Scabrous controls ommatidial rotation in the Drosophila compound eye

Establishment of planar polarity in the Drosophila compound eye requires precise 90 degrees rotation of the ommatidial clusters during development. We found that the morphogenetic furrow controls the stop of ommatidial rotation at 90 degrees by emitting signals to posterior ommatidial clusters. One such signal, Scabrous, is synthesized in the furrow cells and transported in vesicles to ommatidial row 6-8. Scabrous vesicles are transported through actin-based cellular extensions but not transcytosis. Scabrous functions nonautonomously to control the stop of ommatidial rotation by suppressing nemo activity in the second 45 degrees rotation. We propose that the morphogenetic furrow regulates precise ommatidial rotation by transporting Scabrous and perhaps other factors through actin-based cellular extensions.     

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.     

Drosophila Myosin II, Zipper, is essential for ommatidial rotation

The adult Drosophila retina is a highly polarized epithelium derived from a precursor tissue that is initially symmetric across its dorsoventral axis. Specialized 90 degrees rotational movements of subsets of cells, the ommatidial precursors, establish mirror symmetry in the retinal epithelium. Myosin II, or Zipper (Zip), a motor protein, regulates the rate at which ommatidia rotate: in zip mutants, the rate of rotation is significantly slowed. Zip is concentrated in the cells that we show to be at the likely interface between rotating and non-rotating cells: the boundary between differentiated and undifferentiated cells. Zip is also robust in newly added ommatidial cells, consistent with our model that the machinery that drives rotation should shift to newly recruited cells as they are added to the growing ommatidium. Finally, cell death genes and canonical Wnt signaling pathway members genetically modify the zip phenotype.     

Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90 degrees, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Here, we demonstrate a requirement for epidermal growth factor receptor (Egfr) signalling in rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. We propose that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, we have evidence that there is an error-correction mechanism operating during pupal stages to reposition inappropriately orientated ommatidia. Our results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling.     

The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.     

The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development

The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium.     

The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye

Rhabdomeres of the Drosophila melanogaster canoemisl mutant ommatidia were twisted, branched, and often fused to each other. A considerable proportion of rhabdomeres were found to have fallen below the retinal basement membrane. Electron-microscopic observations of the mutant ommatidia revealed that microvilli, the subcellular structures composing the rhabdome, were normal. As was the case with partial loss-of-function mutations in the canoe locus, overexpression of the wild-type canoe transgene driven by the heat shock promoter or sevenless enhancer in the wild-type canoe background caused malformation of the rhabdomeres in the adult ommatidia. Immunolabeling of the Canoe protein in the pupal retinae showed that it was accumulated in adherens junctions in photoreceptor rhabdomeres at high concentrations, as well as in pigment cells, bristle cells, and the interjunctional region of photoreceptors at a lower level. In the canoe mutant ommatidia, the Canoe protein concentration was dramatically decreased in adherens junctions, while it was maintained at a level comparable with the wild-type flies in the interjunctional region. Since Canoe or its mammalian homolog AF-6 is known to bind to F-actin and Ras, we suggest the possibility that Canoe couples Ras signaling with cytoskeleton, thereby supporting the straight elongation of rhabdomeres required for development of a regular array of ommatidia.     

Dpp and Hh signaling in the Drosophila embryonic eye field.

We have analyzed the function of the Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. As reported previously, Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). We discuss the similarities between vertebrate systems and Drosophila with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling.     

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.     

Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway

echinoid (ed) encodes an cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the EGFR signaling pathway during Drosophila eye development. Based on our previous genetic mosaic and epistatic analysis, we proposed that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes EGFR signaling. In this report, we demonstrate that Ed functions as a homophilic adhesion molecule and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting EGFR signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. In addition, Ed and Nrg colocalize in the Drosophila eye and are efficiently co-immunoprecipitated. Together, our results suggest a model in which Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes EGFR signaling.     

Long-range signaling systems controlling glial migration in the Drosophila eye

The Drosophila compound eye comprises about 750 individual ommatidia arranged into an almost crystalline array. The eye is not needed for viability and thus served as a favorite model organ to decipher many signaling systems controlling diverse aspects such as cell fate allocation or cell-cycle control. Here, we review that the Drosophila eye can also serve to study the interaction between neurons and glial cells. In the Drosophila eye, all glial cells originate from the brain lobes and need to migrate onto the larval eye disc as neurogenesis is initiated during the third instar stage. Although we do have a relatively good understanding of the sequential progression of neurogenesis in the eye disc, we are still at the beginning in our dissection of the molecular pathways orchestrating the coordinated development of neurons and glial cells.     

EGF receptor signaling: putting a new spin on eye development

The coordinated polarization of cells within an epithelium is required for the development and function of some tissues. Recent work has shown that the EGF receptor signaling pathway plays a key role in establishing epithelial polarity in the compound eye of Drosophila.     

EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm

Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Here we use time-lapse analysis of Drosophila embryogenesis to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses. Moreover, although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation.