D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system we have identified a mutation in the D-cbl gene which causes hyperactivation of the Egfr pathway. Cbl proteins are known to downregulate activated receptors. We find that the abnormal Egfr activation is ligand dependent. Our results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of D-cbl to ensure the absence of signaling in the ventral most follicle cells.     

The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.     

The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila

During Drosophila oogenesis, localization of the transforming growth factor alpha (TGFalpha)-like signaling molecule Gurken to the oocyte membrane is required for polarity establishment of the egg and embryo. To test Gurken domain functions, full-length and truncated forms of Gurken were expressed ectopically using the UAS/Gal4 expression system, or in the germline using the endogenous promoter. GrkDeltaC, a deletion of the cytoplasmic domain, localizes to the oocyte membrane and can signal. GrkDeltaTC, which lacks the transmembrane and cytoplasmic domains, retains signaling ability when ectopically expressed in somatic cells. However, in the germline, the GrkDeltaTC protein accumulates throughout the oocyte cytoplasm and cannot signal. In addition, we found that several strong gurken alleles contain point mutations in the transmembrane region. We conclude that secretion of Gurken requires its transmembrane region, and propose a model in which the gene cornichon mediates this process.     

Regulation of somatic myosin activity by protein phosphatase 1β controls Drosophila oocyte polarization

The Drosophila body axes are established in the oocyte during oogenesis. Oocyte polarization is initiated by Gurken, which signals from the germline through the epidermal growth factor receptor (Egfr) to the posterior follicle cells (PFCs). In response the PFCs generate an unidentified polarizing signal that regulates oocyte polarity. We have identified a loss-of-function mutation of flapwing, which encodes the catalytic subunit of protein phosphatase 1β (PP1β) that disrupts oocyte polarization. We show that PP1β, by regulating myosin activity, controls the generation of the polarizing signal. Excessive myosin activity in the PFCs causes oocyte mispolarization and defective Notch signaling and endocytosis in the PFCs. The integrated activation of JAK/STAT and Egfr signaling results in the sensitivity of PFCs to defective Notch. Interestingly, our results also demonstrate a role of PP1β in generating the polarizing signal independently of Notch, indicating a direct involvement of somatic myosin activity in axis formation.     

Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis.

We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) alpha-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.     

Alpha-endosulfine, a potential regulator of insulin secretion, is required for adult tissue growth control in Drosophila

Alpha-endosulfine is a small protein that has been proposed to regulate ion channel activity and insulin secretion, but in vivo studies have been lacking. We have previously established the Drosophila ovary as a model system in which to study adult tissue growth regulation, and demonstrated a role of the insulin pathway in the proliferative response of ovarian cells to nutritional changes. Here, we find that the Drosophila alpha-endosulfine (dendos) gene, whose protein is expressed in germline and somatic cells of the ovary, as well as in the brain and certain regions of the intestine, is also required for this response. This requirement is non-cell autonomous, which is consistent with a role of dendos in secretion of Drosophila insulin-like peptides (DILPs), required for the proliferative response to nutritional changes. Our results show that dendos is also required for a distinct process in oogenesis, namely, the osmotic regulation of stage 14 oocytes, and that this requirement is cell autonomous, consistent with the role in ion channel regulation suggested by studies of the mammalian homologues.     

Cct1, a phosphatidylcholine biosynthesis enzyme, is required for Drosophila oogenesis and ovarian morphogenesis

Patterning of the Drosophila egg requires cooperation between the germline cells and surrounding somatic follicle cells. In order to identify genes involved in follicle cell patterning, we analyzed enhancer trap lines expressed in specific subsets of follicle cells. Through this analysis, we have identified tandem Drosophila genes homologous to CTP: phosphocholine cytidylyltransferase (CCT), the second of three enzymes in the CDP-choline pathway, which is used to synthesize phosphatidylcholine. Drosophila Cct1 is expressed at high levels in three specific subsets of follicle cells, and this expression is regulated, at least in part, by the TGF-beta and Egfr signaling pathways. Mutations in Cct1 result in a number of defects, including a loss of germline stem cell maintenance, mispositioning of the oocyte, and a shortened operculum, suggesting that Cct1 plays multiple roles during oogenesis. In addition, Cct1 mutants display a novel branched ovariole phenotype, demonstrating a requirement for this gene during ovarian morphogenesis. These data provide the first evidence for a specific role for CCT, and thus for phosphatidylcholine, in patterning during development.     

