: Journal Article,

Two lineage boundaries and EN1 coordinate AER formation
Kimmel, RA; Turnbull, DH; Blanquet, V; Wurst, W; Loomis, CA; Joyner, AL
2000 JUN 1 ;222(1):227-227, Developmental biology (Orlando)
— id: 54559, year: 2000, vol: 222, page: 227, stat: Journal Article,

Two Pax2/5/8-binding sites in Engrailed2 are required for proper initiation of endogenous mid-hindbrain expression
Li Song D; Joyner AL
2000 Feb;90(2):155-165, Mechanisms of development
During early brain development mouse Engrailed2 (En2) is expressed in a broad band across most of the mid-hindbrain region. Evidence from gene expression data, promoter analysis in transgenic mice and mutant phenotype analysis in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical role in regulating En2 mid-hindbrain expression. Previously, we identified two Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficient to directed reporter gene expression to the early mid-hindbrain region and showed that the two Pax2/5/8-binding sites are essential for the mid-hindbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the context of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced with the bacterial neo gene by homologous recombination in mouse embryonic stem cells. After transmitting the mutation into mice, the neo gene was removed by breeding with transgenic mice expressing cre from a CMV promoter. Embryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression indistinguishable from that seen in wild type embryos. Furthermore, the mutants did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expression of the endogenous En2 gene. Interestingly, a comparison of the lacZ RNA and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbrain region by persisting longer than the mRNA. The transgene expression directed by the 1.0 kb enhancer fragment therefore does not mimic the entire broad domain of En2 expression. Taken together, these two studies demonstrate that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression
— id: 27859, year: 2000, vol: 90, page: 155, stat: Journal Article,

Production and analysis of ES cell aggregation chimeras
Nagy, Andras; Rossant, Janet; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5217, year: 2000, vol: , page: 177, stat: Chapter,

Production of chimeras by blastocyst and morula injection of targeted ES cells
Papaioannou, Virginia; Johnson, Randall; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5218, year: 2000, vol: , page: 133, stat: Chapter,

Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation
Park HL; Bai C; Platt KA; Matise MP; Beeghly A; Hui CC; Nakashima M; Joyner AL
2000 Apr;127(8):1593-1605, Development
The secreted factor Sonic hedgehog (SHH) is both required for and sufficient to induce multiple developmental processes, including ventralization of the CNS, branching morphogenesis of the lungs and anteroposterior patterning of the limbs. Based on analogy to the Drosophila Hh pathway, the multiple GLI transcription factors in vertebrates are likely to both transduce SHH signaling and repress Shh transcription. In order to discriminate between overlapping versus unique requirements for the three Gli genes in mice, we have produced a Gli1 mutant and analyzed the phenotypes of Gli1/Gli2 and Gli1/3 double mutants. Gli3(xt) mutants have polydactyly and dorsal CNS defects associated with ectopic Shh expression, indicating GLI3 plays a role in repressing Shh. In contrast, Gli2 mutants have five digits, but lack a floorplate, indicating that it is required to transduce SHH signaling in some tissues. Remarkably, mice homozygous for a Gli1(zfd )mutation that deletes the exons encoding the DNA-binding domain are viable and appear normal. Transgenic mice expressing a GLI1 protein lacking the zinc fingers can not induce SHH targets in the dorsal brain, indicating that the Gli1(zfd )allele contains a hypomorphic or null mutation. Interestingly, Gli1(zfd/zfd);Gli2(zfd/+), but not Gli1(zfd/zfd);Gli3(zfd/+) double mutants have a severe phenotype; most Gli1(zfd/zfd);Gli2(zfd/+) mice die soon after birth and all have multiple defects including a variable loss of ventral spinal cord cells and smaller lungs that are similar to, but less extreme than, Gli2(zfd/zfd) mutants. Gli1/Gli2 double homozygous mutants have more extreme CNS and lung defects than Gli1(zfd/zfd);Gli2(zfd/+) mutants, however, in contrast to Shh mutants, ventrolateral neurons develop in the CNS and the limbs have 5 digits with an extra postaxial nubbin. These studies demonstrate that the zinc-finger DNA-binding domain of GLI1 protein is not required for SHH signaling in mouse. Furthermore, Gli1 and Gli2, but not Gli1 and Gli3, have extensive overlapping functions that are likely downstream of SHH signaling
— id: 11795, year: 2000, vol: 127, page: 1593, stat: Journal Article,

Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and HdhQ111 knock-in mice
Wheeler VC; White JK; Gutekunst CA; Vrbanac V; Weaver M; Li XJ; Li SH; Yi H; Vonsattel JP; Gusella JF; Hersch S; Auerbach W; Joyner AL; MacDonald ME
2000 Mar 1;9(4):503-513, Human molecular genetics
Huntington's disease (HD) is caused by an expanded N-terminal glutamine tract that endows huntingtin with a striatal-selective structural property ultimately toxic to medium spiny neurons. In precise genetic models of juvenile HD, HdhQ92 and HdhQ111 knock-in mice, long polyglutamine segments change huntingtin's physical properties, producing HD-like in vivo correlates in the striatum, including nuclear localization of a version of the full-length protein predominant in medium spiny neurons, and subsequent formation of N-terminal inclusions and insoluble aggregate. These changes show glutamine length dependence and dominant inheritance with recruitment of wild-type protein, critical features of the altered HD property that strongly implicate them in the HD disease process and that suggest alternative pathogenic scenarios: the effect of the glutamine tract may act by altering interaction with a critical cellular constituent or by depleting a form of huntingtin essential to medium spiny striatal neurons
— id: 35038, year: 2000, vol: 9, page: 503, stat: Journal Article,

Gene trap strategies in ES cells
Wurst, Wolfgand; Gossler, Achim; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 2000,
— id: 5219, year: 2000, vol: , page: 207, stat: Chapter,

Investigation of the role of BMP's in mammalian forebrain development
Corbin, Joshua G; Turnbull, Daniel H; Joyner, Alex; Fishell, Gord
1999 Oct 23-28;25(1-2):525-525, Abstracts (Society for Neuroscience)
— id: 15868, year: 1999, vol: 25, page: 525, stat: Journal Article,

Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent mechanisms
Epstein DJ; McMahon AP; Joyner AL
1999 Jan;126(2):281-292, Development
The axial midline mesoderm and the ventral midline of the neural tube, the floor plate, share the property of being a source of the secreted protein, Sonic hedgehog (Shh), which has the capacity to induce a variety of ventral cell types along the length of the mouse CNS. To gain insight into the mechanisms by which Shh transcription is initiated in these tissues, we set out to identify the cis-acting sequences regulating Shh gene expression. As an approach, we have tested genomic clones encompassing 35 kb of the Shh locus for their ability to direct a lacZ reporter gene to the temporally and spatially restricted confines of the Shh expression domains in transgenic mice. Three enhancers were identified that directed lacZ expression to distinct regions along the anteroposterior axis including the ventral midline of the spinal cord, hindbrain, rostral midbrain and caudal diencephalon, suggesting that multiple transcriptional regulators are required to initiate Shh gene expression within the CNS. In addition, regulatory sequences were also identified that directed reporter expression to the notochord, albeit, under limited circumstances. Sequence analysis of the genomic clones responsible for enhancer activity from a variety of organisms, including mouse, chicken and human, have identified highly conserved binding sites for the hepatocyte nuclear factor 3 (Hnf3) family of transcriptional regulators in some, but not all, of the enhancers. Moreover, the generation of mutations in the Hnf3-binding sites showed their requirement in certain, but not all, aspects of Shh reporter expression. Taken together, our results support the existence of Hnf3-dependent and -independent mechanisms in the direct activation of Shh transcription within the CNS and axial mesoderm
— id: 7326, year: 1999, vol: 126, page: 281, stat: Journal Article,

Huntingtin is required during embryogenesis
Hilditch-Maguire, PA; Calzonetti, T; Joyner, AL; MacDonald, ME
1999 ;10(2):1198-1198, Molecular biology of the cell
— id: 104596, year: 1999, vol: 10, page: 1198, stat: Journal Article,

En1 plays multiple roles in vertebrate limb development
Kimmel, R; Loomis, C; Losos, K; Turnbull, D; Joyner, A
1999 JUN 1 ;210(1):228-228, Developmental biology (Orlando)
— id: 54019, year: 1999, vol: 210, page: 228, stat: Journal Article,

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate
Liu A; Losos K; Joyner AL
1999 Nov;126(21):4827-4838, Development
The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression
— id: 6260, year: 1999, vol: 126, page: 4827, stat: Journal Article,

Fgf8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate
Liu, A; Losos, K; Joyner, A
1999 JUN 1 ;210(1):227-227, Developmental biology (Orlando)
— id: 54018, year: 1999, vol: 210, page: 227, stat: Journal Article,

Gli genes in development and cancer
Matise MP; Joyner AL
1999 Dec 20;18(55):7852-7859, Oncogene
With the realization that many proto-oncogenes and tumor suppressor genes are expressed and have important functions during mammalian development, it is clear that cancer often involves the inappropriate activation of genetic pathways used during normal development. A signaling cascade that has been of considerable interest to both developmental and cancer biologists involves the Hedgehog (Hh) family of secreted proteins. To date, the only transcription factors shown to be directly downstream of Hh are the zinc-finger containing proteins Cubitus interruptus (Ci) and Gli, in flies and vertebrates, respectively. The identification of many of the genes and proteins involved in Hh signaling has come largely from genetic and biochemical studies in Drosophila. Ci mediates Hh signaling through a Hh-dependent set of protein modifications that alter the activity of Ci on Hh target genes. Recent evidence suggests vertebrate Gli proteins may be similarly regulated. The interest in this pathway has taken on added importance with the identification of mutations in Hh pathway genes, including Gli genes, in several human developmental disorders and cancers. We discuss models for how Gli proteins mediate Hh signaling in both vertebrate development and cancers
— id: 8594, year: 1999, vol: 18, page: 7852, stat: Journal Article,

Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord
Matise MP; Lustig M; Sakurai T; Grumet M; Joyner AL
1999 Aug;126(16):3649-3659, Development
Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants
— id: 6162, year: 1999, vol: 126, page: 3649, stat: Journal Article,

A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer
Millet S; Campbell K; Epstein DJ; Losos K; Harris E; Joyner AL
1999 Sep 9;401(6749):161-164, Nature
The mid/hindbrain (MHB) junction can act as an organizer to direct the development of the midbrain and anterior hindbrain. In mice, Otx2 is expressed in the forebrain and midbrain and Gbx2 is expressed in the anterior hindbrain, with a shared border at the level of the MHB organizer. Here we show that, in Gbx2-/- mutants, the earliest phenotype is a posterior expansion of the Otx2 domain during early somite stages. Furthermore, organizer genes are expressed at the shifted Otx2 border, but not in a normal spatial relationship. To test whether Gbx2 is sufficient to position the MHB organizer, we transiently expressed Gbx2 in the caudal Otx2 domain and found that the Otx2 caudal border was indeed shifted rostrally and a normal appearing organizer formed at this new Otx2 border. Transgenic embryos then showed an expanded hindbrain and a reduced midbrain at embryonic day 9.5-10. We propose that formation of a normal MHB organizer depends on a sharp Otx2 caudal border and that Gbx2 is required to position and sharpen this border
— id: 56473, year: 1999, vol: 401, page: 161, stat: Journal Article,

