Differential Activity of Ras1 during Patterning of the Drosophila Dorsoventral Axis

In Drosophila, the Ras1 gene is required downstream of receptor tyrosine kinases for correct eye development, embryonic patterning, wing vein formation, and border cell migration. Here we characterize a P-element allele of Ras1, Ras1(5703), that affects viability, eye morphogenesis, and early and late stages of oogenesis. Flies transheterozgyous for Ras1(5703) and existing EMS-induced Ras1 alleles are viable and exhibit a range of eye and eggshell defects. Differences in the severity of these phenotypes in different tissues suggest that there are allele-specific effects of Ras1 in development. Analysis of rescue constructs demonstrates that these differential phenotypes are due to loss of function in Ras1 alone and not due to effects on neighboring genes. Females mutant at the Ras1 locus lay eggs with reduced or missing dorsal eggshell structures. We observe dominant interactions between Ras1 mutants and other dorsoventral pathway mutants, including Egfr(top) and gurken. Ras1 is also epistatic to K10. Unlike Egfr(top) and gurken mutants, however, Ras1 females are moderately fertile, laying eggs with ventralized eggshells that can hatch normal larvae. These results suggest that Ras1 may have a different requirement in the patterning of the eggshell axis than in the patterning of the embryonic axis during 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.     

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.     

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.     

A novel follicle-cell-dependent dominant female sterile allele, StarKojak, alters receptor tyrosine kinase signaling in Drosophila

We describe a new dominant allele, StarKojak, that alters receptor tyrosine kinase signaling in the follicle cells and in the eyes in Drosophila. We isolated StarKojak in a screen for follicle-cell-dependent dominant female sterile mutations. We show that StarKojak and revertants of StarKojak do not complement Star loss-of-function mutations. We propose that StarKojak is a novel type of allele of Star that has both dominant gain-of-function phenotypes early in development and dominant loss-of-function phenotypes later in development. Star encodes a putative transmembrane protein that has previously been shown to be a critical component of the epidermal growth factor receptor tyrosine kinase signaling pathway. Early in oogenesis, Star mRNA expression is higher in StarKojak egg chambers than in wild-type egg chambers, consistent with its gain-of-function phenotype. Later in oogenesis, Star mRNA expression is lower in StarKojak follicle cells than in wild-type follicle cells, consistent with its loss-of-function phenotype. By genetically analyzing StarKojak and its revertants, we present evidence that Star is involved in anterior-posterior axis formation both in the female germline cells and in the somatic follicle cells. We also demonstrate that at least part of the dominant female sterile phenotype of StarKojak is restricted to the posterior-pole follicle cells. We propose that Star functions by processing pro-Gurken to mature Gurken, which is thereby released in the region between the oocyte and the follicle cells and binds to the epidermal growth factor receptor in the follicle cells.     

Genetic variation for dorsal-ventral patterning of the Drosophila melanogaster eggshell

Patterning of the insect eggshell is an excellent system for exploring the molecular basis of phenotypic variation. In Drosophila melanogaster, two dorsal-anterior respiratory appendages are produced in response to signaling through the Epidermal growth factor receptor (Egfr). Previous work implicates Egfr pathway function in both intraspecific variation for dorsal appendage spacing (DAS) on the eggshell, as well as interspecific differences in dorsal appendage number and location. To test the hypothesis that genetic variation in Egfr contributes to variation in eggshell patterning, we have made use of naturally occurring intraspecific variation for DAS as a model quantitative trait. We found that there is substantial segregating genetic variation for DAS in D. melanogaster, and have tested for associations with 289 common polymorphisms in the Egfr locus. A marginal association was seen with two polymorphic sites in Egfr; however, we failed to replicate these findings in a second population, or in a modified quantitative complementation test designed to specifically test the effects of the putative polymorphisms. Therefore, we conclude that the polymorphisms we have identified in Egfr do not contribute to variation in DAS, and further work is required to understand the genetic architecture of this trait.     

Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6

Epidermal Growth Factor-receptor (Egfr) signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Here we provide evidence for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90 degrees rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90 degrees. Here we show that rlt is a rotation-specific allele of the inhibitory Egfr ligand Argos and that modulation of Egfr activity shows defects in ommatidial rotation. Our data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. We provide further evidence for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway.     

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.     

A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.     

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.     

The CNS midline cells control the spitz class and Egfr signaling genes to establish the proper cell fate of the Drosophila ventral neuroectoderm.

The spitz class genes, pointed (pnt), rhomboid frho), single-minded (sim), spitz (spi)and Star (S), as well as the Drosophila epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis showed that the expression of all the spitz class and Egfrsignaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. It was shown that overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. On the other hand, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the EGFR signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling EGFR activity through the regulation of the expression of spitz class genes and Egfr signaling genes.     

Mutations modulating the Argos-regulated signaling pathway in Drosophila eye development

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.     

A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed.     

A Screen for Genes That Function Downstream of Ras1 during Drosophila Eye Development

Cell-fate specification of the R7 photoreceptor cell is controlled by the sevenless receptor tyrosine kinase (SevRTK) and Ras1, the Drosophila homologue of mammalian H-ras, K-ras and N-ras oncogenes. An activated form of Ras1 expressed under control of the sevenless enhancer/promoter (sev-Ras1(V12)) induces production of supernumerary R7 photoreceptor cells, which causes the eye to become rough in appearance. To isolate mutations in genes functioning downstream of Ras1, we carried out a screen for dominant suppressors and enhancers of this rough eye phenotype. Approximately 850,000 mutagenized flies were screened, and 282 dominant suppressors and 577 dominant enhancers were isolated. Mutations in the Drosophila homologues of Raf, MEK, MAPK, type I Geranylgeranyl Transferase and Protein Phosphatase 2A were isolated, as were mutations in several novel signaling genes. Some of these mutant genes appear to be general signaling factors that function in other Ras1 pathways, while one seems to be more specific for photoreceptor development. At least two suppressors appear to function either between Ras1 and Raf or in parallele to Raf.     

Sprouty, an intracellular inhibitor of Ras signaling

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty- eyes have excess photoreceptors, cone cells, and pigment cells. Sprouty's function is, however, more widespread. We show that it also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, we show by biochemical analysis that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. Our results indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction.     

The Rap1 GTPase functions as a regulator of morphogenesis in vivo.

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. We show that Ras1-mediated signaling pathways in Drosophila are not influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. Our experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo.