The mature eggs of many organisms are partitioned into domains that contain different informational molecules. After fertilization, these molecules become active and direct the future body pattern of the embryo. We are analyzing the genes and mechanisms used during oogenesis of Drosophila to build asymmetries into the egg. Our laboratory has found that two genes, gurken (grk) and torpedo (top), participate in a cell signaling process that conveys patterning information from the germline to the surrounding follicle cells. The top gene encodes the Drosophila homolog of the vertebrate epidermal growth factor receptor (EGFR). The grk gene encodes a protein with homology to transforming growth factor α. Understanding the activation of this signaling process and analyzing the subsequent patterning events have been the major focus of our recent work. In addition, we have started to use the follicle cell epithelium as a model system to study morphogenesis and polarity development in a single-layer epithelium.
grk RNA Localization
In mid-oogenesis, RNA is localized to the future dorsal side of the egg, in proximity to the oocyte nucleus. This localization provides a molecular asymmetry in the egg that is used to establish the dorsoventral axis of egg and embryo. We are analyzing the process that localizes grk RNA to the future dorsal side.
Mutations in several genes, in particular squid and fs(1)K10, as well as Hrb27c/Hrp48, result in mislocalization of grk mRNA to an anterior ring within the oocyte instead of the restricted accumulation at the normal dorsal-anterior site. The mislocalized grk RNA in these mutations is efficiently translated, which results in a dorsalized egg phenotype due to ectopic activation of EGFR. The squid gene encodes a heterogeneous nuclear ribonucleoprotein (hnRNP) homologous to human hnRNP A1 and A2. We found that the Squid (Sqd) proteins can be cross-linked to the grk RNA. Sqd presumably binds to grk RNA in the nucleus and facilitates its regulated export as well as its localization. In addition, Sqd is required to keep unlocalized RNA from being translated and thus ectopically activating EGFR. In screens for interactors of Sqd, we have isolated a number of proteins that are candidates for acting as components in a grk RNA transport and/or translational control complex. Among these we have analyzed the contributions of Cup and poly(A)-binding protein (pAbp). These proteins mediate the translational regulation of the grk RNA, repressing translation while the RNA is not localized and activating translation once the RNA has reached its destination.
To study the dynamic localization of grk mRNA in vivo, we have used a fluorescently tagged viral coat protein that can bind to a series of RNA stem loops that are inserted into the RNA of choice. We have introduced these stem loops into the grk mRNA and have made in vivo studies of the fluorescently tagged, localized RNA. This has revealed that at earlier stages of oogenesis the grk mRNA is dynamically localized in the posterior of the oocyte, whereas at mid-to-late stages of oogenesis the RNA is much more stably anchored at the dorsal-anterior corner of the oocyte.
Meiotic DNA Repair and Gurken Production
In earlier screens for oogenesis-specific mutations, we and others identified a number of mutations that cause an eggshell phenotype reminiscent of mutations in grk or Egfr. We characterized three of the corresponding genes: okra (okr), spindle-B (spn-B), and spindle-D (spn-D). Surprisingly, these genes encode proteins that function in repair of double-strand breaks (DSBs) in the DNA and are required for recombination during meiosis (okr: Rad54; spn-B and spn-D: Rad51, or RecA-like proteins). Mutations in these genes affect the frequency of meiotic recombination and chromosome disjunction and result in the failure to accumulate high levels of Grk (as well as other developmentally important proteins) in the oocyte.
Mutations in these DNA-repair proteins have been studied in detail in yeast, where it has been shown that in the absence of repair of DSBs, a checkpoint is activated that halts further progression through meiosis. We therefore reasoned that in Drosophila oogenesis a similar checkpoint might be activated and that in addition to affecting progression through meiosis, the checkpoint might also halt or slow down translation of specific proteins in the oocyte cytoplasm. As predicted by our model, in double mutants between either okr or spn-B and mutations that are presumed to act in the checkpoint process, the developmental phenotypes are suppressed and Grk protein accumulation is again comparable to wild-type oogenesis. We further found that Vasa, a protein with homology to the translational regulator eIF4A, is modified in response to meiotic checkpoint activity. Recently, we have found that Drosophila hus-1 and Brca2 homologs also act in the meiotic checkpoint.
We also discovered that RNA interference (RNAi) processes in the germline can trigger the checkpoint response. We molecularly analyzed the genes zucchini, squash, and cutoff from our mutant collection. These genes, we found, act in concert with aubergine to down-regulate the levels of retrotransposon RNAs in the germline of Drosophila by a specialized RNAi process that is only partially understood. In the mutants, high levels of retrotransposon RNAs trigger a checkpoint response similar to that seen in the meiotic repair mutants. We are continuing to analyze this novel pathway.
Follicle Cell Response to EGFR Activation and Epithelial Cell Polarity
The follicle cells receive the grk-encoded signal from the oocyte through the activation of EGFR. To understand the cellular consequences of this signaling process, we have been conducting screens to identify genes that act in this pathway. We have isolated a number of mutations that cause the follicle cells to lose their normal epithelial morphology and/or undergo abnormal morphogenesis. Among these new mutations are several alleles of the gene crag (calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein). Follicle cells mutant for crag lose epithelial integrity: they are irregular in shape, lose apical-basal polarity, become multilayered, and frequently become invasive. We showed that crag mutant follicle cells accumulate basement membrane proteins on both the apical and the basal side of the epithelium. We propose that Crag plays a unique role in organizing epithelial architecture by regulating the polarized secretion of basement membrane proteins.
EGFR and Notch Cooperate in Follicle Cell Differentiation
The follicle cells at the posterior of the egg chamber respond to early EGFR activation by assuming a posterior follicle cell fate. However, they fail to do so unless they also activate Notch, a transmembrane receptor protein. In our screens we identified Rabconnectin-3α and -3β (Rbcn-3A and -3B) as two regulators of Notch signaling in Drosophila. We showed that Notch is transported to the surface of mutant cells and that signaling is disrupted after the S2 cleavage. Interestingly, the yeast homolog of Rbcn-3A, Rav1, regulates the V-ATPase proton pump responsible for acidifying intracellular organelles. We found that, similarly, Rbcn-3A and -3B appear to regulate acidification. Moreover, we identified mutants in VhaAC39, a V-ATPase subunit, and showed that they phenocopy Rbcn-3A and Rbcn-3B mutants. This demonstrates that Rbcn-3 affects Notch signaling and trafficking by regulating V-ATPase function, which implies that the acidification of an intracellular compartment in the receiving cells is crucial for signaling.
Grants from the National Institutes of Health provided partial support for this research.
As of May 30, 2012
