Holger Knaut, Ph.D.

Developmental neurotoxicity testing: recommendations for developing alternative methods for the screening and prioritization of chemicals
Crofton, Kevin M; Mundy, William R; Lein, Pamela J; Bal-Price, Anna; Coecke, Sandra; Seiler, Andrea E M; Knaut, Holger; Buzanska, Leonora; Goldberg, Alan
2011 ;28(1):9-15, Altex : Alternatives to animal experimentation
Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in critical need of alternative methods to current animal testing protocols and guidelines. An immediate goal is to develop test methods that are capable of screening large numbers of chemicals. This document provides recommendations for developing alternative DNT approaches that will generate the type of data required for evaluating and comparing predictive capacity and efficiency across test methods and laboratories. These recommendations were originally drafted to stimulate and focus discussions of alternative testing methods and models for DNT at the TestSmart DNT II meeting (http://caat.jhsph.edu/programs/workshops/dnt2.html) and this document reflects critical feedback from all stakeholders that participated in this meeting. The intent of this document is to serve as a catalyst for engaging the research community in the development of DNT alternatives and it is expected that these recommendations will continue to evolve with the science
— id: 141797, year: 2011, vol: 28, page: 9, stat: Journal Article,

Neurovascular niche in the ventricular zone of the adult zebrafish telencephalon
Kishimoto N.; Shimizu K.; Nagai H.; Asakawa K.; Urasaki A.; Knaut H.; Nonaka S.; Kawakami K.; Sawamoto K.
2011 ;71:e74-e74, Neuroscience research
In the adult mammalian brain, newborn cells generated in the subventricular zone migrate towards the olfactory bulb (OB) through the rostral migratory stream (RMS). We have previously reported that adult zebrafish also possesses a niche for neural stem cells in the telencephalic ventricular zone (TVZ), in which neuronal precursor cells (NPCs) are generated and migrate into the OB via the RMS. However, the cellular and molecular mechanisms underlying the formation of such structure and migration of these NPCs in the migratory stream remain uncovered. Here, we show that blood vessels precisely outline the migratory stream of the NPCs in the adult zebrafish brain. In this region, NPCs migrate along the blood vessels into the OB. In addition, we show that Sdf1 and Cxcr4 are expressed in the blood vessels and NPCs in the TVZ, respectively.Wefound that perturbation of the Sdf1/Cxcr4 signaling resulted in the dispersion of NPCs from the migratory stream without affecting cell proliferation in the TVZ, eventually leading to the decreased number of mature neurons in the OB. Thus, our data suggest that the Sdf1/Cxcr4 chemokine signaling plays an important role in the neurovascular niche to maintain the RMS structure within the adult zebrafish TVZ for the migration of NPCs to the OB
— id: 137091, year: 2011, vol: 71, page: e74, stat: Journal Article,

miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration
Staton, Alison A; Knaut, Holger; Giraldez, Antonio J
2011 Mar;43(3):204-211, Nature genetics
microRNAs (miRNAs) function as genetic rheostats to control gene output. Based on their role as modulators, it has been postulated that miRNAs canalize development and provide genetic robustness. Here, we uncover a previously unidentified regulatory layer of chemokine signaling by miRNAs that confers genetic robustness on primordial germ cell (PGC) migration. In zebrafish, PGCs are guided to the gonad by the ligand Sdf1a, which is regulated by the sequestration receptor Cxcr7b. We find that miR-430 regulates sdf1a and cxcr7 mRNAs. Using target protectors, we demonstrate that miR-430-mediated regulation of endogenous sdf1a (also known as cxcl12a) and cxcr7b (i) facilitates dynamic expression of sdf1a by clearing its mRNA from previous expression domains, (ii) modulates the levels of the decoy receptor Cxcr7b to avoid excessive depletion of Sdf1a and (iii) buffers against variation in gene dosage of chemokine signaling components to ensure accurate PGC migration. Our results indicate that losing miRNA-mediated regulation can expose otherwise buffered genetic lesions leading to developmental defects
— id: 134143, year: 2011, vol: 43, page: 204, stat: Journal Article,

