In most organisms, primordial germ cells (PGCs) are set-aside early during embryogenesis. Subsequently, PGCs migrate through the embryo, associate with somatic gonadal cells and form the embryonic gonad. Here, PGCs become germline stem cells that eventually give rise to sperm and egg. We developed assay systems in Drosophila that allow us to conduct large-scale genetic screens to identify factors required for (1) PGC formation and specification, (2) PGC migration, (3) stem cell maintenance and differentiation.
1. Germ cell formation and transcriptional silencing
The molecular mechanisms that set aside germ cells and somatic cells in Drosophila are very different: somatic cells form as a polarized epithelium while germ cells develop by budding within the specialized germplasm. Our goal is to characterize the function and architecture of germplasm. In genetic studies, we focus on Tudor and Germ cell-less, two proteins known to affect germ cell formation. We use high-resolution imaging and biochemical co-purification of proteins and RNAs known to localize to the germplasm to characterize the physical relationship between germplasm components. We identified 14 maternally synthesized RNAs that are localized to germ plasm and protected from degradation as germ cells form. In addition to characterizing the function of these “germ genes”, we use these RNAs to derive a “RNA regulatory code” that mediates localization and translational control.
Another aspect that distinguishes early germ cells from somatic cells is that germ cells are transcriptionally silent. We showed that germ cells lacking the polar granule component (pgc) gene transcribe genes normally expressed only in adjacent somatic cells. Our analysis of RNA polymerase II activity and histone methylation in wild-type and mutant germ cells is consistent with pgc blocking transcription at the elongation stage. Our present goal is to determine the underlying mechanism.
2. Germ cell migration.
PGCs form at the posterior pole of the Drosophila embryo and are carried inside the embryo during gastrulation in juxtaposition to the posterior midgut. Subsequently, PGCs migrate actively through the midgut epithelium and navigate along the midgut toward the mesoderm, where they associate with somatic gonadal precursors to form the embryonic gonad. At least three signaling pathways regulate germ cell migration: a) The G protein coupled receptor tre1 controls transepithelial migration through the posterior midgut. b) Wunen and Wunen 2, two lipid phosphate phosphatase, affect germ cell repulsion and survival. c) The isoprenylation via the HMGCoA reductase pathway regulates the production of a germ cell attractant. Complementary to genetic and biochemical studies we are using live imaging to develop a cellular view of germ cell chemoattraction and repulsion.
3. Germline stem cells
The germ line is an ideal system to study germ line stem cell (GSC) maintenance and differentiation. We are interested in understanding how PGCs populate the gonad, how they are prevented from differentiation during larval stages and how, as GSC, they are maintained in the adult. During larval stages, PGCs and the ovary grow in size. We recently found that a negative feedback loop between germ cells and somatic cells regulates germ cell number. In this loop, PGCs express the EGF receptor ligand Spitz and regulate the survival of somatic cells. These in turn produce a growth inhibitory signal that limits PGC division. This mechanism may assure that a sufficient number of PGCs occupy the niche and become GSCs. We further found that the same signals prevented GSCs and PGCs from differentiation. These results also showed that the entire early embryonic gonad represses stem cell differentiation while at later stages this repressive activity restricts to the niche. Future experiments are aimed to analyze somatic signals that influence stem cell behavior and to identify a common repertoire of genes regulating PGC and GSC behavior.
