Germ line stem cell development in Drosophila
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 understand how the architecture of germplasm contributes to germ cell functions. In genetic studies, we focus on Tudor and Germ cell-less, two proteins known to affect germ cell formation. We identified more than 50 maternally synthesized RNAs that are localized to germplasm and protected from degradation as primordial germ cells (PGCs) form. In addition to characterizing the function of these “germ genes” in germ cell formation, specification and migration, we are developing approaches to identify evolutionary conserved “RNA regulatory codes” that mediates localization and translation of these RNAs specifically during germ line development. Coincident with germ cell specification, early Drosophila PGCs become transcriptionally silent. During subsequent embryonic development, transcription in germ cells resumes and reaches high activity as PGCs reach the somatic gonad. We have begun a systematic analysis of the mechanisms underlying germ cell transcription.
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 and in vitro migration assays to identify the molecules involved in germ cell chemotaxis and survival.
3. Germ line stem cells
The germ line is an ideal system to study stem cell 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 germ line stem cells (GSCs), they are maintained in the adult. Most recently we have focused on analyzing how transcriptional regression mechanisms and RNA regulation via the piRNA pathways regulate GSC differentiation and gonad development. We are using large-scale genomic approaches to follow changes in gene and protein expression and observe changes in chromatin structure during germ line development. The goal is to correlate these global changes with specific regulatory steps required for germ line fate specification, control of genome integrity, GSC establishment and differentiation.