Clearing the path for germ cells
Knaut, Holger; Schier, Alexander F
2008 Feb 8;132(3):337-339, Cell
The chemokine SDF-1a and its receptor CXCR4b guide germ cell migration in zebrafish by activating downstream signaling events. Boldajipour et al. (2008) now report that a second SDF-1a receptor, CXCR7, is also required for guided migration but does not function as a signaling receptor, and instead sequesters SDF-1a. These results highlight the importance of ligand clearance during guided cell migration
— id: 76155, year: 2008, vol: 132, page: 337, stat: Journal Article,

Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies
Coecke, Sandra; Goldberg, Alan M; Allen, Sandra; Buzanska, Leonora; Calamandrei, Gemma; Crofton, Kevin; Hareng, Lars; Hartung, Thomas; Knaut, Holger; Honegger, Paul; Jacobs, Miriam; Lein, Pamela; Li, Abby; Mundy, William; Owen, David; Schneider, Steffen; Silbergeld, Ellen; Reum, Torsten; Trnovec, Tomas; Monnet-Tschudi, Florianne; Bal-Price, Anna
2007 Jun;115(6):924-931, Environmental health perspectives
This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work
— id: 90758, year: 2007, vol: 115, page: 924, stat: Journal Article,

Cxcl12/Cxcr4 chemokine signaling is required for placode assembly and sensory axon pathfinding in the zebrafish olfactory system
Miyasaka, Nobuhiko; Knaut, Holger; Yoshihara, Yoshihiro
2007 Jul;134(13):2459-2468, Development
Positioning neurons in the right places and wiring axons to the appropriate targets are essential events for establishment of neural circuits. In the zebrafish olfactory system, precursors of olfactory sensory neurons (OSNs) assemble into a compact cluster to form the olfactory placode. Subsequently, OSNs differentiate and extend their axons to the presumptive olfactory bulb with high precision. In this study, we aim to elucidate the molecular mechanism underlying these two developmental processes. cxcr4b, encoding a chemokine receptor, is expressed in the migrating olfactory placodal precursors, and cxcl12a (SDF-1a), encoding a ligand for Cxcr4b, is expressed in the abutting anterior neural plate. The expression of cxcr4b persists in the olfactory placode at the initial phase of OSN axon pathfinding. At this time, cxcl12a is expressed along the placode-telencephalon border and at the anterior tip of the telencephalon, prefiguring the route and target of OSN axons, respectively. Interfering with Cxcl12a/Cxcr4b signaling perturbs the assembly of the olfactory placode, resulting in the appearance of ventrally displaced olfactory neurons. Moreover, OSN axons frequently fail to exit the olfactory placode and accumulate near the placode-telencephalon border in the absence of Cxcr4b-mediated signaling. These data indicate that chemokine signaling contributes to both the olfactory placode assembly and the OSN axon pathfinding in zebrafish
— id: 90759, year: 2007, vol: 134, page: 2459, stat: Journal Article,

Assembly of trigeminal sensory ganglia by chemokine signaling
Knaut, Holger; Blader, Patrick; Strahle, Uwe; Schier, Alexander F
2005 Sep 1;47(5):653-666, Neuron
Sensory neurons with related functions form ganglia, but how these precisely positioned clusters are assembled has been unclear. Here, we use the zebrafish trigeminal sensory ganglion as a model to address this question. We find that some trigeminal sensory neurons are born at the position where the ganglion is assembled, whereas others are born at a distance and have to migrate against opposing morphogenetic movements to reach the site of ganglion assembly. Loss of Cxcr4b-mediated chemokine signaling results in the formation of mispositioned ganglia. Conversely, ectopic sources of the chemokine SDF1a can attract sensory neurons. Transplantation experiments reveal that neuron-neuron interaction and the adhesion molecules E- and N-Cadherin also contribute to ganglion assembly. These results indicate that ganglion formation depends on the interplay of birthplace, chemokine attraction, cell-cell interaction, and cadherin-mediated adhesion
— id: 58739, year: 2005, vol: 47, page: 653, stat: Journal Article,

Towing of sensory axons by their migrating target cells in vivo
Gilmour, Darren; Knaut, Holger; Maischein, Hans-Martin; Nusslein-Volhard, Christiane
2004 May;7(5):491-492, Nature neuroscience
Many pathfinding axons must locate target fields that are themselves positioned by active migration. A hypothetical method for ensuring that these migrations are coordinated is towing, whereby the extension of axons is entirely dependent on the migration of their target cells. Here we combine genetics and time-lapse imaging in the zebrafish to show that towing by migrating cells is a bona fide mechanism for guiding pathfinding axons in vivo
— id: 90760, year: 2004, vol: 7, page: 491, stat: Journal Article,

