Germ cells are the stem cells for the next generation. Set aside during embryogenesis, they have the potential to generate a new organism through the fusion of sperm and egg. We are interested in understanding how germ cells are specified in the early embryo, how they migrate through the embryo to reach the somatic part of the gonad, and how they become stem cells that continue to produce egg and sperm throughout adulthood. We study germ cell development in Drosophila, where large-scale genetic analysis can be combined with sophisticated imaging techniques to identify and characterize factors that regulate germline development.
Setting Germ Cells Aside
Drosophila germ cells are formed at the posterior pole of the embryo in a highly specialized, maternally synthesized cytoplasm, the germplasm. We are interested in how the germplasm instructs germ cell fate. Polar granules, electron-dense particles, are the hallmark of germplasm. Our goal is to characterize structural and functional units within the polar granule. For this analysis, we have focused on Tudor protein, which is composed of 11 Tudor "domains" and affects polar granule architecture. The germplasm also harbors a large number of RNAs that likely play specific roles in germ cell specification, migration, and germ cell fate. Among these, nanos (nos), germ cell less (gcl), and polar granule component (pgc) RNAs become enriched in the germplasm during oogenesis. As nuclei approach the germplasm, RNA "islands" surround each nucleus (Figure 1). These three genes have important roles during early germline development: Gcl protein is required for germ cell formation, Nos protein is required for germ cell fate and migration, and Pgc protein is required for transcriptional silencing in germ cells. Despite their similar RNA distribution pattern, protein production by these three RNAs is strikingly different: nos RNA is translated as it reaches the germplasm, gcl is translated as nuclei migrate into the germplasm, and pgc is translated once germ cells are formed. Prashanth Rangan, a postdoctoral fellow in our lab, found that the 3'-untranslated region (3'-UTR) of these RNAs instructs not only the localization pattern but also the time when the RNAs are translated.
Following the Germ Cell Path
Primordial germ cells (PGCs) form next to somatic cells that give rise to the posterior midgut. During gastrulation, germ cells are carried into the embryo inside the blind end of the posterior midgut. Here germ cells start their active migration. We have identified mutations in genes that affect different stages of migration. A G protein–coupled receptor (GPCR) that acts in PGCs controls the directed migration through the midgut epithelium (Figure 2). PGCs fail to migrate through the posterior midgut in embryos lacking this GPCR and remain "trapped in the gut" (giving the gene that affects this step its name, trapped in endoderm 1 [tre1]). Live imaging studies reveal that Tre1 mediates the transition of germ cells to individualized migratory behavior by modulating the adhesion protein E-cadherin.
During the next migration step, germ cells migrate along the midgut toward the nearby mesoderm, where they sort into bilateral groups before reaching the somatic gonad. Using real-time imaging, Hiroko Sano in our lab found that PGCs migrate directionally toward the somatic gonad (Movie 1), mostly guided by repellent activity of the wunen (wun) and wunen-2 (wun2) genes. PGCs migrate aimlessly in embryos that lack Wun and Wun2 activity (Movie 2). The wunen genes encode homologs of mammalian lipid phosphate phosphatases (LPPs), a family of transmembrane proteins that carry an extracellular enzymatic region that hydrolyzes phospholipids. Both genes are transcribed in tissues flanking the PGC migratory path, and germ cells avoid these tissues.
Andrew Renault in our lab found that Wun and Wun2 are also required in germ cells, where they affect germ cell survival. The opposing effects on germ cell survival depend on whether Wun and Wun2 are expressed in the germ cells or the soma. This led us to propose the following model: In the soma, Wun and Wun2 deplete a survival and attractant phospholipid factor for germ cells, thereby creating a gradient of phospholipid. In the germ cells, Wun and Wun2 mediate hydrolysis of the phospholipid, and uptake of the lipid is required for germ cell survival and possibly for directed migration.
Finally, germ cells are attracted to the somatic component of the gonad and associate with it. This requires hmgcr, the gene encoding the Drosophila homolog of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). Normally, hmgcr is expressed at high levels in the somatic gonad. Genetic analysis revealed that mutations in enzymes acting downstream of HMG-CoAR and required for isoprenoid synthesis show PGC migration defects similar to hmgcr mutant embryos. Our studies suggest that isoprenylation may be the rate-limiting step in the production of the germ cell attractant. Sara Ricardo, a postdoctoral fellow in our lab, has developed an in vivo attraction assay to determine the nature of the attractant. (Our studies on germ cell migration are supported by a grant from the National Institutes of Health.)
Making the Next Generation
Once PGCs reach the somatic gonad, they divide mitotically during larval development to produce germline stem cells (GSCs), the source of continuous egg and sperm production throughout adulthood. GSCs reside in a specialized somatic niche. The niche forms during late larval stages and directs the asymmetric divisions of GSCs to rejuvenate and to generate a differentiating cystoblast throughout adulthood (Figure 3).
Lilach Gilboa, a postdoctoral fellow in our lab, asked how PGC growth is regulated during larval growth to assure that a sufficient pool of PGCs is present to fill all niches. First, she found that animals that start with fewer embryonic germ cells show increased PGC proliferation. Next, she showed that epidermal growth factor receptor (EGFR) activation in somatic cells that surround the germ cells sets a limit for germ cell proliferation: Loss of EGFR function leads to too many PGCs, while constitutive activation of the receptor leads to a decrease in PGC numbers. The EGFR ligand Spitz is expressed in the germ cells, and signaling to the receptor is required for the survival of a specific subset of somatic gonadal cells that surround the PGCs.
These and additional experiments led us to propose that an inhibitory feedback mechanism controls homeostasis during the growth of the gonad. In this model, activation of the EGFR by PGCs produces a survival signal for the surrounding somatic cells. This signal also activates a feedback inhibitor for germ cell proliferation. When germ cell numbers are low, little of this inhibitor is present and PGCs proliferate; as PGC numbers increase, more somatic cells survive and PGC proliferation slows. We do not yet know the nature of the inhibitory signal. One hypothesis is that direct physical contact between germ cells and somatic signals may limit growth or access to a growth signal. Alternatively, the somatic cells may directly produce an inhibitor of germ cell proliferation.