EHD1 - An EH-domain-containing protein with a specific expression pattern
Mintz, L; Galperin, E; Pasmanik-Chor, M; Tulzinsky, S; Bromberg, Y; Kozak, CA; Joyner, A; Fein, A; Horowitz, M
1999 JUL 1 ;59(1):66-76, Genomics
A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well, as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles, Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes,and in specific retinal layers. Results of ia situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches-and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 ERD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly con-served between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis. (C) 1999 Academic Press
— id: 53997, year: 1999, vol: 59, page: 66, stat: Journal Article,

Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis
Nakashima, M; Toyono, T; Akamine, A; Joyner, A
1999 Feb;80(2):185-189, Mechanisms of development
We have cloned and characterized a new member of the bone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta) superfamily, growth differentiation factor 11 (Gdf11), from rat incisor pulp RNA by reverse transcription-polymerase chain reaction using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is most closely related to Gdf8, being 90% identical to the mouse gene. Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole-mount embryos demonstrated Gdf11 is first strongly expressed in restricted domains at 8.5 days post coitus (dpc) when it is highest in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminally-differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain
— id: 111697, year: 1999, vol: 80, page: 185, stat: Journal Article,

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse
Wheeler VC; Auerbach W; White JK; Srinidhi J; Auerbach A; Ryan A; Duyao MP; Vrbanac V; Weaver M; Gusella JF; Joyner AL; MacDonald ME
1999 Jan;8(1):115-122, Human molecular genetics
The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species
— id: 57082, year: 1999, vol: 8, page: 115, stat: Journal Article,

A progressive nuclear phenotype in precise genetic mouse models of Huntington's disease
Wheeler, Vanessa C.; White, Jacqueline K.; Gutekunst, Claire-Anne; Auerbach, Wojtek; Hersch, Steven; Joyner, Alexandra L.; Gusella, James F.; MacDonald, Marcy E.
1999 ;25(1-2):21-21, Abstracts (Society for Neuroscience)
— id: 104597, year: 1999, vol: 25, page: 21, stat: Journal Article,

Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development
Hanks MC; Loomis CA; Harris E; Tong CX; Anson-Cartwright L; Auerbach A; Joyner A
1998 Nov;125(22):4521-4530, Development
The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally
— id: 7339, year: 1998, vol: 125, page: 4521, stat: Journal Article,

A mouse model of Greig cephalapolysyndactyly syndrome: the extra-toes' mutation contains an intragenic deletion of the Gli3 gene (vol 3, pg 241, 1993)
Hui, CC; Joyner, AL
1998 ;19(4):404-404, Nature genetics
— id: 104598, year: 1998, vol: 19, page: 404, stat: Journal Article,

Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells
Liu A; Joyner AL; Turnbull DH
1998 Jul;75(1-2):107-115, Mechanisms of development
A basic limitation of the study of development in the mouse is the inaccessibility of the embryos, which are encased in the maternal uterus. We demonstrate the first use of ultrasound backscatter microscopy for guiding injections of cells and other agents into early stage mouse embryos. Cells were injected into the mouse neural tube cavity as early as 9.5 days post coitus (E9.5), and into the developing limb buds as early as E10.5. Furthermore, a cell-line engineered to express the secreted factor Sonic Hedgehog (Shh) was injected into early developing mouse brains or limbs. The Shh-expressing cells were found to induce ectopic expression of the Shh target genes Patched and Hnf3beta in the dorsal brain, and to alter digit patterning in the anterior limb bud. These results show that gene misexpression studies can be performed in mouse embryos using ultrasound-guided injection of transfected cells or retroviruses. In combination with the many available mouse mutants, this method offers a new approach for analyzing genetic interactions through gain-of-function studies performed in mutant mouse backgrounds
— id: 57334, year: 1998, vol: 75, page: 107, stat: Journal Article,

Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs
Loomis CA; Kimmel RA; Tong CX; Michaud J; Joyner AL
1998 Mar;125(6):1137-1148, Development
The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. We have previously shown that the limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. Our studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and further show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme. We suggest a model whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER
— id: 7664, year: 1998, vol: 125, page: 1137, stat: Journal Article,

Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system
Matise MP; Epstein DJ; Park HL; Platt KA; Joyner AL
1998 Aug;125(15):2759-2770, Development
Induction of the floor plate at the ventral midline of the neural tube is one of the earliest events in the establishment of dorsoventral (d/v) polarity in the vertebrate central nervous system (CNS). The secreted molecule, Sonic hedgehog, has been shown to be both necessary and sufficient for this induction. In vertebrates, several downstream components of this signalling pathway have been identified, including members of the Gli transcription factor family. In this study, we have examined d/v patterning of the CNS in Gli2 mouse mutants. We have found that the floor plate throughout the midbrain, hindbrain and spinal cord does not form in Gli2 homozygotes. Despite this, motoneurons and ventral interneurons form in their normal d/v positions at 9.5 to 12.5 days postcoitum (dpc). However, cells that are generated in the region flanking the floor plate, including dopaminergic and serotonergic neurons, were greatly reduced in number or absent in Gli2 homozygous embryos. These results suggest that early signals derived from the notochord can be sufficient for establishing the basic d/v domains of cell differentiation in the ventral spinal cord and hindbrain. Interestingly, the notochord in Gli2 mutants does not regress ventrally after 10.5 dpc, as in normal embryos. Finally, the spinal cord of Gli1/Gli2 zinc-finger-deletion double homozygous mutants appeared similar to Gli2 homozygotes, indicating that neither gene is required downstream of Shh for the early development of ventral cell fates outside the ventral midline
— id: 7678, year: 1998, vol: 125, page: 2759, stat: Journal Article,

Gli2 mutant mouse embryos have defects in ventral midline cell induction and commissural axon guidance
Matise, Michael P; Park, Heidi L; Epstein, Douglas J; Joyner, Alexandra L
1998 Nov 7-12;24(1-2):1280-1280, Abstracts (Society for Neuroscience)
— id: 15924, year: 1998, vol: 24, page: 1280, stat: Journal Article,

Role of OTX2/GBX2 interactions in localization of the midbrain-hindbrain organizer
Millet, Sandrine; Campbell, Kenneth; Epstein, Douglas J; Bothe, Gerald W M; Joyner, Alexandra L
1998 Nov 7-12;24(1-2):533-533, Abstracts (Society for Neuroscience)
— id: 15928, year: 1998, vol: 24, page: 533, stat: Journal Article,

Engrailed-1 expression and regulatory role during skin appendage development
Mohan, S; Tong, CX; Perone, J; Chubak, B; Kimmel, R; Joyner, AL; Loomis, CA
1998 APR ;110(4):603-603, Journal of investigative dermatology
— id: 53525, year: 1998, vol: 110, page: 603, stat: Journal Article,

Genetic analysis of the role of Gli genes during embryogenesis in double mutant mice
Park, H; Platt, K; Matise, M; Joyner, A
1998 JUN 1 ;198(1):217-217, Developmental biology (Orlando)
— id: 53427, year: 1998, vol: 198, page: 217, stat: Journal Article,

Physiological and molecular characterization of interneurons in the developing spinal cord
Wenner P; Matise MP; Joyner A; O'Donovan MJ
1998 Nov 16;860:425-427, Annals of the New York Academy of Sciences
— id: 57081, year: 1998, vol: 860, page: 425, stat: Journal Article,

Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system
Zinyk DL; Mercer EH; Harris E; Anderson DJ; Joyner AL
1998 May 21;8(11):665-668, Current biology. CB
The mouse midbrain-hindbrain constriction is centrally involved in patterning of the midbrain and anterior hindbrain (cerebellum), as revealed by recent genetic studies using mice and embryological studies in chick (reviewed in [1,2]). This region can act as an organizer region to induce midbrain and cerebellar development. Genes such as Engrailed-1, Pax-2 and Pax-5, which are expressed in the embryonic cells that will form the midbrain and the cerebellum, are required for development of these regions. Fate-mapping experiments at early somite stages in chick have revealed that the cerebellar primordium is located both anterior and posterior to the midbrain-hindbrain constriction, whereas midbrain precursors lie more anteriorly. Fate mapping in mice has been complicated by the inaccessibility of the postimplantation embryo. Here, we report the use of a new in vivo approach involving the Cre-IoxP site-specific recombination system [3] to map the fate of cells in the mouse midbrain-hindbrain constriction. We show that cells originating in the mouse dorsal midbrain-hindbrain constriction during embryonic days 9-12 contribute significantly to the medial cerebellum and colliculi. Our data demonstrate the feasibility of using a recombinase-based lineage-tracing system for fate mapping in the mouse
— id: 57295, year: 1998, vol: 8, page: 665, stat: Journal Article,

Identification of a potential interacting partner of the mouse Engrailed-1 protein in vertebrate limb patterning
Briegel, KJ; Ehrlich, JS; Joyner, AL
1997 ;186(2):B110-B110, Developmental biology (Orlando)
— id: 104599, year: 1997, vol: 186, page: B110, stat: Journal Article,

Genetic control of early midhindbrain development
Campbell, K; Epstein, DJ; Joyner, AL
1997 ;27(3):260-260, Behavior genetics
— id: 104600, year: 1997, vol: 27, page: 260, stat: Journal Article,

Untitled
Gruss, P; Jaeckle, H; Joyner, A; McGinnis, W; Stern, C
1997 JAN ;61(1-2):3-3, Mechanisms of development
— id: 53269, year: 1997, vol: 61, page: 3, stat: Journal Article,

Role of En1 in vertebrate limb patterning
Kimmel, RA; Loomis, CA; Joyner, AL
1997 ;186(2):B230-B230, Developmental biology (Orlando)
— id: 104601, year: 1997, vol: 186, page: B230, stat: Journal Article,

Pattern deformities and cell loss in Engrailed-2 mutant mice suggest two separate patterning events during cerebellar development
Kuemerle, B; Zanjani, H; Joyner, A; Herrup, K
1997 ;17(20):7881-7889, Journal of neuroscience
Null alleles of the mouse Engrailed-2 gene, a molecular homolog of the fly gene engrailed, have demonstrable effects on the anteroposterior (A/P) patterning of cerebellum as reflected in the disruption of the normal process of foliation of the cerebellar cortex and the alteration of transgene expression boundaries in the adult. Engrailed-2 also affects the transient mediolateral (M/L) pattern of En-1 and Wnt-7b expression seen in late embryogenesis. We have examined three markers of cerebellar compartmentation in En-2 mutant mice: the Zebrin II and Ppath monoclonal antibodies and the transgene L7lacZ. In En-2 mutants, the normal temporal pattern of expression is preserved for all three markers, although the size and spatial location of various bands differ from those of the wild type. Unlike the foliation abnormalities, the M/L pattern disturbances we have found occur in nearly all cerebellar regions. Cell counts reveal that all major cell types of the olivocerebellar circuit are reduced by 30-40%. We propose that these results are best explained by a model in which the Engrailed-2 gene is involved in the early specification of the cerebellar field including the number of progenitors. Because each of these progenitors gives rise to a clone of defined size, Engrailed-2 helps specify adult cell number. We further postulate that the configuration of the seven Zebrin bands as well as the shapes and locations of the cerebellar lobules are set up by a second patterning event that occurs after neurogenesis is complete. :
— id: 104602, year: 1997, vol: 17, page: 7881, stat: Journal Article,