A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor
Knaut, Holger; Werz, Christian; Geisler, Robert; Nusslein-Volhard, Christiane
2003 Jan 16;421(6920):279-282, Nature
Germ cells preserve an individual's genetic information and transmit it to the next generation. Early in development germ cells are set aside and undergo a specialized developmental programme, a hallmark of which is the migration from their site of origin to the future gonad. In Drosophila, several factors have been identified that control germ-cell migration to their target tissues; however, the germ-cell chemoattractant or its receptor have remained unknown. Here we apply genetics and in vivo imaging to show that odysseus, a zebrafish homologue of the G-protein-coupled chemokine receptor Cxcr4, is required specifically in germ cells for their chemotaxis. odysseus mutant germ cells are able to activate the migratory programme, but fail to undergo directed migration towards their target tissue, resulting in randomly dispersed germ cells. SDF-1, the presumptive cognate ligand for Cxcr4, shows a similar loss-of-function phenotype and can recruit germ cells to ectopic sites in the embryo, thus identifying a vertebrate ligand-receptor pair guiding migratory germ cells at all stages of migration towards their target
— id: 90761, year: 2003, vol: 421, page: 279, stat: Journal Article,

Production of maternal-zygotic mutant zebrafish by germ-line replacement
Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F
2002 Nov 12;99(23):14919-14924, Proceedings of the National Academy of Sciences of the United States of America
We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies
— id: 39385, year: 2002, vol: 99, page: 14919, stat: Journal Article,

An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish
Knaut, Holger; Steinbeisser, Herbert; Schwarz, Heinz; Nusslein-Volhard, Christiane
2002 Mar 19;12(6):454-466, Current biology. CB
BACKGROUND: In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization. RESULTS: To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, we tested different vasa homologs for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish. CONCLUSIONS: Our results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline
— id: 90762, year: 2002, vol: 12, page: 454, stat: Journal Article,

Analyse der Produkte des Vasa-Gens bei Keimzellspezifizierung von Zebrafischen
Knaut, Holger
[S.l. : s.n.], 2001,
Thesis -- Tubingen Universitat, 2001
— id: 1943, year: 2001, vol: , page: , stat: ,

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification
Knaut, H; Pelegri, F; Bohmann, K; Schwarz, H; Nusslein-Volhard, C
2000 May 15;149(4):875-888, Journal of cell biology
Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment
— id: 90763, year: 2000, vol: 149, page: 875, stat: Journal Article,

A radiation hybrid map of the zebrafish genome
Geisler, R; Rauch, G J; Baier, H; van Bebber, F; Bross, L; Dekens, M P; Finger, K; Fricke, C; Gates, M A; Geiger, H; Geiger-Rudolph, S; Gilmour, D; Glaser, S; Gnugge, L; Habeck, H; Hingst, K; Holley, S; Keenan, J; Kirn, A; Knaut, H; Lashkari, D; Maderspacher, F; Martyn, U; Neuhauss, S; Neumann, C; Nicolson, T; Pelegri, F; Ray, R; Rick, J M; Roehl, H; Roeser, T; Schauerte, H E; Schier, A F; Schonberger, U; Schonthaler, H B; Schulte-Merker, S; Seydler, C; Talbot, W S; Weiler, C; Nusslein-Volhard, C; Haffter, P
1999 Sep;23(1):86-89, Nature genetics
Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb
— id: 90765, year: 1999, vol: 23, page: 86, stat: Journal Article,

A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization
Pelegri, F; Knaut, H; Maischein, H M; Schulte-Merker, S; Nusslein-Volhard, C
1999 Dec 16-30;9(24):1431-1440, Current biology. CB
BACKGROUND: In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow
— id: 90764, year: 1999, vol: 9, page: 1431, stat: Journal Article,

The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence
Kim, J H; Ryan, M G; Knaut, H; Hille, R
1996 Mar 22;271(12):6771-6780, Journal of biological chemistry
The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the 'very rapid' EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay
— id: 90766, year: 1996, vol: 271, page: 6771, stat: Journal Article,