Drosophila Compound Eye Research Articles

Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates.

Although development of the adult Drosophila compound eye is very well understood, little is known about development of photoreceptors (PRs) in the simple larval eye. We show here that the larval eye is composed of 12 PRs, four of which express blue-sensitive rhodopsin5 (rh5) while the other eight contain green-sensitive rh6. This is similar to the 30:70 ratio of adult blue and green R8 cells. However, the stochastic choice of adult color PRs and the bistable loop of the warts and melted tumor suppressor genes that unambiguously specify rh5 and rh6 in R8 PRs are not involved in specification of larval PRs. Instead, primary PR precursors signal via EGFR to surrounding tissue to develop as secondary precursors, which will become Rh6-expressing PRs. EGFR signaling is required for the survival of the Rh6 subtype. Primary precursors give rise to the Rh5 subtype. Furthermore, the combinatorial action of the transcription factors Spalt, Seven-up, and Orthodenticle specifies the two PR subtypes. Therefore, even though the larval PRs and adult R8 PRs express the same rhodopsins (rh5 and rh6), they use very distinct mechanisms for their specification.

Screen for Genetic Modifiers of stbm Reveals that Photoreceptor Fate and Rotation Can Be Genetically Uncoupled in the Drosophila Eye

BackgroundPolarity of the Drosophila compound eye arises primarily as a consequence of two events that are tightly linked in time and space: fate specification of two photoreceptor cells, R3 and R4, and the subsequent directional movement of the unit eyes of the compound eye, or ommatidia. While it is thought that these fates dictate the direction of ommatidial rotation, the phenotype of mutants in the genes that set up this polarity led to the hypothesis that these two events could be uncoupled.Methodology/Principal FindingsTo definitively demonstrate these events are genetically separable, we conducted a dominant modifier screen to determine if genes, when misexpressed, could selectively enhance subclasses of mutant ommatidia in which the direction of rotation does not follow the R3/R4 cell fates, yet not affect the number of ommatidia in which rotation follows the R3/R4 cell fates. We identified a subset of P element lines that exhibit this selective enhancement. We also identified lines that behave in the opposite manner: They enhance the number of ommatidia that rotate in the right direction, but do not alter the number of ommatidia that rotate incorrectly with respect to the R3/R4 fates.Conclusions/SignificanceThese results indicate that fate and direction of rotation can be genetically separated, and that there are genes that act between R3/R4 fate specification and direction of ommatidial rotation. These data affirm what has been a long-standing assumption about the genetic control of ommatidial polarity.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.

Microscopy of the Drosophila facet eye: Vademecum for standardized fixation, embedding, and sectioning

We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.

Smoothened, thickveins and the genetic control of cell cycle and cell fate in the developing Drosophila eye

The Hedgehog and Decapentaplegic pathways have several well-characterized functions in the developing Drosophila compound eye, including initiation and progression of the morphogenetic furrow. Other functions involve control of cell cycle and cell survival as well as cell type specification. Here we have used the mosaic clone analysis of null mutations of the smoothened and thickveins genes (which encode the receptors for these two signals) both alone and in combination, to study cell cycle and cell fate in the developing eye. We conclude that both pathways have several, but differing roles in furrow induction and cell fate and survival, but that neither directly affects cell type specification.

Genetic and biochemical analysis of the role of Egfr in the morphogenetic furrow of the developingDrosophilaeye

A key event in patterning the developing Drosophila compound eye is the progressive restriction of the transcription factor Atonal in the morphogenetic furrow. The Atonal pattern evolves from expression in all cells to an over-dispersed pattern of single founder cells (the future R8 photoreceptors). This restriction involves Notch-mediated lateral inhibition. However, there have been inconsistent data on a similar proposed role for the Egf receptor (Egfr). Experiments using a conditional Egfr mutation (Egfr(tsla)) suggested that Egfr does not regulate Atonal restriction, whereas experiments using Egfr-null mosaic Minute+ clones suggested that it does. Here, we have re-examined both approaches. We report that the lesion in Egfr(tsla) is a serine to phenylalanine change in a conserved extracellular ligand-binding domain. We show by biochemical and genetic approaches that the Egfr(tsla) protein is rapidly and completely inactivated upon shift to the non-permissive temperature. We also find that on temperature shift the protein moves from the cell surface into the cell. Finally, we report a flaw in the Egfr-null mosaic Minute+ clone approach. Thus, we demonstrate that Egfr does not play a role in the initial specification or spacing of ommatidial founder cells.