Engrailed-1 (En1) plays multiple roles in patterning of the distal limb and development of the overlying skin
Loomis, CA; Tong, XC; Zeitler, E; Kimmel, RA; Paulson, M; Hanks, M; Joyner, AL
1997 APR ;108(4):241-241, Journal of investigative dermatology
— id: 53217, year: 1997, vol: 108, page: 241, stat: Journal Article,

Expression patterns of developmental control genes in normal and Engrailed-1 mutant mouse spinal cord reveal early diversity in developing interneurons
Matise MP; Joyner AL
1997 Oct 15;17(20):7805-7816, Journal of neuroscience
The vertebrate spinal cord has long served as a useful system for studying the pattern of cell differentiation along the dorsoventral (d/v) axis. In this paper, we have defined the expression of several classes of genes expressed in restricted d/v domains in the intermediate region (IR) of the mouse spinal cord, in which most interneurons are generated. From this analysis, we have found that spinal cord interneurons and their precursors express unique combinations of transcription factors and Notch ligands at the onset of their differentiation. The domains of expression of a number of different classes of genes share similar boundaries, indicating that there could be a basic subdivision of the ventral IR into four distinct regions. This differential gene expression suggests that spinal cord interneurons acquire unique identities early in their development and that Notch signaling mechanisms may participate in the determination of cell fate along the d/v axis. Gene expression studies in Engrailed-1 (En-1) mutants showed that En-1-expressing and other closely positioned classes of neurons do not require the homeodomain protein En-1 for their early pattern of differentiation. Rather, it is suggested that En-1 may function to distinguish a subset of interneurons during the later maturation of the spinal cord
— id: 7213, year: 1997, vol: 17, page: 7805, stat: Journal Article,

Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development
Mo R; Freer AM; Zinyk DL; Crackower MA; Michaud J; Heng HH; Chik KW; Shi XM; Tsui LC; Cheng SH; Joyner AL; Hui C
1997 Jan;124(1):113-123, Development
The correct patterning of vertebrate skeletal elements is controlled by inductive interactions. Two vertebrate hedgehog proteins, Sonic hedgehog and Indian hedgehog, have been implicated in skeletal development. During somite differentiation and limb development, Sonic hedgehog functions as an inductive signal from the notochord, floor plate and zone of polarizing activity. Later in skeletogenesis, Indian hedgehog functions as a regulator of chondrogenesis during endochondral ossification. The vertebrate Gli zinc finger proteins are putative transcription factors that respond to Hedgehog signaling. In Drosophila, the Gli homolog cubitus interruptus is required for the activation of hedgehog targets and also functions as a repressor of hedgehog expression. We show here that Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 (C.-c. Hui and A. L. Joyner (1993). Nature Genet. 3, 241-246) mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development. The role of Gli2 and Gli3 in Hedgehog signaling during skeletal development is discussed
— id: 35041, year: 1997, vol: 124, page: 113, stat: Journal Article,

Functional and molecular characterization of Gli1 in mouse development
Park, HL; Platt, K; Joyner, AL
1997 ;186(2):B257-B257, Developmental biology (Orlando)
— id: 104603, year: 1997, vol: 186, page: B257, stat: Journal Article,

Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice
Platt KA; Michaud J; Joyner AL
1997 Mar;62(2):121-135, Mechanisms of development
The three mouse Gli genes are putative transcription factors which are the homologs of cubitus interruptus (ci) in Drosophila. Along with the gene patched (Ptc), ci has been implicated in the hedgehog (Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, we compared its expression with that of Ptc and Hh family members in mouse. We found that Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. We also show that Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo
— id: 56979, year: 1997, vol: 62, page: 121, stat: Journal Article,

Ultrasound-guided injections into the mouse embryonic brain
Turnbull, DH; Olsson, M; Fishell, G; Joyner, AL
1997 ;62(2):1485-1488, Proceedings (IEEE Ultrasonics Symposium)
The authors have modified a 40-50 MHz ultrasound imaging system to allow image-guided injections into targeted brain regions of living mouse embryos. The injection technique is described, and example injections are shown. The significance of this research in basic biological science and the potential impact on future attempts at in utero therapy are described
— id: 104604, year: 1997, vol: 62, page: 1485, stat: Journal Article,

Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function
Wassarman KM; Lewandoski M; Campbell K; Joyner AL; Rubenstein JL; Martinez S; Martin GR
1997 Aug;124(15):2923-2934, Development
Analysis of mouse embryos homozygous for a loss-of-function allele of Gbx2 demonstrates that this homeobox gene is required for normal development of the mid/hindbrain region. Gbx2 function appears to be necessary at the neural plate stage for the correct specification and normal proliferation or survival of anterior hindbrain precursors. It is also required to maintain normal patterns of expression at the mid/hindbrain boundary of Fgf8 and Wnt1, genes that encode signaling molecules thought to be key components of the mid/hindbrain (isthmic) organizer. In the absence of Gbx2 function, isthmic nuclei, the cerebellum, motor nerve V, and other derivatives of rhombomeres 1-3 fail to form. Additionally, the posterior midbrain in the mutant embryos appears to be extended caudally and displays abnormalities in anterior/posterior patterning. The failure of anterior hindbrain development is presumably due to the loss of Gbx2 function in the precursors of the anterior hindbrain. However, since Gbx2 expression is not detected in the midbrain it seems likely that the defects in midbrain anterior/posterior patterning result from an abnormal isthmic signaling center. These data provide genetic evidence for a link between patterning of the anterior hindbrain and the establishment of the mid/hindbrain organizer, and identify Gbx2 as a gene required for these processes to occur normally
— id: 35040, year: 1997, vol: 124, page: 2923, stat: Journal Article,

Early mesencephalon/metencephalon patterning and development of the cerebellum
Wassef M; Joyner AL
1997 ;5(1):3-16, Perspectives on developmental neurobiology
Experimental studies in chick and analysis of mouse mutants have provided a framework for studying the early developmental processes involved in specifying the cerebellar anlage. Fate mapping studies in chick have shown that at early stages the cerebellum derives from cells in the mesencephalon and metencephalon (mes-met). Transplantation studies in chick have implicated the mes-met junction (isthmus) as a source of secreted factors that organize development of the entire mes-met, perhaps by stimulating proliferation and specifying positional values across the region. Fgf-8 has been implicated as a major factor involved in the isthmus organizing activity. Gene expression studies indicate that the anterior and posterior expression domains of the homeobox genes Otx-2 and Gbx-2, respectively, are the earliest indication of a division of the brain. Furthermore, the Otx-2/Gbx-2 expression border later resides at the mes-met junction. Genetic studies in mouse have shown that Otx-2 and Gbx-2 are required for normal development of cells on both sides of the border. In addition, mutations affecting the secreted factor Wnt-1, which is expressed anterior to the Otx-2/Gbx-2 expression border and the homeodomain transcription factors, Engrailed-1,2 and Pax-2,5 that have broad overlapping expression domains in the mes-met, result in deletions of mes-met structures. Taken together, these studies suggest that specification of the cerebellar territory requires a hierarchy of complex cellular and genetic interactions that gradually subdivide the brain into smaller regions
— id: 57279, year: 1997, vol: 5, page: 3, stat: Journal Article,

Inheritance of the Huntington's disease CAG repeat in mice
Wheeler, VC; Ryan, A; Auerbach, A; Calzonetti, T; Duyao, MP; White, JK; Vrbanac, V; Allen, MJ; Gusella, JF; Srinidhi, J; Joyner, AL; MacDonald, ME
1997 OCT ;61(4):A323-A323, American journal of human genetics
— id: 53611, year: 1997, vol: 61, page: A323, stat: Journal Article,

Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion
White JK; Auerbach W; Duyao MP; Vonsattel JP; Gusella JF; Joyner AL; MacDonald ME
1997 Dec;17(4):404-410, Nature genetics
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion that lengthens a glutamine segment in the novel huntingtin protein. To elucidate the molecular basis of HD, we extended the polyglutamine tract of the mouse homologue, Hdh, by targetted introduction of an expanded human HD CAG repeat, creating mutant HdhneoQ50 and HdhQ50 alleles that express reduced and wild-type levels of altered huntingtin, respectively. Mice homozygous for reduced levels displayed characteristic aberrant brain development and perinatal lethality, indicating a critical function for Hdh in neurogenesis. However, mice with normal levels of mutant huntingtin did not display these abnormalities, indicating that the expanded CAG repeat does not eliminate or detectably impair huntingtin's neurogenic function. Thus, the HD defect in man does not mimic complete or partial Hdh inactivation and appears to cause neurodegenerative disease by a gain-of-function mechanism
— id: 35039, year: 1997, vol: 17, page: 404, stat: Journal Article,

Huntingtin function is required for mouse brain development and is not impaired by the Huntington's disease CAG expansion mutation
White, JK; Auerbach, W; Calzonetti, T; Weaver, M; Vrbanac, V; Vonsattel, JP; Gusella, JF; Joyner, AL; MacDonald, ME
1997 OCT ;61(4):A324-A324, American journal of human genetics
— id: 53612, year: 1997, vol: 61, page: A324, stat: Journal Article,

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia
Chen M; Tomkins DJ; Auerbach W; McKerlie C; Youssoufian H; Liu L; Gan O; Carreau M; Auerbach A; Groves T; Guidos CJ; Freedman MH; Cross J; Percy DH; Dick JE; Joyner AL; Buchwald M
1996 Apr;12(4):448-451, Nature genetics
Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease
— id: 35044, year: 1996, vol: 12, page: 448, stat: Journal Article,

An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro
Forrester LM; Nagy A; Sam M; Watt A; Stevenson L; Bernstein A; Joyner AL; Wurst W
1996 Feb 20;93(4):1677-1682, Proceedings of the National Academy of Sciences of the United States of America
We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways
— id: 35045, year: 1996, vol: 93, page: 1677, stat: Journal Article,

Impaired motor learning performance in cerebellar En-2 mutant mice
Gerlai R; Millen KJ; Herrup K; Fabien K; Joyner AL; Roder J
1996 Feb;110(1):126-133, Behavioral neuroscience
Mice homozygous for a null mutation in their En-2 gene exhibit cerebellar neuroanatomical alterations including absence and misplacements of specific fissures and size reduction. The present study investigated cerebellar function by comparing the behavior of age-matched homozygous and heterozygous En-2 mutant and wild-type mice. Motor function of the mutants was found normal in several situations. Habituation to novelty in the open field was not significantly different in mutants. However, in a motor learning paradigm, the rotating rod, the performance of homozygous mutant mice improved significantly less than that of the heterozygous mice which were also significantly impaired compared to wild-type mice. Unlike other cerebellar mutants in which severe motor or sensory defects are obvious, the En-2 mouse model offers a unique tool to study the role of cerebellum in complex behavioral phenomena, including motor learning, without confounding effects
— id: 35046, year: 1996, vol: 110, page: 126, stat: Journal Article,

Engrailed, Wnt and Pax genes regulate midbrain--hindbrain development
Joyner AL
1996 Jan;12(1):15-20, Trends in genetics
The mouse Engrailed, Wnt and Pax genes, which are homologues of Drosophila segmentation genes, have provided a critical genetic entry point for dissecting the molecular and cellular control of mesencephalon and metencephalon development in vertebrates. Mutant phenotypes and gene expression data suggest that six members of these gene families are required for early formation of these brain regions. Ectopic transplantation studies have shown that the midbrain-hindbrain-junction protein can act as an organizer and recruit certain host cells to re-establish parts of the entire region. Taken together, these studies indicate that the mesencephalon and metencephalon develop as one independent unit, and that the genetic network regulating development of this region involves conserved genes that control segmentation in Drosophila. By analogy, segmentation of the rest of the brain might best be described in terms of 'genetic units' defined by genetic and transplantation data
— id: 7076, year: 1996, vol: 12, page: 15, stat: Journal Article,