Genetic link between beta-sarcoglycan and the Egfr signaling pathway

Sarcoglycans are a multimeric, integral membrane protein complex that is part of the dystrophin glycoprotein complex. Previous findings suggest that the dystrophin glycoprotein complex plays roles not only in maintaining the mechanical structure of the cell membrane but also in signal transduction. To evaluate the functions of sarcoglycans, we here took advantage of Drosophila, which is useful for screening genetic interactions. Morphological aberrancy was observed in the adult compound eyes of Drosophila beta-sarcolgycan (dscgbeta) knockdown flies. We also detected genetic interactions between dscgbeta and Egfr related genes, such as rhomboid-1, rhomboid-3, and mirror. Furthermore two extra cell types with strong expression of Rhomboid were found in the ommatidia of dscgbeta knockdown pupal retina. These cells exhibited phosphorylation of ERKA, suggesting that Egfr signaling is activated via Rhomboid. Through these in vivo analyses, we conclude that dscgbeta negatively regulates the Egfr signaling pathway.     

Formation, architecture and polarity of female germline cyst in Xenopus

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis. We found that the germline cyst in Xenopus contains 16 cells that are similar in general architecture and molecular composition to the cyst in Drosophila. Nest cells are connected by cytoplasmic bridges that contain ring canal-like structures. The nest cells contain a structure similar to the Drosophila fusome that that is probably involved in anchoring of the centrioles and organization of the primary mitochondrial cloud (PMC) around the centriole. We also find that in contrast to other organisms, in Xenopus, apoptosis is a rare event within the developing ovary. Our studies indicate that the processes responsible for the formation of female germline cysts and the establishment of germ cell polarity are highly conserved between invertebrates and vertebrates. The dissimilarities between Drosophila and Xenopus and the uniqueness of each system probably evolved through modifications of the same fundamental design of the germline cyst.     

Smiling Gurken gradient-an expansion of the Gurken gradient

Morphogen gradients provide unique positional information within a tissue. Cells that are sensitive to the concentration of the morphogen integrate this signal and develop an appropriately distinct cell fate. A morphogen gradient is usually generated by a restricted source and shaped by the speed of diffusion and stability of the signaling molecule. In addition, the availability of receptor and Heparan Sulfate Proteoglycans (HSPGs) help to shape the gradient. We have shown that over-expression of Dally-like protein (Dlp) causes an expansion of Gurken distribution and a loss of cell fates which are specified by high levels of epidermal growth factor receptor (Egfr) signaling. In this article, we discuss how D-Cbl mediated Egfr endocytosis and the levels of Dlp affect the shape of the Gurken gradient.     

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis.     

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.     

Smiling Gurken gradient: An expansion of the Gurken gradient

Morphogen gradients provide unique positional information within a tissue. Cells that are sensitive to the concentration of the morphogen integrate this signal and develop an appropriately distinct cell fate. A morphogen gradient is usually generated by a restricted source and shaped by the speed of diffusion and stability of the signaling molecule. In addition, the availability of receptor and Heparan Sulfate Proteoglycans (HSPGs) help to shape the gradient. We have shown that overexpression of Dally-like protein (Dlp) causes an expansion of Gurken distribution and a loss of cell fates which are specified by high levels of epidermal growth factor receptor (Egfr) signaling. In this article, we discuss how D-Cbl mediated Egfr endocytosis and the levels of Dlp affect the shape of the Gurken gradient.