A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila.     

Delta/Notch and Boss/Sevenless signals act combinatorially to specify the Drosophila R7 photoreceptor.

The R7 photoreceptor, a unique cell type within the Drosophila ommatidium, was initially proposed to be specified by two distinct signals from neighboring cells, one from the R8 photoreceptor and another from the R1/6 photoreceptor pair. The R8-to-R7 signal is the transmembrane ligand Bride of Sevenless (Boss), which is received by the receptor tyrosine kinase Sevenless (Sev) and transduced via Ras activation within the presumptive R7 cell. However, the identity of the R1/6-to-R7 signal has remained elusive. Here, we present evidence that the transmembrane ligand Delta (Dl), expressed by the R1/6 pair, activates the receptor Notch (N) in the presumptive R7 cell and constitutes the postulated R1/6-to-R7 signal required in combination with the Boss/Sev signal to specify the R7 fate.     

Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the sevenless receptor tyrosine kinase

The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (DOS) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that DOS becomes tyrosine phosphorylated during SEV signaling and collaborates with the protein tyrosine phosphatase CSW. We have investigated this possibility by identifying tyrosine residues 801 and 854 of DOS as the phosphorylated binding sites for the CSW SH2 domains. We show that these sites become phosphorylated in response to SEV activation and that phosphorylation of both sites is required to allow CSW to bind DOS. Mutant DOS proteins in which either Y801 or Y854 of DOS has been changed to phenylalanine are unable to function during signaling by SEV and other receptor tyrosine kinases. In contrast, we find that a mutant DOS protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide DOS function during SEV signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for DOS during signaling by SEV and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit CSW.     

Positive and negative signaling mechanisms in the regulation of photoreceptor induction in the developingDrosophila retina

An ommatidium of aDrosophila compound eye contains eight photoreceptor cells, R1–R8. The fates of the photoreceptors are determined exclusively by inductive interactions between neuronal precursors in the cell cluster from which the ommatidium is formed. R7 induction has been extensively analysed at the molecular level. Activation of a membrane receptor tyrosine kinase (Sevenless) in the R7 precursor by a ligand (Bride of sevenless) present on the surface of R8 triggers a transduction cascade mediated by Ras, establishing the R7 fate of this cell. Other Sev-expressing cells are prevented from taking on the R7 fate by several different mechanisms. Pokkuri-mediated repression represents one such regulatory mechanism. The positive and negative signaling pathways operating in the fate determination of other photoreceptor cells are also discussed.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Specification of cell fate in the developing eye of Drosophila.

Determination of cell fate in the developing eye of Drosophila depends on a precise sequence of cellular interactions which generate the stereotypic array of ommatidia. In the eye imaginal disc, an initially unpatterned epithelial sheath of cells, the first step in this process may be the specification of R8 photoreceptor cells at regular intervals. Genes such as Notch and scabrous, known to be involved in bristle development, also participate in this process, suggesting that the specification of ommatidial founder cells and the formation of sensory organs in the adult epidermis may involve a similar mechanism, that of lateral inhibition. The subsequent steps of ommatidial assembly, following R8 assignment, involve a different mechanism: Undetermined cells read their position based on the contacts they make with neighbors that have already begun to differentiate. The development of the R7 photoreceptor cell, one of the eight photoreceptor cells in the ommatidium, is best understood. An important role seems to be played by sevenless, a receptor tyrosine kinase on the surface of the R7 precursor. It transmits the positional information--most likely encoded by the boss protein on the neighboring R8 cell membrane--into the cell via its tyrosine kinase, which activates a signal transduction cascade. Constitutive activation of the sevenless kinase by overexpression of an N-terminally truncated form results in the diversion of other ommatidial cells into the R7 pathway suggesting that activation of the sevenless signalling pathway is sufficient to specify R7 development. Genetic dissection of this pathway should therefore identify components of a signalling cascade activated by a tyrosine kinase.     

The little R cell that could

Drosophila eye development provides an excellent model system to study the role of inter-cellular signaling in the specification of unique cell fates. Behavioral screens by Benzer and his colleagues led to the identification of a gene, Sevenless, a receptor tyrosine kinase (RTK) receptor, required for the specification of the UV sensitive R7 cell. Genetic analysis further showed that the Ras/Raf/MAPK pathway function downstream of Sevenless in the specification of R7 fate. Signaling mediated by another RTK, EGFR and Notch have also been shown to function in either an antagonistic or a synergistic manner in the specification of cell fate during eye development. In some instances, these pathways are linked in a sequential manner by the regulation of the expression of Notch ligand, Delta by EGFR, while in others, these pathways function in a combinatorial fashion on enhancer elements to control target gene expression. In this review, we highlight the elegant genetic strategies used by several laboratories in early elucidation of the Sevenless pathway which helped link the RTK receptor to the Ras/Raf/MAPK cascade and discuss how EGFR and Notch signaling pathways are used in a reiterative manner and by combining in different modes, generate sufficient diversity required for the specification of unique cell fates.     

R7 photoreceptor specification requires Notch activity

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment [1,2]. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase which is activated by a ligand from R8 [3]. Here, we report that the specification of R7 requires a second signal that activates Notch. We found that a Notch target gene is expressed in R7 shortly after recruitment. When Notch activity was reduced, the cell was misrouted to an R1/R6 fate. Conversely, when activated Notch was present in the R1/R6 cells, it caused them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. We propose two models: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signalling, and a combinatorial model in which Notch and Ras signalling would act together to regulate genes that determine cell fate.     

MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.     

Patrilocal* cell-cell interactions: sevenless captures its bride

Development of the R7 neuron in the compound eye of Drosophila requires an inductive interaction between the R8 photoreceptor cell and the bipotential R7 precursor cell. Two transmembrane proteins mediate this induction: sevenless (sev), a receptor tyrosine kinase expressed on the apical surface of the R7 precursor cell, and its ligand, the bride of sevenless protein (boss) on the apical membrane of the R8 neuron. The boss protein, with its large extracellular domain and seven transmembrane segments, is an unusual ligand for a receptor tyrosine kinase, and its internalization into the R7 cell following interaction with sev is particularly intriguing.     

Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.     

Apical Accumulation of the Sevenless Receptor Tyrosine Kinase During Drosophila Eye Development Is Promoted by the Small GTPase Rap1

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other corequired signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell–cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.     

SOCS36E specifically interferes with Sevenless signaling during Drosophila eye development

During the development of multicellular organisms the fate of individual cells is specified with great precision and reproducibility. Although classical genetic approaches led to the identification of many of the signaling pathways contributing to cell fate specification, they have provided little insight into the mechanisms that ensure robustness and reproducibility. We have used the specification of the R7 photoreceptor cells controlled by the Sevenless receptor tyrosine kinase (Sev) pathway to screen for modulators of pathway activity and to uncover the mechanisms underlying the robustness of cell fate decisions. Here we provide genetic evidence that the Drosophila SOCS36E adaptor protein containing an SH2 domain and a SOCS box acts as an attenuator of Sev signaling. Overexpression of Socs36E strongly suppresses the specification of extra R7 photoreceptor cells in response to constitutive activation of Sev, and loss of Socs36E function suppresses the loss of R7 cells when Sev activity is impaired. In a wild-type background, however, loss and gain of Socs36E function exhibits little effect on R7 specification. We also show that SH2 domain of SOCS36E is essential for this function in inhibiting Sev action and that Socs36E expression is suppressed by high Sev pathway activity. In our model, only the cell able to activate high levels of receptor tyrosine kinase signaling will repress SOCS36E expression, reduce the negative effect on Sev signaling and allow this cell to differentiate into R7. In contrast, the remaining cells fail to receive high signaling, and thus maintain high levels of SOCS36E. This represses residual Sev activity and blocks R7 development. Therefore, Socs36E constitutes a novel partially redundant feedback mechanism that contributes to the robustness of R7 specification. The SOCS family of adaptor proteins may have evolved as modulators of specific signaling pathways that contribute to the robustness and precision of cell fate specification.     

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.     

Signal transduction and the fate of the R7 photoreceptor in Drosophila

The sevenless protein tyrosine kinase receptor plays a central role in the pathway of cell fate induction that determines the development of the R7 photoreceptor in the Drosophila eye. In the last year we have learned much about the probable ligand for sevenless and have begun to dissect the signal transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cell fate.     

Signal transduction and the fate of the R7 photoreceptor in Drosophila.

The sevenless protein tyrosine kinase receptor plays a central role in the pathway of cell fate induction that determines the development of the R7 photoreceptor in the Drosophila eye. In the last year we have learned much about the probable ligand for sevenless and have begun to dissect the signal transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cell fate.     

semang affects the development of a subset of cells in the Drosophila compound eye

semang (sag), a mutation isolated as a suppressor of Drosophila Src42A, has previously been shown to affect some receptor tyrosine kinase mediated embryonic processes. Here we show that sag specifically affects the development of R1, R6 and R7 photoreceptor cells in a cell-autonomous manner. These cells are absent in the mutant at the time when they normally appear in the ommatidial pre-clusters. Genetic analyses suggest that sag functions downstream of, or parallel to, Mapk and Yan in the photoreceptor differentiation pathway. The autonomous requirement of sag for R1/R6/R7 development could be explained by a selective impairment of the late, but not early, rounds of Egfr-induced precursor cell assembly by the sag mutations. Egfr signaling is highly regulated by autocrine or paracrine mechanisms in different cells. Knowing that the photoreceptor cluster formation is a complex process involving dynamic changes in cell-cell contact, our hypothesis is that the sag alleles affected certain special aspects of Egfr-signaling that are unique for the recruitment of R1/R6/R7 cells.     

The bride of sevenless and sevenless interaction: internalization of a transmembrane ligand.

During Drosophila retinal development, the R8 photo-receptor neuron induces a neighboring cell to assume an R7 cell fate through cell contact. This is mediated by the transmembrane protein bride of sevenless (boss) on the surface of the R8 cell, which binds the sevenless tyrosine kinase receptor (sev) on the surface of the R7 precursor cell. The boss protein, which contains a large extracellular domain, seven transmembrane segments, and a C-terminal cytoplasmic domain, has an exceptional structure for a ligand of a receptor tyrosine kinase. Using a panel of antibodies directed to various cytoplasmic and extracellular epitopes, we demonstrate that the entire boss protein from its extreme N-terminus to its extreme C-terminus is internalized by sev-expressing tissue culture cells and by the R7 precursor cell in the developing eye imaginal disc. The receptor-mediated transfer of a transmembrane ligand represents a novel mechanism for protein transfer between developing cells.