The mouse Engrailed-1 gene and ventral limb patterning
Loomis CA; Harris E; Michaud J; Wurst W; Hanks M; Joyner AL
1996 Jul 25;382(6589):360-363, Nature
During vertebrate limb development, positional information must be specified along three distinct axes. Although much progress has been made in our understanding of the molecular interactions involved in anterior-posterior and proximal-distal limb patterning, less is known about dorsal-ventral patterning. The genes Wnt-7a and Lmx-1, which are expressed in dorsal limb ectoderm and mesoderm, respectively, are thought to be important regulators of dorsal limb differentiation. Whether a complementary set of molecules controls ventral limb development has not been clear. Here we report that Engrailed-1, a homeodomain-containing transcription factor expressed in embryonic ventral limb ectoderm, is essential for ventral limb patterning. Loss of Engrailed-1 function in mice results in dorsal transformations of ventral paw structures, and in subtle alterations along the proximal-distal limb axis. Engrailed-1 seems to act in part by repressing dorsal differentiation induced by Wnt-7a, and is essential for proper formation of the apical ectodermal ridge
— id: 56881, year: 1996, vol: 382, page: 360, stat: Journal Article,

The role of Engrailed-1 in epidermal appendage formation and skin patterning
Loomis, CA; Michaud, J; Hanks, M; Joyner, AL
1996 APR ;106(4):171-171, Journal of investigative dermatology
— id: 52997, year: 1996, vol: 106, page: 171, stat: Journal Article,

Targeted inactivation of the mouse Huntington's disease gene homolog Hdh
MacDonald ME; Duyao M; Calzonetti T; Auerbach A; Ryan A; Barnes G; White JK; Auerbach W; Vonsattel JP; Gusella JF; Joyner AL
1996 ;61(2):627-638, Cold Spring Harbor symposia on quantitative biology
— id: 35047, year: 1996, vol: 61, page: 627, stat: Journal Article,

Engrailed-1 is expressed in a subset of spinal interneurons during mouse development
Matise, Michael P.; Joyner, Alexandra L.
1996 ;22(1-3):527-527, Abstracts (Society for Neuroscience)
— id: 104605, year: 1996, vol: 22, page: 527, stat: Journal Article,

From screens to genes: prospects for insertional mutagenesis in zebrafish
Schier AF; Joyner AL; Lehmann R; Talbot WS
1996 Dec 15;10(24):3077-3080, Genes & development
— id: 12447, year: 1996, vol: 10, page: 3077, stat: Journal Article,

Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene
Scrocchi LA; Brown TJ; MaClusky N; Brubaker PL; Auerbach AB; Joyner AL; Drucker DJ
1996 Nov;2(11):1254-1258, Nature medicine
Glucagon-like peptide 1 (GLP1) is postulated to regulate blood glucose and satiety, but the biological importance of GLP1 as an incretin and neuropeptide remains controversal. The regulation of nutrient-induced insulin secretion is dependent on the secretion of incretins, gut-derived peptides that potentiate insulin secretion from the pancreatic islets. To ascertain the relative physiological importance of GLP1 as a regulator of feeding behavior and insulin secretion, we have generated mice with a targeted disruption of the GLP1 receptor gene (GLP1R). These GLP1R-/- mice are viable, develop normally but exhibit increased levels of blood glucose following oral glucose challenge in association with diminished levels of circulating insulin. It is surprising that they also exhibit abnormal levels of blood glucose following intraperitoneal glucose challenge. Intracerebroventricular administration of GLP1 inhibited feeding in wild-type mice but not in GLP1R-/- mice; however, no evidence for abnormal body weight or feeding behavior was observed in GLP1R-/- mice. These observations demonstrate that GLP1 plays a central role in the regulation of glycemia; however, disruption of GLP1/GLP1R signaling in the central nervous system is not associated with perturbation of feeding behavior or obesity in vivo
— id: 35042, year: 1996, vol: 2, page: 1254, stat: Journal Article,

Diabetes in mice with a targeted disruption of the GLP-1 receptor gene
Scrocchi, L; Auerbach, AB; Joyner, AL; Drucker, DJ
1996 ;45(24):71-71, Diabetes
— id: 104606, year: 1996, vol: 45, page: 71, stat: Journal Article,

Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene
Song DL; Chalepakis G; Gruss P; Joyner AL
1996 Feb;122(2):627-635, Development
The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution
— id: 6964, year: 1996, vol: 122, page: 627, stat: Journal Article,

Regulation of engrailed-2 expression during early brain development
Song, DL; Chalepakis, G; Gruss, P; Auerbach, W; Joyner, AL
1996 DEC ;7(3):3729-3729, Molecular biology of the cell
— id: 53362, year: 1996, vol: 7, page: 3729, stat: Journal Article,

The Engrailed-2 homeobox gene and patterning of spinocerebellar mossy fiber afferents
Vogel MW; Ji Z; Millen K; Joyner AL
1996 Oct 23;96(1-2):210-218, Brain research. Developmental brain research
The mouse Engrailed-2 gene, En-2, appears to be involved in cerebellar pattern formation. Homozygous null mutants for En-2 have abnormal foliation patterns in the posterior half of the cerebellum and there are changes in Purkinje and granule cell gene expression in some posterior folia, possibly reflecting changes in cell identity. We have examined the distribution of spinocerebellar mossy fiber terminals in homozygous En-2hd null mutants to determine if En-2 is involved in regulating the pattern of afferent connectivity in the cerebellum. Spinocerebellar mossy fiber terminals were labeled following WGA-HRP injections in the lumbar region of 5 homozygous En-2hd mutants and 4 heterozygous controls. The distribution of spinocerebellar mossy fiber terminals was consistently altered in lobules VIII and IX of the En-2hd mutants. The principal changes were a reduction in the number of mossy fiber terminal fields in the dorsal aspect of lobule VIII and the dorsal midline field in lobule IX was fused into a single compartment. The results suggest that the deletion of En-2 expression does not transform lobule identity, at least with respect to afferent fiber positional information cues. However, the changes in foliation and afferent connectivity in the En-2 mutant support a broad role for the En-2 gene in cerebellar patterning
— id: 35043, year: 1996, vol: 96, page: 210, stat: Journal Article,

ESTABLISHMENT OF A MOUSE MODEL FOR FANCONI-ANEMIA BY GENE TARGETING
CHEN, M; AUERBACH, W; LIU, L; GAN, O; CARREAU, M; AUERBACH, A; GROVES, T; GUIDOS, C; TOMKINS, D; DICK, JE; JOYNER, AL; BUCHWALD, M
1995 ;57(4):1208-1208, American journal of human genetics
— id: 104607, year: 1995, vol: 57, page: 1208, stat: Journal Article,

MOLECULAR-GENETICS OF FANCONI-ANEMIA
CHEN, M; CUMMING, R; KRASNOSHTEIN, F; SAVOIA, A; LIGHTFOOT, J; SANTOS, C; PARKER, L; WONG, J; JOYNER, A; BUCHWALD, M
1995 MAR 10 ;14(1-6):272-272, Journal of cellular biochemistry
— id: 86747, year: 1995, vol: 14, page: 272, stat: Journal Article,

Inactivation of the mouse Huntington's disease gene homolog Hdh
Duyao MP; Auerbach AB; Ryan A; Persichetti F; Barnes GT; McNeil SM; Ge P; Vonsattel JP; Gusella JF; Joyner AL; et al.
1995 Jul 21;269(5222):407-410, Science
Huntington's disease (HD) is a dominant neurodegenerative disorder caused by expansion of a CAG repeat in the gene encoding huntingtin, a protein of unknown function. To distinguish between 'loss of function' and 'gain of function' models of HD, the murine HD homolog Hdh was inactivated by gene targeting. Mice heterozygous for Hdh inactivation were phenotypically normal, whereas homozygosity resulted in embryonic death. Homozygotes displayed abnormal gastrulation at embryonic day 7.5 and were resorbing by day 8.5. Thus, huntingtin is critical early in embryonic development, before the emergence of the nervous system. That Hdh inactivation does not mimic adult HD neuropathology suggests that the human disease involves a gain of function
— id: 35048, year: 1995, vol: 269, page: 407, stat: Journal Article,

Genomic imprinting of Mash2, a mouse gene required for trophoblast development
Guillemot F; Caspary T; Tilghman SM; Copeland NG; Gilbert DJ; Jenkins NA; Anderson DJ; Joyner AL; Rossant J; Nagy A
1995 Mar;9(3):235-242, Nature genetics
The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19
— id: 35049, year: 1995, vol: 9, page: 235, stat: Journal Article,

Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2 [see comments]
Hanks M; Wurst W; Anson-Cartwright L; Auerbach AB; Joyner AL
1995 Aug 4;269(5224):679-682, Science
The related mouse Engrailed genes En-1 and En-2 are expressed from the one- and approximately five-somite stages, respectively, in a similar presumptive mid-hindbrain domain. However, mutations in En-1 and En-2 produce different phenotypes. En-1 mutant mice die at birth with a large mid-hindbrain deletion, whereas En-2 mutants are viable, with cerebellar defects. To determine whether these contrasting phenotypes reflect differences in temporal expression or biochemical activity of the En proteins, En-1 coding sequences were replaced with En-2 sequences by gene targeting. This rescued all En-1 mutant defects, demonstrating that the difference between En-1 and En-2 stems from their divergent expression patterns
— id: 7925, year: 1995, vol: 269, page: 679, stat: Journal Article,

Jin tagetingu
Joyner, Alexandara L; Noda, Tetsuo
Tokyo : Medikarusaiensuintanashonaru, 1995,
— id: 1641, year: 1995, vol: , page: , stat: ,

A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum
Millen KJ; Hui CC; Joyner AL
1995 Dec;121(12):3935-3945, Development
To gain insight into the molecular genetic basis of cerebellar patterning, the expression patterns of many vertebrate homologues of Drosophila segment polarity genes were examined during normal and abnormal cerebellar development, including members of the En, Wnt, Pax, Gli and Dvl gene families. Five of these genes were found to show transient, spatially restricted patterns of expression. Strikingly, expression of En-2, En-1, Wnt-7B and Pax-2 defined eleven similar sagittal domains at 17.5 dpc, reminiscent of the transient sagittal domains of expression of Purkinje cell markers which have been implicated in cerebellar afferent patterning. Postnatally, transient anterior/posterior differences in expression were observed for En-2, En-1, Gli and Wnt-7B dividing the cerebellum into anterior and posterior regions. The expression patterns of these genes were altered in cerebella of En-2 homozygous mutant mice, which show a cerebellar foliation patterning defect. Strikingly, four of the Wnt-7B expression domains that are adjacent to the En-2 domains are lost in En-2 mutant embryonic cerebella. These studies provide the first evidence of a potential network of regulatory genes that establish spatial cues in the developing cerebellum by dividing it into a grid of positional information required for patterning foliation and afferents. Taken together with previous gene expression studies, our data suggests that eleven sagittal domains and at least two anterior/posterior compartments are the basic elements of spatial information in the cerebellum
— id: 6938, year: 1995, vol: 121, page: 3935, stat: Journal Article,