Slingshot cofilin phosphatase localization is regulated by Receptor Tyrosine Kinases and regulates cytoskeletal structure in the developing Drosophila eye

Animal development requires that positional information act on the genome to control cell fate and cell shape. The primary determinant of animal cell shape is the cytoskeleton and thus the mechanisms by which extracellular signals influence the cytoskeleton are crucial for morphogenesis. In the developing Drosophila compound eye, localized polymerization of actin functions to constrict the apical surface of epithelial cells, both at the morphogenetic furrow and later to maintain the coherence of the nascent ommatidia. As elsewhere, actin polymerization in the developing eye is regulated by ADF/cofilin (‘Twinstar’, or ‘Tsr’ in Drosophila), which is activated by Slingshot (Ssh), a cofilin phosphatase. Here we show that Ssh does act in the developing eye to limit actin polymerization in the assembling ommatidia, but not in the morphogenetic furrow. While Ssh does control cell shape, surprisingly there are no direct or immediate consequences for cell type. Ssh protein becomes apically concentrated in cells that express elevated levels of the Sevenless (Sev) receptor-tyrosine kinase (RTK), even those which receive no ligand. We interpret this as a non-signal driven, RTK-dependent localization of Ssh to allow for locally increased actin filament turnover. We suggest that there are two modes of actin remodeling in the developing eye: a non-RTK, non-Ssh mediated mechanism in the morphogenetic furrow, and an RTK and Ssh-dependent mode during ommatidial assembly.

Cell‐type specific utilization of multiple negative feedback loops generates developmental constancy

Signaling pathways generally contain multiple negative regulators that are induced by the signal they repress, constructing negative feedback loops. Although such negative regulators are often expressed in a tissue- or cell-type specific manner during development, little is known about the significance of their differential expression patterns and possible interactions. We show the role and interplay of two cell-type specific negative feedback loops during specification of photoreceptor neurons in the Drosophila compound eye, a process that occurs via epidermal growth factor (EGF)-mediated sequential induction through the activation of the Ras/MAPK signaling pathway. Inducing cells secreting EGF express a negative regulator Sprouty (SPRY) that lowers Ras/MAPK signaling activity, and as a consequence reduces the signal-dependent expression of a secreted EGF inhibitor, Argos (AOS). Induced cells in turn express an orphan nuclear receptor Seven-up (SVP), which represses SPRY expression thereby allowing expression and secretion of AOS, preventing further induction. When this intricate system fails, as in spry mutants, sequential induction is no longer constant and the number of photoreceptor neurons becomes variable. Thus, cell-type specific utilization of multiple negative feedback loops not only confers developmental robustness through functional redundancy, but is a key component in generating consistent patterning.

Isolation of gene sets affected specifically by polyglutamine expression: implication of the TOR signaling pathway in neurodegeneration

Transcriptional dysregulation as a result of sequestration of essential transcription factors into protein aggregates formed by polyglutamine (polyQ) expansions can lead to late-onset progressive neurodegeneration. DNA microarray analysis of Drosophila expressing polyQ in the compound eye over time revealed large numbers of transcriptional changes at the earliest stages of the disease including repression of the transient receptor potential calcium channels in a polyQ-induced cell death specific manner. While significant differences in expression profiles were found between the Drosophila compound eye and polyQ-sensitive neural cells, a number of possible key overlapping regulators were extracted. Among these, PDK1 was shown to act as a mediator for polyQ-toxicity, suggesting the involvement of the TOR pathway in polyQ-induced neurodegeneration.

Mutations in Drosophila sec15 Reveal a Function in Neuronal Targeting for a Subset of Exocyst Components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

Cell-Cycle Control during Development: Taking it up a Notch

The signals that coordinate cellular proliferation with G1 arrest and differentiation have long been of interest. Two papers in this issue of Developmental Cell show that the conserved Hedgehog and Notch signaling pathways regulate cell division during development of the Drosophila compound eye.

Novel dominant-negative mutation within the six domain of the conserved eye specification gene sine oculis inhibits eye development in Drosophila.

The development of the compound eye of Drosophila is controlled, in part, by the concerted actions of several nuclear proteins that form an intricate regulatory system. One member of this network is sine oculis (so), the founding member of the Six gene family. Mutations within so affect the entire visual system, including the compound eye. The vertebrate homologs Six3 and Six6 also appear to play crucial roles in retinal formation. Mutations in Six3 inhibit retinal formation in chickens and fish, whereas those in Six6 are the underlying cause of bilateral anophthalmia in humans. Together, these phenotypes suggest a conserved role for the Six genes in eye development. In this report, we describe the effects of a dominant-negative mutation of sine oculis on the development of the compound eye of Drosophila. The mutation resides within the Six domain and may have implications for eye development and disease.

The Ste20-like kinase misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing drosophila eye

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. Here, we show that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic- D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic- D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. We propose that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells.

Targeted Mutagenesis of the Farnesylation Site of Drosophila Gγe Disrupts Membrane Association of the G Protein βγ Complex and Affects the Light Sensitivity of the Visual System

Activation of phototransduction in the compound eye of Drosophila is mediated by a heterotrimeric G protein that couples to the effector enzyme phospholipase Cbeta. The gamma subunit of this G protein (Ggammae) as well as gamma subunits of vertebrate transducins contain a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) with a consensus sequence for protein farnesylation. To examine the function of Ggammae farnesylation, we mutated the farnesylation site and overexpressed the mutated Ggammae in Drosophila. Mass spectrometry of overexpressed Ggammae subunits revealed that nonmutated Ggammae is modified by farnesylation, whereas the mutated Ggammae is not farnesylated. In the transgenic flies, mutated Ggammae forms a dimeric complex with Gbetae, with the consequence that the fraction of non-membrane-bound Gbetagamma is increased. Thus, farnesylation of Ggammae facilitates the membrane attachment of the Gbetagamma complex. We also expressed human Ggammarod in Drosophila photoreceptors. Despite similarities in the primary structure between the transducin gamma subunit and Drosophila Ggammae, we observed no interaction of human Ggammarod with Drosophila Gbetae. This finding indicates that human Ggammarod and Drosophila Ggammae provide different interfaces for the interaction with Gbeta subunits. Electroretinogram recordings revealed a significant loss of light sensitivity in eyes of transgenic flies that express mutated Ggammae. This loss in light sensitivity reveals that post-translational farnesylation is a critical step for the formation of membrane-associated Galphabetagamma required for transmitting light activation from rhodopsin to phospholipase Cbeta.

Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis.

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye, which often lack the full complement of photoreceptors and support cells, undergo apoptosis during mid-pupation. We have found that this cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. We estimate that approximately 6-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, we show that the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. We consider the implications of these findings for eye development and function in Drosophila and other organisms.

Spatial pattern of nonmuscle myosin-II distribution during the development of the Drosophila compound eye and implications for retinal morphogenesis

Nonmuscle myosin-II is a motor protein that drives cell movement and changes in cell shape during tissue and organ development. This study has determined the dynamic changes in myosin-II distribution during Drosophila compound eye morphogenesis. In photoreceptor neurons, myosin-II is undetectable at the apical domain throughout the first half of pupal life, at which time this membrane domain is involuted into the epithelium and progresses toward the retinal floor. Myosin-II is deployed at the apical surface at about 60% of pupal development, once the developing rhabdomeres reach the retinal floor. Subsequently, myosin-II becomes restricted to two stripes at the sides of the developing rhabdomere, adopting its final position within the visual cells R1–6; here, myosin-II is associated with a set of actin filaments that extend alongside the rhabdomeres. At the midpupal stage, myosin-II is also incorporated into stress-fiber-like arrays within the basal endfeet of the pigment cells that then change their shape. This spatiotemporal pattern of myosin-II localization and the morphological defects observed in the eyes of a myosin-II mutant suggest that the myosin-II/F-actin system is involved in the alignment of the rhabdomeres within the retina and in the flattening of the retinal floor. The observation that the myosin-II/F-actin arrays are incomplete or disorganized in R7/R8 and in rhodopsin-1-null R1–6 suggests further that the establishment and stability of this cytoskeletal system depend on rhodopsin-1 expression.

Golgi analysis of tangential neurons in the lobula plate of Drosophila melanogaster.

The lobula plate (LP), which is the third order optic neuropil of flies, houses wide-field neurons which are exquisitely sensitive to motion. Among Diptera, motion-sensitive neurons of larger flies have been studied at the anatomical and physiological levels. However, the neurons of Drosophila lobula plate are relatively less explored. As Drosophila permits a genetic analysis of neural functions, we have analysed the organization of lobula plate of Drosophila melanogaster. Neurons belonging to eight anatomical classes have been observed in the present study. Three neurons of the horizontal system (HS) have been visualized. The HS north (HSN) neuron, occupying the dorsal lobula plate is stunted in its geometry compared to that of larger flies. Associated with the HS neurons, thinner horizontal elements known as h-cells have also been visualized in the present study. Five of the six known neurons of the vertical system (VS) have been visualized. Three additional neurons in the proximal LP comparable in anatomy to VS system have been stained. We have termed them as additional VS (AVS)-like neurons. Three thinner tangential cells that are comparable to VS neurons, which are elements of twin vertical system (tvs); and two cells with wide dendritic fields comparable to CH neurons of Diptera have been also observed. Neurons comparable to VS cells but with 'tufted' dendrites have been stained. The HSN and VS1-VS2 neurons are dorsally stunted. This is possibly due to the shape of the compound eye of Drosophila which is reduced in the fronto-dorsal region as compared to larger flies.

Cell-cycle regulation and cell-type specification in the developing Drosophila compound eye.

During nervous system development stem cell daughters must exit the proliferative cycle to adopt specific neural and glial fates and they must do so in the correct positions. Cell proliferation in the central nervous system occurs in neuroepithelia such as the neural retina and the ventricular zones. As cells are assigned specific fates they migrate out of the plane of the epithelium to form higher layers. Recent evidence from the Drosophila compound eye suggests that a novel mode of Ras pathway regulation may be crucial in both cell-cycle exit and neural patterning: "MAP Kinase cytoplasmic hold".

Genetic control of retinal specification and determination in Drosophila

The Drosophila compound eye has long served as an outstanding model system to study many processes, including cell fate specification, cell division, cell growth and cell death. In addition, exploring the molecular basis of eye specification in Drosophila has identified a set of nuclear factors that trigger the conversion of a group of multipotent epithelial cells into eye primordia. These nuclear factors act in complex networks to regulate retinal specification and appear to be conserved throughout phylogeny. Finally, evidence suggests that these nuclear networks have been co-opted to specify cell fates in other tissues. We review the latest developments in the field of retinal specification in Drosophila and discuss several future directions that remain open for investigation.