The Drosophila EGF receptor (DER) is required for the specification of diverse cell fates throughout development. We have examined how the activation of DER controls the development of vein and intervein cells in the Drosophila wing. The data presented here indicate that two distinct events are involved in the determination and differentiation of wing cells. (1) The establishment of a positive feedback amplification loop, which drives DER signaling in larval stages. At this time, rhomboid (rho), in combination with vein, initiates and amplifies the activity of DER in vein cells. (2) The late downregulation of DER activity. At this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Together, these temporal and spatial changes in the activity of DER constitute an autoregulatory network that controls the definition of vein and intervein cell types.
We have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.
Cells commonly use multiprotein kinase cascades to signal information from the cell membrane to the nucleus. Several conserved signaling pathways related to the mitogen activated protein kinase (MAPK) pathway allow cells to respond to normal developmental signals as well as signals produced under stressful conditions. Genetic and molecular studies in Drosophila melanogaster over the last several years have related that components of stress signaling pathways, namely the Jun kinase (JNK) and p38 kinase signaling modules, are functionally conserved and participate in numerous processes during normal development. Specifically, the JNK pathway is required for morphogenetic movements in embryogenesis and generation of tissue polarity in the adult. The role of the p38 pathway in generation of axial polarity during oogenesis has been inferred from phenotypic analysis of mutations in the Drosophila homolog of DMKK3. In addition to their requirement for normal development, cell culture and genetic investigations point to a role for both the JNK and p38 pathways in regulation of the immune response in the fly. This review details the known components of stress signaling pathways in Drosophila and recent insights into how these pathways are used and regulated during development and homeostasis.
As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.
The JAK/STAT signaling pathway is required for many processes including cytokine signaling, hematopoiesis, gliagenesis, and Drosophila segmentation. In this report we present evidence demonstrating that the JAK/STAT pathway is also central to the establishment of planar polarity during Drosophila eye development. We show that a localized source of the pathway ligand, Unpaired, is present at the midline of the developing eye, which is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror.
In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. Using a heterologous system, we show that Dsh is recruited selectively to the membrane by Fz but not DFz2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. We propose that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation.
We have identified a gene, DRhoGEF2, which encodes a putative guanine nucleotide exchange factor belonging to the Dbl family of oncogenes. DRhoGEF2 function is essential for the coordination of cell shape changes during gastrulation. In the absence of maternal DRhoGEF2 gene activity, mesodermal and endodermal primordia fail to invaginate. The phenotype seen in DRhoGEF2 mutants is more severe than the defects associated with mutations in two previously identified gastrulation genes, folded gastrulation and concertina, suggesting that DRhoGEF2 acts in a signaling pathway independent of these genes. Expression of dominant-negative DRhoA during gastrulation results in phenocopies of the DRhoGEF2 mutant, suggesting that a signaling cascade involving DRhoGEF2 and the small GTPase DRhoA is responsible for the regulation of cell shape changes during early Drosophila morphogenesis.
In vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired (upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a segmentally restricted manner in the early Drosophila embryo.
The small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras )in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.
We have developed a 'directed mosaic' system in Drosophila by using the GAL4 system to control the expression of the yeast recombinase, FLP, in a spatial and temporal fashion. By directing FLP expression, we show that it is possible to efficiently and specifically target loss-of-function studies for vital loci to the developmental pathway of interest. A simple F1 adult phenotypic screen demonstrated that most adult tissues can be analyzed with this approach. Using GAL4 lines expressed during oogenesis, we have refined the system to examine the roles of vital loci in the development of the follicular epithelium. We have identified essential genes involved in egg chamber organization, cell migration and cell shape. Further, we have used this technique to gain insights into the role of the Drosophila EGF receptor pathway in establishing the egg axes. Finally, using different UAS-FLP, GAL4 and existing FRT lines, we have built stocks that permit the analysis of approximately 95% of the genome in follicular mosaics.
In Drosophila, specification of embryonic terminal cells is controlled by the Torso receptor tyrosine kinase. Here, we analyze the molecular basis of positive (Y630) and negative (Y918) phosphotyrosine (pY) signaling sites on Torso. We find that the Drosophila homolog of RasGAP associates with pY918 and is a negative effector of Torso signaling. Further, we show that the tyrosine phosphatase Corkscrew (CSW), which associates with pY630, specifically dephosphorylates the negative pY918 Torso signaling site, thus identifying Torso to be a substrate of CSW in the terminal pathway. CSW also serves as an adaptor protein for DRK binding, physically linking Torso to Ras activation. The opposing actions of CSW and RasGAP modulate the strength of the Torso signal, contributing to the establishment of precise boundaries for terminal structure development.
Hedgehog (Hh) proteins act through both short-range and long-range signalling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. Here we identify a new Drosophila gene, named tout-velu, that is required for diffusion of Hedgehog. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Our results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, lead us to propose that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog.
The ability to create mosaic animals allows the phenotypic analysis of patches of groups of genetically different cells that develop in a wild type environment. In Drosophila, a variety of techniques have been developed over the years to generate mosaics, and in this chapter, I review the techniques that our laboratory has developed. These include the "Dominant Female Sterile" technique which allows the analysis of gene functions to oogenesis and embryogenesis; the "Gal4-UAS" technique which allows the control of where and when specific genes are expressed; and, the "Positive Marked Mutant Lineages" technique which allows clones of cells to express a specific reporter gene.
Within the last three years, Frizzled receptors have risen from obscurity to celebrity status owing to their functional identification as receptors for the ubiquitous family of secreted WNT signaling factors. However, the founding member of the Frizzled family, Drosophila Frizzled (FZ), was cloned almost a decade ago because of its role in regulating cell polarity within the plane of an epithelium. In this review, we consider the role of FZ in this intriguing context. We discuss recent progress towards elucidating mechanisms for the intracellular specification of planar polarity, and further review evidence for models of global polarity regulation at the tissue level. The data suggest that a genetic 'cassette', encoding a set of core signaling components, could pattern hair, bristle and ommatidial planar polarity in Drosophila, and that additional tissue-specific factors might explain the diversity of signal responses. Recently described examples from the nematode and frog suggest that the developmental control of cell polarity by FZ receptors might represent a functionally conserved signaling mechanism.