Ultrasound backscatter microscope analysis of early mouse embryonic brain development
Turnbull DH; Bloomfield TS; Baldwin HS; Foster FS; Joyner AL
1995 Mar 14;92(6):2239-2243, Proceedings of the National Academy of Sciences of the United States of America
The history of developmental and genetic analysis in the mouse has made it the model of choice for studying mammalian embryogenesis. Presently lacking is a simple technique for efficiently analyzing early mouse mutant phenotypes in utero. We demonstrate application of a real-time imaging method called ultrasound backscatter microscopy for visualizing mouse early embryonic neural tubes and hearts. This method was used to study live embryos in utero between 9.5 and 11.5 days of embryogenesis, with a spatial resolution close to 50 microns. Ultrasound backscatter microscope images of cultured embryos made it possible to visualize the heart chambers. This noninvasive imaging method was also used for analyzing a neural tube defect. The midhindbrain deletion associated with a null mutation of the Wnt-1 protooncogene was easily recognizable on ultrasound backscatter microscope images of 10.5- and 11.5-day embryos. Computer-generated volumetric renderings of the neural tube cavities were made from three-dimensional image data. This allowed a much clearer definition of the Wnt-1 mutant phenotype. These imaging techniques should be of considerable use in studying mouse development in utero
— id: 56723, year: 1995, vol: 92, page: 2239, stat: Journal Article,

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice
Wurst W; Rossant J; Prideaux V; Kownacka M; Joyner A; Hill DP; Guillemot F; Gasca S; Cado D; Auerbach A; et al.
1995 Feb;139(2):889-899, Genetics
We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes
— id: 49333, year: 1995, vol: 139, page: 889, stat: Journal Article,

THE USE OF A SITE-SPECIFIC RECOMBINATION SYSTEM FOR FATE MAPPING IN MICE
ZINYK, DL; MERCER, EH; ANDERSON, DJ; JOYNER, AL
1995 ;139(2):346-346, Journal of cellular biochemistry
— id: 104608, year: 1995, vol: 139, page: 346, stat: Journal Article,

Essential role of Mash-2 in extraembryonic development
Guillemot F; Nagy A; Auerbach A; Rossant J; Joyner AL
1994 Sep 22;371(6495):333-336, Nature
The outer layer of the blastocyst, or trophectoderm, is the first cell lineage to differentiate in the mouse embryo, but little is known about the genetic control of its development. Lineage-specific transcription factors may be important in lineage specification, and the product of the Mash-2 gene fulfils the criteria for such a factor. Mash-2 is a mammalian member of the achaete-scute family which encodes basic-helix-loop-helix transcription factors and is strongly expressed in the extraembryonic trophoblast lineage. Mash-2 transcripts are found in the female germ line and in the embryo throughout preimplantation development, but are highly expressed later only in the ectoplacental cone, the chorion and their derivatives in the placenta. Mash-2 transcripts are not found in primary and secondary giant cells, yolk sac or allantois at any post-implantation stage, and are present only transiently and at low levels in the embryo during gastrulation. To analyse the role of Mash-2 in development, we have used gene targeting to generate mice having no Mash-2 function. We report here that Mash-2-/- embryos die from placental failure at 10 days postcoitum. In mutant placentas, spongiotrophoblast cells and their precursors are absent and chorionic ectoderm is reduced. We have rescued this placental mutant phenotype by constructing chimaeras with tetraploid wild-type embryos which contribute almost exclusively to extraembryonic tissues. Mash-2-/- embryos developed normally and adult Mash-2-/- mice were viable, demonstrating that Mash-2 has no major role in the embryo itself. Mash-2 is the first transcription factor shown to play a critical part in the development of the mammalian trophoblast lineage
— id: 35051, year: 1994, vol: 371, page: 333, stat: Journal Article,

Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development
Hui CC; Slusarski D; Platt KA; Holmgren R; Joyner AL
1994 Apr;162(2):402-413, Developmental biology (Orlando)
Three mouse genes, Gli, Gli-2, and Gli-3, which share a similar zinc finger domain with the products of the Drosophila segment polarity gene cubitus interruptus and the Caenorhabditis elegans sex-determining gene tra-1 were cloned and characterized. The expression patterns during postimplantation development of the three genes were analyzed by Northern blot, whole-mount, and section in situ hybridizations. Expression was first detected during gastrulation in both the ectoderm and mesoderm. Later in development, their expression became more restricted in various ectoderm- and mesoderm-derived tissues and was not detectable after completion of organogenesis. Interestingly, in the developing neural tube, Gli showed a narrow ventral domain of expression, whereas Gli-2 and Gli-3 showed a broad and dorsally restricted domain. Expression of these three Gli genes in various ectoderm- and mesoderm-derived tissues suggests that they play multiple roles during postimplantation development. Consistent with this hypothesis, a naturally occurring Gli-3 mutation, the mouse extra-toes mutant; shows defects in both mesoderm- and ectoderm-derived tissues
— id: 35053, year: 1994, vol: 162, page: 402, stat: Journal Article,

Gene targeting and development of the nervous system
Joyner AL; Guillemot F
1994 Feb;4(1):37-42, Current opinion in neurobiology
Gene targeting provides a means of directly assaying the function of specific genes during mouse nervous system development. Generation of targeted mutant mice has provided the first evidence of developmental roles for genes whose function was suggested based on their expression, but for which appropriate assay systems were lacking. In other cases, where gene function was known, targeted mutations have revealed in which cell population, and at what developmental stage, particular genes are first indispensable. The existing targeted mutants suggest that an early mechanism of pattern formation in mammals involves regional control of proliferation or survival of neural precursors, and that later general functions, such as the control of differentiation of precursors, may be performed by different genes in distinct neural lineages. As many genes display complex temporal and spatial patterns of expression, analysis of the full range of functions of such genes will require the generation of a series of alleles, including stage- and tissue-specific mutations
— id: 35055, year: 1994, vol: 4, page: 37, stat: Journal Article,

MASH-1: a marker and a mutation for mammalian neural crest development
Lo L; Guillemot F; Joyner AL; Anderson DJ
1994 ;2(2):191-201, Perspectives on developmental neurobiology
The ability to generate mice containing null mutations in any cloned gene promises new insights into the molecular control of mammalian neural crest development. This approach has recently been applied to MASH-1, a transcription factor in the bHLH family that is a mammalian homologue of the Drosophila proneural genes achaete-scute. In wild-type embryos, this gene is expressed early in the development of the autonomic nervous system, in apparent precursors of sympathetic, parasympathetic, and enteric neurons (as well as in restricted regions of the central nervous system). A null mutation in the MASH-1 gene eliminates sympathetic and parasympathetic neurons and enteric neurons of the foregut (esophagus); however, enteric neurons of the stomach and hindgut are only partially affected. Analysis with other markers indicates that the mutation acts after neural crest cells have localized in the anlagen of the autonomic nervous system to prevent neuronal differentiation. The differentiation of autonomic glia appears unaffected. Thus, MASH-1 provides one of the most specific mutations affecting neural development in mammals, as well as a valuable marker to study the early segregation of neural crest cell lineages
— id: 35056, year: 1994, vol: 2, page: 191, stat: Journal Article,

Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants
Millen KJ; Wurst W; Herrup K; Joyner AL
1994 Mar;120(3):695-706, Development
The cerebellum is an ideal system to study pattern formation in the central nervous system because of its simple cytoarchitecture and regular organization of folds and neural circuitry. Engrailed-2 (En-2) is expressed in a spatially restricted broad band around the mesencephalic-metencephalic junction, a region from which the cerebellum is derived. Mice homozygous for a targeted deletion of the En-2 homeobox, En-2hd, previously have been shown to have an altered adult cerebellar foliation pattern. To address whether the En-2hd allele was hypomorphic, we generated a putative null mutation that makes an N-terminal deletion (ntd). Mice homozygous for this new mutation, En-2ntd, display an identical cerebellar patterning defect, suggesting that both alleles represent null alleles. We also examined the developmental profile of En-2 homozygous mutant cerebellar foliation. This revealed a complex phenotype of general developmental delay and abnormal formation of specific fissures with the most severe morphological disruptions being limited to the posterior region of the cerebellum. The expression of two transgenes, which express lacZ in lobe-specific patterns in the cerebellum, also was found to be altered in En-2 homozygotes, suggesting possible lobe transformations. Finally, during embryogenesis there was a clear delay in fusion of the cerebellar rudiments at the midline by 15.5 d.p.c. This and the expression pattern of En-2 suggests that although cerebellar foliation is largely a postnatal process, the patterning of the cerebellum may begin during embryogenesis and that En-2 plays a critical role in this early process
— id: 35054, year: 1994, vol: 120, page: 695, stat: Journal Article,

Expression of mouse LSP1/S37 isoforms. S37 is expressed in embryonic mesenchymal cells
Misener VL; Hui C; Malapitan IA; Ittel ME; Joyner AL; Jongstra J
1994 Dec;107 ( Pt 12)(3):3591-3600, Journal of cell science
Mouse LSP1 is a 330 amino acid intracellular F-actin binding protein expressed in lymphocytes and macrophages but not in non-hematopoietic tissues. A 328 amino acid LSP1-related protein, designated S37, is expressed in murine bone marrow stromal cells, in fibroblasts, and in a myocyte cell line. The two proteins differ only at their N termini, the first 23 amino acid residues of LSP1 being replaced by 21 different residues in S37. The presence of different amino termini suggests that the LSP1 and S37 proteins are encoded by transcripts arising through alternative exon splicing. Here we report the genomic organization of the Lsp1 gene and show that the distinct N termini of LSP1 and S37 are encoded by two alternatively used exons, each containing a translational start codon. We also demonstrate that alternative 3' acceptor sites are used in the splicing of exon 5. This results in LSP1 and S37 transcripts that either do or do not contain 18 bp encoding the 6 amino acids HLIRHQ of the acidic domain. Therefore, the Lsp1 gene encodes four protein isoforms: full-length LSP1 and S37 proteins, designated LSP1-I and S37-I and the same proteins without the HLIRHQ sequence, designated LSP1-II and S37-II. By in situ hybridization analysis we show that the S37 isoforms are expressed in mesenchymal tissue, but not in adjacent epithelial tissue, of several developing organs during mouse embryogenesis. This, together with our finding that S37 is an F-actin binding protein, suggests that S37 is a cytoskeletal protein of mesenchymal cells, which may play a role in mesenchyme-induced epithelial differentiation during organogenesis
— id: 35050, year: 1994, vol: 107 ( Pt 12), page: 3591, stat: Journal Article,

Idenshi tagettingu = Gene targeting
Sedivy, John M; Joyner, Alexandra L; Nakagawa, Hachiro
Kyoto : Kagaku Dojin, 1994,
— id: 1642, year: 1994, vol: , page: , stat: ,

Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum
Wurst W; Auerbach AB; Joyner AL
1994 Jul;120(7):2065-2075, Development
During mouse development, the homeobox-containing gene En-1 is specifically expressed across the mid-hindbrain junction, the ventral ectoderm of the limb buds, and in regions of the hindbrain, spinal cord, somites and somite-derived tissues. To address the function of En-1 during embryogenesis, we have generated mice homozygous for a targeted deletion of the En-1 homeobox. En-1 mutant mice died shortly after birth and exhibited multiple developmental defects. In the brains of newborn mutants, most of the colliculi and cerebellum were missing and the third and fourth cranial nerves were absent. A deletion of midhindbrain tissue was observed as early as 9.5 days of embryonic development and the phenotype resembles that previously reported for Wnt-1 mutant mice. In addition, patterning of the forelimb paws and sternum was disrupted, and the 13th ribs were truncated. The results of these studies suggest a cell autonomous role for En-1 in generation and/or survival of mid-hindbrain precursor cells and also a non-cell autonomous role in signalling normal development of the limbs and possibly sternum
— id: 35052, year: 1994, vol: 120, page: 2065, stat: Journal Article,

Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system
Guillemot F; Joyner AL
1993 Aug;42(3):171-185, Mechanisms of development
The Drosophila Achaete-Scute Complex genes encode transcriptional regulators belonging to the basic-helix-loop-helix family which control early steps of development of the central and peripheral nervous systems. We have isolated two mouse homologues of Achaete-Scute Complex genes, Mash-1 and Mash-2, by using the conservation of the basic-helix-loop-helix domain in this family. In this article, we analyse the expression of Mash-1 from its onset during neurulation to adult stages by RNA in situ hybridization on whole mounts and sections. As was observed for the rat Mash-1 protein, mouse Mash-1 RNA expression is restricted to cells of the developing central and peripheral nervous systems. We have observed three successive phases in the distribution of Mash-1 transcripts in the developing central nervous system. Initially, between embryonic day 8.5 and 10.5, Mash-1 transcripts are found in restricted domains in the neuroepithelium of the midbrain and ventral forebrain, as well as in the spinal cord. Between embryonic day 10.5 and 12.5, Mash-1 expression pattern changes from a restricted to a widespread one. Mash-1 transcripts are then found at variable levels in the ventricular zone in all regions of the brain. From embryonic day 12.5 to post-natal stages, Mash-1 is also expressed in cells outside of the ventricular zone throughout the brain. In addition, Mash-1 is expressed during development of the olfactory epithelium and neural retina. Overall, its expression pattern suggest that Mash-1 plays a role at early stages of development of specific neural lineages in most regions of the central nervous system and of several lineages in the peripheral nervous system. We have also compared the expression of Mash-1 and mouse Notch because their Drosophila homologues have been shown to interact genetically. The two genes show very similar expression patterns, both spatially and temporally, in the early developing brain and in the retina, suggesting that both genes may participate in the development of the same neural lineages
— id: 35059, year: 1993, vol: 42, page: 171, stat: Journal Article,

Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons
Guillemot F; Lo LC; Johnson JE; Auerbach A; Anderson DJ; Joyner AL
1993 Nov 5;75(3):463-476, Cell
The mouse Mash-1 gene, like its Drosophila homologs of the achaete-scute complex (AS-C), encodes a transcription factor expressed in neural precursors. We created a null allele of this gene by homologous recombination in embryonic stem cells. Mice homozygous for the mutation die at birth with apparent breathing and feeding defects. The brain and spinal cord of the mutants appear normal, but their olfactory epithelium and sympathetic, parasympathetic, and enteric ganglia are severely affected. In the olfactory epithelium, neuronal progenitors die at an early stage, whereas the nonneuronal supporting cells are present. In sympathetic ganglia, the mutation arrests the development of neuronal precursors, preventing the generation of sympathetic neurons, but does not affect glial precursor cells. These observations suggest that Mash-1, like its Drosophila homologs of the AS-C, controls a basic operation in development of neuronal progenitors in distinct neural lineages
— id: 35057, year: 1993, vol: 75, page: 463, stat: Journal Article,

TRANSCRIPTIONAL ACTIVITY OF THE MOUSE ENGRAILED PROTEINS
HANKS, M; JONES, F; JOYNER, A
1993 ;75(3):196-196, Journal of cellular biochemistry
— id: 104609, year: 1993, vol: 75, page: 196, stat: Journal Article,

A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene
Hui CC; Joyner AL
1993 Mar;3(3):241-246, Nature genetics
Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder affecting limb and craniofacial development. Recently, the human GLI3 gene has been proposed to be a candidate gene for GCPS. Here we describe the molecular characterization of extra-toes (Xt), which is a mouse model of GCPS. The Xt heterozygotes show craniofacial defects and a polydactyly phenotype similar to GCPS. We show that a deficiency of Gli3 expression in the XtJ mutant is due to a deletion within the 3' end of the gene. Furthermore, structures affected in the mouse mutant and human syndrome were found to correlate with expression domains of Gli3 in mouse. These results strongly suggest that the deficiency of GLI3 function leads to GCPS
— id: 35060, year: 1993, vol: 3, page: 241, stat: Journal Article,

Gene targeting : a practical approach
Joyner, Alexandra L.
Oxford ; New York : IRL Press at Oxford University Press, c1993,
— id: 690, year: 1993, vol: , page: , stat: ,

Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death
Klein R; Smeyne RJ; Wurst W; Long LK; Auerbach BA; Joyner AL; Barbacid M
1993 Oct 8;75(1):113-122, Cell
We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145trkB, a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkBTK(-/-), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkBTK (-/-) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp145trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system
— id: 35058, year: 1993, vol: 75, page: 113, stat: Journal Article,

GERMLINE TARGETING OF THE MOUSE TRKB GENE
KLEIN, R; WURST, W; LONG, LK; JOYNER, AL; BARBACID, M
1993 ;75(1):234-234, Journal of cellular biochemistry
— id: 104610, year: 1993, vol: 75, page: 234, stat: Journal Article,

Regional assignment of the human homeobox-containing gene EN1 to chromosome 2q13-q21
Kohler A; Logan C; Joyner AL; Muenke M
1993 Jan;15(1):233-235, Genomics
The human homeobox-containing genes EN1 and EN2 are closely related to the Drosophila pattern formation gene engrailed (en), which may be important in brain development, as shown by gene expression studies during mouse embryogenesis. Here, we have refined the localization of EN1 to human chromosome 2q13-q21 using a mapping panel of rodent/human cell hybrids containing different regions of chromosome 2 and a lymphoblastoid cell line with an interstitial deletion, del(2)(q21-q23.2). This regional assignment of EN1 increases to 22 the number of currently known genes on human chromosome 2q that have homologs on the proximal region of mouse chromosome 1
— id: 35063, year: 1993, vol: 15, page: 233, stat: Journal Article,

Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts
Logan C; Khoo WK; Cado D; Joyner AL
1993 Mar;117(3):905-916, Development
An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was capable of directing lacZ expression in an En-2-specific manner both temporally and spatially during embryogenesis and in the adult. lacZ expression was confined in the embryo to cells within the mid/hindbrain and mandibular arch regions and in the adult to cells of the molecular and granular layers of the cerebellum, and within the pons and colliculi regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior-posterior compartments. Analysis of the expression pattern of this transgene, in fetal and adult mice lacking a functional En-2 protein, provided evidence that the En-2 gene in mouse is not autoregulated. Deletion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5' of the transcribed sequences, which independently directed expression in the embryo to either the mid/hindbrain region or mandibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogenic enhancer thus far characterized. These En-2-specific regulatory regions can now be used in a biochemical analysis to identify proteins important in anterior-posterior patterning of the vertebrate CNS and in the specification of muscle identity as well as in a mutational analysis to direct expression of other developmentally important genes to these regions
— id: 35061, year: 1993, vol: 117, page: 905, stat: Journal Article,

Targeted mutagenesis in mice : a video guide
Pedersen, Roger A; Papaioannou, VE; Joyner, Alexandra L; Rossant, Janet
[Cold Spring Harbor NY] : Cold Spring Harbor Press, 1993,
— id: 1639, year: 1993, vol: , page: , stat: ,

Culture and transplantation of the mammalian circadian pacemaker
Ralph MR; Joyner AL; Lehman MN
1993 ;8 Suppl(3):S83-S87, Journal of biological rhythms
In transplantation studies using the tau mutation in the golden hamster, it has been demonstrated that suprachiasmatic nucleus (SCN) pacemaker cells and mechanisms of communication with the host brain are retained even after tissue dissociation and maintenance for many weeks in primary cell culture. Brain grafts of cultured SCN cells are capable of restoring overt rhythms of locomotor activity, and preliminary studies where cells from two tau genotypes are combined in a single graft demonstrate that pacemaker cells may communicate with each other to produce coherent rhythms with intermediate periods. The opportunity is presented, therefore, to study pacemaker-pacemaker communication in circadian chimeras produced by SCN transplantation. Immunocytochemical analysis of graft-host interactions requires the positive identification of host versus donor cells. Although grafted blocks of tissue are easily recognized during immunocytochemical analysis, implants of dissociated and cultured cells may be more diffusely located and are not as readily identified. Unless distinct strain- or species-specific markers are available, it is difficult to identify connections that may carry timing information to the host organism. We have taken an anatomical approach that utilizes cell-labeling techniques for hamster tissue along with foreign protein expression in transgenic mice to identify patterns of communication among graft and host cells, focusing specifically on SCN-SCN communication. The data indicate the usefulness of these transgenes as markers in transplantation studies where communication between graft and host is addressed
— id: 35062, year: 1993, vol: 8 Suppl, page: S83, stat: Journal Article,

IDENTIFICATION OF CIS-ACTING ELEMENTS OF THE MOUSE EN-2 GENE
SONG, DL; LOGAN, C; CADO, G; KHOO, W; JOYNER, AL
1993 ;8(3):209-209, Journal of cellular biochemistry
— id: 104611, year: 1993, vol: 8, page: 209, stat: Journal Article,

Production of targeted embryonic stem cell clones
Wurst, Wolfgang; Joyner, Alexandra L.
Gene targeting : a practical approach New York : Oxford University Press, 1993,
— id: 5220, year: 1993, vol: , page: 33, stat: Chapter,

PROBLEMS WITH CELL MARKERS AND EMBRYO CULTURES
BUCKINGHAM, ME; MCMAHON, AP; GOODFELLOW, PN; BEDDINGTON, RSP; HASTIE, H; HOGAN, BL; MCLAREN, AL; LAWSON, CA; JOYNER, AL; SMITH, JC; SOLTER, D; SAXEN, L; KAUFMAN, MH; COPP, AJ; LOVELLBADGE, R
1992 ;165(3):50-55, CIBA Foundation symposium
— id: 104612, year: 1992, vol: 165, page: 50, stat: Journal Article,

The gene trap approach in embryonic stem cells: the potential for genetic screens in mice
Joyner AL; Auerbach A; Skarnes WC
1992 ;165(5):277-288, CIBA Foundation symposium
The gene trap approach in embryonic stem cells was developed as a means to screen for genes expressed during early postimplantation development in the mouse. We have validated the approach by showing that lacZ from the integrated vector is activated by splicing to endogenous exons and expressed in embryos in patterns that mimic those of the endogenous genes. These insertions can produce developmental defects in homozygous mice. The results indicate that a large screen of gene trap cell lines on the basis of embryonic lacZ expression is feasible and should provide a new source of genes, mouse mutants and mouse strains that express lacZ in particular domains and lineages. The gene trap approach could be extended to a smaller screen for genes based on mutant phenotypes
— id: 35067, year: 1992, vol: 165, page: 277, stat: Journal Article,

Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions
Logan C; Hanks MC; Noble-Topham S; Nallainathan D; Provart NJ; Joyner AL
1992 ;13(5):345-358, Developmental genetics
We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways
— id: 35068, year: 1992, vol: 13, page: 345, stat: Journal Article,

FUNCTIONAL REDUNDANCY
MCLAREN, AL; GOODFELLOW, PN; SOLTER, D; HOGAN, BLM; JAENISCH, R; KRUMLAUF, R; MCMAHON, AP; COPP, AJ; JOYNER, AL; EVANS, M
1992 ;165(5):298-300, CIBA Foundation symposium
— id: 104613, year: 1992, vol: 165, page: 298, stat: Journal Article,

The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum
McMahon AP; Joyner AL; Bradley A; McMahon JA
1992 May 15;69(4):581-595, Cell
Mice homozygous for null alleles of the putative signaling molecule Wnt-1 have a reproducible phenotype: loss of the midbrain and adjacent cerebellar component of the metencephalon. By examining embryonic expression of the mouse engrailed (En) genes, from 8.0 to 9.5 days postcoitum, we demonstrate that Wnt-1 primarily regulates midbrain development. The midbrain itself is required for normal development of the metencephalon. Thus, the observed neonatal phenotype is explained by a series of early events, within 48 hr of neural plate induction, that leads to a complete loss of En domains in the anterior central nervous system. Wnt-1 and a related gene, Wnt-3a, are coexpressed from early somite stages in dorsal aspects of the myelencephalon and spinal cord. We suggest that functional redundancy between these two genes accounts for the lack of a caudal central nervous system phenotype
— id: 35065, year: 1992, vol: 69, page: 581, stat: Journal Article,

COMPILING INFORMATION ON DEVELOPMENTAL GENE-EXPRESSION
MCMAHON, AP; HERRMANN, BG; HOGAN, BLM; EVANS, M; GOODFELLOW, PN; KRUMLAUF, R; JOYNER, AL; BEDDINGTON, RSP; SOLTER, D; MCLAREN, AL; BUCKINGHAM, ME; WILKINSON, DG
1992 ;165(5):235-236, CIBA Foundation symposium
— id: 104614, year: 1992, vol: 165, page: 235, stat: Journal Article,

A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung
Moens CB; Auerbach AB; Conlon RA; Joyner AL; Rossant J
1992 May;6(5):691-704, Genes & development
The N-myc proto-oncogene encodes a putative transcription factor that has been postulated to be involved in the control of differentiation in a number of lineages at various stages during mammalian embryogenesis. We have generated a leaky mutation in N-myc by gene targeting in embryonic stem cells. In this allele, the neo(r) gene was inserted into the first intron of N-myc, in such a way that alternative splicing around this insertion could result in the generation of a normal N-myc transcript in addition to a mutant transcript. Mice homozygous for this mutation died immediately after birth owing to an inability to oxygenate their blood. Histological examination revealed a marked underdevelopment in the lung airway epithelium, resulting in a decreased respiratory surface area. Analysis of N-myc expression in wild-type and homozygous mutant embryonic lungs suggests that N-myc is required for the proliferation of the lung epithelium in response to local inductive signals emanating from the lung mesenchyme. Homozygous mutant embryos were slightly smaller than normal and also had a marked reduction in spleen size, whereas other tissues that normally express N-myc appeared to be unaffected by the mutation. Molecular analysis revealed that normal N-myc transcripts were found in tissues from homozygous mutant embryos. Different tissues expressed the normal N-myc transcript at different levels relative to those observed in wild-type embryos, with the lowest levels being observed in the lungs. These results illustrate one way in which gene targeting can be used to generate partial loss-of-function mutations and support the importance of generating a series of alleles at a given locus to elucidate the various different functions of a gene during development
— id: 35066, year: 1992, vol: 6, page: 691, stat: Journal Article,

Gene targeting
Sedivy, John M; Joyner, Alexandra L
New York : W.H. Freeman, 1992,
— id: 1638, year: 1992, vol: , page: , stat: ,

A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice
Skarnes WC; Auerbach BA; Joyner AL
1992 Jun;6(6):903-918, Genes & development
We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration
— id: 35064, year: 1992, vol: 6, page: 903, stat: Journal Article,

Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum
Davis CA; Holmyard DP; Millen KJ; Joyner AL
1991 Feb;111(2):287-298, Development
We have raised an antiserum, designated alpha Enhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, alpha Enhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and hindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombomeres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes
— id: 35071, year: 1991, vol: 111, page: 287, stat: Journal Article,

Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development
Joyner AL
1991 Dec;13(12):649-656, Bioessays
Mouse embryonic stem cell lines offer an attractive route for introducing rare genetic alternations into the gene pool since the cells can be pre-screened in culture and the mutations then transmitted into the germline through chimera production. Two applications of this technique that seem ideally suited for a genetic analysis of development are enhancer and gene trap screens for loci expressed during gastrulation and production of targeted mutations using homologous recombination. These approaches should greatly increase the number of mouse developmental mutants available and help to elucidate the genetic hierarchy controlling embryogenesis
— id: 35069, year: 1991, vol: 13, page: 649, stat: Journal Article,

Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox
Joyner AL; Herrup K; Auerbach BA; Davis CA; Rossant J
1991 Mar 8;251(4998):1239-1243, Science
The two mouse genes, En-1 and En-2, that are homologs of the Drosophila segmentation gene engrailed, show overlapping spatially restricted patterns of expression in the neural tube during embryogenesis, suggestive of a role in regional specification. Mice homozygous for a targeted mutation that deletes the homeobox were viable and showed no obvious defects in embryonic development. This may be due to functional redundancy of En-2 and the related En-1 gene product during embryogenesis. Consistent with this hypothesis, the mutant mice showed abnormal foliation in the adult cerebellum, where En-2, and not En-1, is normally expressed
— id: 35070, year: 1991, vol: 251, page: 1239, stat: Journal Article,

Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes
Gossler A; Joyner AL; Rossant J; Skarnes WC
1989 Apr 28;244(4903):463-465, Science
A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development
— id: 35073, year: 1989, vol: 244, page: 463, stat: Journal Article,

Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells
Joyner AL; Skarnes WC; Rossant J
1989 Mar 9;338(6211):153-156, Nature
A full understanding of the function of genes that control developmental events can be obtained only by a combination of molecular and mutational analysis. One putative developmental gene is the mouse engrailed-like gene En-2, which was isolated by virtue of its extensive homology to Drosophila engrailed, which contributes to the control of segmentation in the developing insect. Our hybridization analysis in situ has revealed that expression of En-2 is restricted to a specific domain of the developing central nervous system from 8 days of development on, indicating a role for the gene in establishing spatial domains in the brain. Unfortunately no En-2 mutations are available to elucidate further its function in development. To this end, we report here the isolation of three pluripotent embryonic stem cell lines in which one copy of the homoeobox-containing gene, En-2, has been altered by homologous recombination
— id: 35077, year: 1989, vol: 338, page: 153, stat: Journal Article,

An HaeIII RFLP for the human homeo box-containing gene EN1
Logan C; Joyner AL
1989 Apr 11;17(7):2877-2877, Nucleic acids research
— id: 35076, year: 1989, vol: 17, page: 2877, stat: Journal Article,

An SstI RFLP for the human homeo box-containing gene EN2
Logan C; Joyner AL
1989 Apr 11;17(7):2878-2878, Nucleic acids research
— id: 35075, year: 1989, vol: 17, page: 2878, stat: Journal Article,

PvuII and RsaI RFLPs for the human homeo box-containing gene EN2
Logan C; Joyner AL
1989 Apr 11;17(7):2879-2879, Nucleic acids research
— id: 35074, year: 1989, vol: 17, page: 2879, stat: Journal Article,

Chromosomal localization of the human homeo box-containing genes, EN1 and EN2
Logan C; Willard HF; Rommens JM; Joyner AL
1989 Feb;4(2):206-209, Genomics
The human homologs of the mouse homeo box-containing genes, En-1 and En-2, which show homology to the Drosophila engrailed gene, have been isolated. The human EN1 gene was mapped to chromosome 2 by analysis of mouse-human somatic cell hybrids. The human EN2 gene was localized to chromosome 7, 7q32-7qter, by analysis of rodent-human somatic cell hybrids and cell lines carrying portions of chromosome 7
— id: 35078, year: 1989, vol: 4, page: 206, stat: Journal Article,

Towards a molecular-genetic analysis of mammalian development
Rossant J; Joyner AL
1989 Aug;5(8):277-283, Trends in genetics
The mouse has been a slower starter than many other organisms in the race to unravel the genetic control of embryonic development. Recent cloning of putative developmental genes combined with new approaches to manipulating the mouse genome seem set, however, to allow the mammalian embryo to move towards the front of the field
— id: 35072, year: 1989, vol: 5, page: 277, stat: Journal Article,

Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development
Davis CA; Joyner AL
1988 Dec;2(12B):1736-1744, Genes & development
We have compared the expression of the murine genes En-1,En-2, and in-1 during development by in situ hybridization. Expression of all three genes was first detected at 8.0 days in overlapping bands of the anterior neural folds. By 12.0 days the expression patterns diverged. En-1 and En-2 were expressed in a similar ring of cells in the central nervous system (CNS) at the midbrain/hindbrain junction. En-1 was also expressed de novo in two lateral stripes extending the length of the hindbrain and spinal cord, in the developing vertebral column, in two lateral stripes of dermatome-derived cells, and in the tail and limb buds. By 12.0 days int-1 expression showed little overlap with the En genes and could not be detected at later stages. At 15.5 days En gene expression was primarily limited to the midbrain/hindbrain in overlapping but nonidentical sets of differentiated cells. In the adult, En-1 and En-2 marked the same sets of cells in the pons, but En-2 alone was detected in the granular layer of the cerebellum. The results are consistent with int-1 and the En genes playing a role early in development in defining spatial domains in the CNS. Later in development the En genes may have an additional function in neurogenesis. En-1 expression in the developing pericordal tube suggests that it may also be involved in vertebral assembly
— id: 35079, year: 1988, vol: 2, page: 1736, stat: Journal Article,

Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain
Davis CA; Noble-Topham SE; Rossant J; Joyner AL
1988 Mar;2(3):361-371, Genes & development
We have examined the pattern of expression of the homeo box-containing gene En-2 during mouse embryogenesis using in situ hybridization. Transcripts were first detected in the neural folds of 8.0-day, 5-somite embryos, and expression continued throughout development into adulthood. Hybridization occurred only in the central nervous system (CNS) and was limited to one band of the neural tube and to parts of those structures that later developed from it; the cerebellum, pons, periaqueductal gray, and colliculi. Expression in the germinal zone of the CNS was uniform within the hybridizing band. However, later in development, once cells had migrated out of the germinal zone, there was a reduction in the extent of hybridization and an increase in its spatial complexity. In the adult, expression of En-2 appeared to be limited to specific groups of neurons. The early, localized expression of En-2 within an apparently homogeneous tissue is consistent with the hypothesis that En-2 plays a role in defining a spatial domain within the developing brain
— id: 35080, year: 1988, vol: 2, page: 361, stat: Journal Article,

En-1 and En-2, two mouse genes with sequence homology to the Drosophila engrailed gene: expression during embryogenesis
Joyner AL; Martin GR
1987 Mar;1(1):29-38, Genes & development
Two mouse genes, En-1 and En-2, have sequence homology to the engrailed (en) and invected (inv) genes of Drosophila (Joyner et al. 1985). Partial nucleotide sequence analyses of genomic and cDNA clones show that the homologous sequences can encode a stretch of 107 amino acids, including a centrally located, 60-amino-acid homeo box. Within the homologous regions of the mouse and Drosophila genes, 78 (73%) of the amino acids are identical. Such extensive conservation of sequence outside the homeo box between vertebrate and Drosophila homeo box-containing genes is thus far unique. En-1 and En-2 are expressed during mouse embryogenesis. Transcripts from both genes were detected in RNA samples from teratocarcinoma cells, which serve as in vitro models for the early embryo, and from embryos at 9.5 through 17.5 days of development. For each gene we observed a unique pattern of changes in the number and relative intensities of transcripts detectable during embryonic development. Transcripts from both genes are represented abundantly in RNA extracted from the posterior portion of the fetal brain, and are much less abundant in RNA from other fetal tissues, including the anterior portion of the brain and the spinal cord. The chromosome map positions of En-1 and En-2 were determined by recombinant inbred strain analyses. Unlike their Drosophila counterparts, they are unlinked: En-1 is in the central portion of the mouse chromosome 1 and En-2 is in the proximal portion of mouse chromosome 5. Both genes map in the vicinity of mutations that are known to cause abnormalities during development
— id: 35083, year: 1987, vol: 1, page: 29, stat: Journal Article,

CORRECTION
JOYNER, AL
1987 ;1(5):521-521, Genes & development
— id: 104615, year: 1987, vol: 1, page: 521, stat: Journal Article,

Establishment of embryonic stem cell lines from preimplantation mouse embryos homozygous for lethal mutations in the t-complex
Martin GR; Silver LM; Fox HS; Joyner AL
1987 May;121(1):20-28, Developmental biology (Orlando)
We have determined the frequency at which embryonic stem cell (ESC) lines can be established from inner cell masses (ICMs) isolated from blastocysts homozygous for lethal mutations in the mouse t-complex. Approximately one-third of the expected number, 3/29, of the ESC lines established from embryos obtained by inter-se mating of +/tw18 mice are homozygous for the tw18 haplotype. These tw18/tw18 ESC lines form a variety of cell types in vitro and in vivo, including mesodermal derivatives such as cartilage and muscle. On the basis of these and data from other studies, we suggest that the normal function of the gene represented by the tw18 lethal allele is required for multiplication/survival of mesodermal precursors in the embryo rather than the specification of the mesodermal lineage, and that the lethal effects of this mutation are expressed in only the highly structured environment of the early postimplantation embryo. In studies of the lethal tw5 haplotype, we found that 2/2 ESC lines obtained are mutant homozygotes. Analysis of these data, in conjunction with the results of our earlier study (Magnuson, T., Epstein, C. J., Silver, L. M., and Martin, G. R. (1982), Nature (London) 298, 750-753), suggests that homozygosity for the genes found in the tw5 haplotype does not reduce cell viability. By contrast, 0/16 ESC lines isolated from embryos obtained from matings of +/t0 mice are mutant homozygotes. Analysis of the genotypes of ICM-derived primary stem cell colonies suggests that t0 homozygous ICM cells are unable to undergo sufficient proliferation in vitro to give rise to ESC lines
— id: 35082, year: 1987, vol: 121, page: 20, stat: Journal Article,

Differential expression of the mouse homeobox-containing gene Hox-1.4 during male germ cell differentiation and embryonic development
Wolgemuth DJ; Viviano CM; Gizang-Ginsberg E; Frohman MA; Joyner AL; Martin GR
1987 Aug;84(16):5813-5817, Proceedings of the National Academy of Sciences of the United States of America
Hox-1.4 is a mouse homeobox-containing gene (initially identified as HBT-1), whose expression appears to be testis-specific in the adult animal. Examination of Hox-1.4 transcripts in RNA from testes of mutant mice deficient in germ cells confirms that Hox-1.4 expression within the testis is germ cell-specific. Enriched populations of spermatogenic cells were used to localize the expression of Hox-1.4 specifically to germ cells that have entered into and progressed beyond the meiotic prophase stage of differentiation and to demonstrate the presence of two different size Hox-1.4 transcripts. Examination of RNA from teratocarcinoma cell cultures and mouse embryos at 10.5-16.5 days of gestation demonstrated the presence of several Hox-1.4 transcripts, which are larger than those present in germ cells. In the midgestation fetus, Hox-1.4 expression is most abundant in the spinal cord
— id: 35081, year: 1987, vol: 84, page: 5813, stat: Journal Article,

Expression of homologous homeo-box-containing genes in differentiated human teratocarcinoma cells and mouse embryos
Hauser CA; Joyner AL; Klein RD; Learned TK; Martin GR; Tjian R
1985 Nov;43(1):19-28, Cell
Human chromosome 17 contains a cluster of at least three distinct homeo box regions, Hu1, Hu2, and Hu5, within a 20 kb stretch of DNA. A mouse homeo box region, Mu1, which maps to chromosome 11, was isolated and found to contain extensive nucleotide homology with a 4 kb region encompassing the Hu1 homeo box. The conservation of these chromosomal regions between man and mouse was confirmed by nucleotide sequence analysis: approximately 380 bp of DNA are more than 90% homologous and the 61 amino acids of the homeo box domain are perfectly conserved. We found that a human teratocarcinoma cell line expresses high levels of Hu1 homeo-box-containing mRNA only after differentiation of the cells following treatment with retinoic acid. In contrast, mouse teratocarcinoma cells did not express Mu1 homeo-box-containing mRNA at any stage of differentiation, whereas the expression of such transcripts was detected in mouse embryos from 10 to 17 days of gestation
— id: 35084, year: 1985, vol: 43, page: 19, stat: Journal Article,

Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene
Joyner AL; Kornberg T; Coleman KG; Cox DR; Martin GR
1985 Nov;43(1):29-37, Cell
Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis
— id: 35085, year: 1985, vol: 43, page: 29, stat: Journal Article,

Comparative chromosome mapping of a conserved homoeo box region in mouse and human
Joyner AL; Lebo RV; Kan YW; Tjian R; Cox DR; Martin GR
1985 Mar 14-20;314(6007):173-175, Nature
Specific genes are assumed to regulate pattern formation in the mammalian embryo, but as yet none has been identified unequivocally. It is possible that such genes in mammals may be identified by virtue of a conserved coding sequence, because many of the Drosophila melanogaster homoeotic and segmentation genes, which have crucial roles in the regulation of segmental pattern formation during embryonic development, contain a 180-base pair (bp) DNA sequence, the homoeo box, and that sequences homologous to the Drosophila homoeo box are also present in 6-10 copies in higher animals, including mammals. Although the assumption that the homoeo box identifies genes responsible for pattern formation in mammals remains to be validated, it is a particularly attractive hypothesis given the strong conservation of homoeo boxes over vast evolutionary distances. Here we report the localization of a human homoeo box region, previously cloned and shown to contain two homoeo boxes within a sequence of 5-kilobases (kb), to the long arm of chromosome 17. We show that two single-copy homoeo box-flanking probes derived from this region strongly hybridize to single-copy restriction fragments in mouse genomic DNA and that these conserved homoeo box-flanking sequences map to mouse chromosome 11. This may be significant as several genes that map to chromosome 17 in human also map to chromosome 11 in the mouse, implying that a segment of mouse chromosome 11 is homologous to a region of human chromosome 17. Taken together, these data suggest that the homoeo box region detected with our probes is highly conserved in human and mouse
— id: 35086, year: 1985, vol: 314, page: 173, stat: Journal Article,

COMPARATIVE CHROMOSOME MAPPING OF A CONSERVED HOMEO BOX REGION IN HUMAN AND MOUSE
JOYNER, AL; LEBO, RV; KAN, YW; TJIAN, R; COX, DR; MARTIN, GR
1985 ;40(1-4):663-663, Cytogenetics & cell genetics
— id: 104616, year: 1985, vol: 40, page: 663, stat: Journal Article,

Retrovirus mediated gene expression and gene transduction
Joyner, Alexandra L
Ottawa : National Library of Canada, 1985,
'Thesis (Ph.D) -- University of Toronto, 1983'
— id: 1640, year: 1985, vol: , page: , stat: ,

Retrovirus transduction: generation of infectious retroviruses expressing dominant and selectable genes is associated with in vivo recombination and deletion events
Joyner AL; Bernstein A
1983 Dec;3(12):2180-2190, Molecular & cellular biology
We describe the generation of infectious retroviruses containing foreign genes by an in vivo recombination-deletion mechanism. Cotransfection into mouse cells of chimeric plasmids carrying a murine retrovirus 5' long terminal repeat and either the thymidine kinase (tk) gene of herpesvirus or the dominant selectable bacterial gene for neomycin resistance (neo), along with a clone of Moloney murine leukemia virus, results in the generation of infectious thymidine kinase or neomycin-resistant viruses. Expression of the selectable marker in these viruses can be regulated by the homologous transcriptional promoter of the gene, by the promoter contained within the Friend spleen focus-forming virus long terminal repeat, or by the simian virus 40 early region promoter. In all cases, the rescued viruses appeared to arise by recombination in vivo with Moloney murine leukemia virus sequences, resulting in the acquisition of the Moloney 3' long terminal repeat and variable amounts of the 3' adjacent Moloney genome. In two of the thymidine kinase constructs where tk was inserted 200 base pairs downstream from the long terminal repeat, the rescued viruses acquired a large part of the murine leukemia virus genome, including the region involved in packaging genomic RNA into virions. The generation of infectious neomycin-resistant virus is associated with deletions of simian virus 40 splicing and polyadenylation sequences. These results demonstrate that nonhomologous recombination and deletion events can take place in animal cells, resulting in the acquisition or removal of cis-acting sequences required for, or inhibitory to, retrovirus infectivity
— id: 35088, year: 1983, vol: 3, page: 2180, stat: Journal Article,

Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors
Joyner AL; Bernstein A
1983 Dec;3(12):2191-2202, Molecular & cellular biology
A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers
— id: 35087, year: 1983, vol: 3, page: 2191, stat: Journal Article,

Molecular identification of a human DNA repair gene following DNA-mediated gene transfer
Rubin JS; Joyner AL; Bernstein A; Whitmore GF
1983 Nov 10-16;306(5939):206-208, Nature
Although it has long been evident that the response of eukaryotes to DNA damaging agents is determined by the effectiveness of a variety of DNA repair systems, there is little detailed knowledge of the nature of these systems or the genes which control them. In humans, a number of hereditary conditions, including xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anaemia, exhibit increased sensitivity to a variety of DNA damaging agents and a predisposition to cancer, suggesting a defect in some aspect of DNA repair. This report describes the identification of a human DNA repair gene following DNA-mediated gene transfer into Chinese hamster ovary (CHO) mutant cells, that like xeroderma pigmentosum cells, are sensitive to a variety of DNA damaging agents and are defective in the initial incision step of DNA repair. The resulting transformants exhibit normal resistance to DNA damaging agents and independent transformants demonstrate a common set of human DNA sequences associated with a human DNA repair gene. These observations provide the basis for the isolation and characterization of the human genes responsible for DNA repair
— id: 35089, year: 1983, vol: 306, page: 206, stat: Journal Article,