Developing Eye Disc Research Articles

Human 14-3-3 gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster

Background14-3-3 proteins are a family of adaptor proteins that participate in a wide variety of cellular processes. Recent evidence indicates that the expression levels of these proteins are elevated in some human tumors providing circumstantial evidence for their involvement in human cancers. However, the mechanism through which these proteins act in tumorigenesis is uncertain.ResultsTo determine whether elevated levels of 14-3-3 proteins may perturb cell growth we overexpressed human 14-3-3 gamma (h14-3-3 gamma) in Drosophila larvae using the heat shock promoter or the GMR-Gal4 driver and then examined the effect that this had on cell proliferation in the eye imaginal discs of third instar larvae. We found that induction of h14-3-3 gamma resulted in the abnormal appearance of replicating cells in the differentiating proneural photoreceptor cells of eye imaginal discs where h14-3-3 gamma was driven by the heat shock promoter. Similarly, we found that driving h14-3-3 gamma expression specifically in developing eye discs with the GMR-Gal4 driver resulted in increased numbers of replicative cells following the morphogenetic furrow. Interestingly, we found that the effects of overexpressing h1433 gamma on eye development were increased in a genetic background where String (cdc25) function was compromised.ConclusionTaken together our results indicate that h14-3-3 gamma can promote abnormal cell proliferation and may act through Cdc25. This has important implications for 14-3-3 gamma as an oncogene as it suggests that elevated levels of 14-3-3 may confer a growth advantage to cells that overexpress it.

Tiling of R7 Axons in the Drosophila Visual System Is Mediated Both by Transduction of an Activin Signal to the Nucleus and by Mutual Repulsion

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-alpha3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals.

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development.

The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Cullins are the major components of a series of ubiquitin ligases that control the degradation of a broad range of proteins. We found that Nedd8 modifies Cul1 in Drosophila. In Drosophila Nedd8 and Cul1 mutants, protein levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are highly accumulated, suggesting that the Cul1-based SCF complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. We further show that two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCF(Slimb) complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent.

Regulators of the morphogenetic furrow.

Unlike other imaginal discs, the Drosophila eye disc has a progressive pattern of differentiation. Photoreceptor clusters begin to form at the posterior margin of the eye disc in the third larval instar, and more anterior rows of clusters then differentiate in succession (Ready et al. 1976). Just prior to their differentiation, cells undergo an apical constriction and apical-basal contraction that produces an indentation in the disc known as the morphogenetic furrow (MF; Ready et al. 1976). Cells anterior to the MF divide actively and appear unpatterned. Just posterior to the MF, cells assemble into evenly spaced rosettes; slightly more posteriorly these transform into arcs, and the arcs then close to produce five-cell preclusters (Wolff and Ready 1991). Concurrently, these cells initiate a program of gene expression resulting in the appearance of neuralspecific markers in a defined sequence in the cells of each cluster (Tomlinson and Ready 1987). Cells in the MF are arrested in the G1 phase of the cell cycle (Thomas et al. 1994); posterior to the MF, cells excluded from the preclusters undergo one more round of division, the second mitotic wave, to generate the remaining cells of each ommatidium (Ready et al. 1976; Wolff and Ready 1991). This orderly and sequential pattern of differentiation, proliferation, and morphogenesis is organized by a set of signaling molecules that also direct many other developmental processes. The expression patterns and interactions of these molecules in the developing eye disc are shown in Fig. 1.

Cell fate specification in the Drosophila eye.

With the passing of the furrow over a uniformly equivalent group of cells, a spectacular array of cell types, each different in structure and function arises in the developing eye disc of Drosophila. A small number of ubiquitously expressed transcription factors combine with an even smaller number of signals emanating from the furrow and the developing clusters and generate cell-specific expression of a panoply of transcription factors (reviewed in Kumar and Moses 1997).The challenge is to understand how the cell-specific transcription factors are placed in their respective cells and how they then function in assigning different identities to each cell type. Here, we have presented our current understanding of this process. The aim is to be eclectic rather than comprehensive, and we apologize in advance to those investigators whose work we have not fully cited.KeywordsEpidermal Growth Factor ReceptorCone CellEpidermal Growth Factor Receptor PathwayCell Fate SpecificationMorphogenetic FurrowThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Homothorax and iroquois-C genes are required for the establishment of territories within the developing eye disc

In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. Here we show that homothorax is required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc only, homothorax and wingless are involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, we show that the iroquois-complex genes are required for dorsal head development antagonizing the function of homothorax in this region of the disc.

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.

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development.

The Drosophila Pax-6 gene eyeless acts high up in the genetic hierarchy involved in compound eye development and can direct the formation of extra eyes in ectopic locations. Here we identify sine oculis and eyes absent as two mediators of the eye-inducing activity of eyeless. We show that eyeless induces and requires the expression of both genes independently during extra eye development. During normal eye development, eyeless is expressed earlier than and is required for the expression of sine oculis and eyes absent, but not vice versa. Based on the results presented here and those of others, we propose a model in which eyeless induces the initial expression of both sine oculis and eyes absent in the eye disc. sine oculis and eyes absent then appear to participate in a positive feedback loop that regulates the expression of all three genes. In contrast to the regulatory interactions that occur in the developing eye disc, we also show that in the embryonic head, sine oculis acts in parallel to eyeless and twin of eyeless, a second Pax-6 gene from Drosophila. Recent studies in vertebrate systems indicate that the epistatic relationships among the corresponding vertebrate homologs are very similar to those observed in Drosophila.

Induction of Drosophila eye development by Decapentaplegic

The Drosophila decapentaplegic (dpp) gene, encoding a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily, controls proliferation and patterning in diverse tissues, including the eye imaginal disc. Pattern formation in this tissue is initiated at the posterior edge and moves anteriorly as a wave; the front of this wave is called the morphogenetic furrow (MF). Dpp is required for proliferation and initiation of pattern formation at the posterior edge of the eye disc. It has also been suggested that Dpp is the principal mediator of Hedgehog function in driving progression of the MF across the disc. In this paper, ectopic Dpp expression is shown to be sufficient to induce a duplicated eye disc with normal shape, MF progression, neuronal cluster formation and direction of axon outgrowth. Induction of ectopic eye development occurs preferentially along the anterior margin of the eye disc. Ectopic Dpp clones situated away from the margins induce neither proliferation nor patterning. The Dpp signalling pathway is shown to be under tight transcriptional and post-transcriptional control within different spatial domains in the developing eye disc. In addition, Dpp positively controls its own expression and suppresses wingless transcription. In contrast to the wing disc, Dpp does not appear to be the principal mediator of Hedgehog function in the eye.

Atonal, rough and the resolution of proneural clusters in the developing Drosophila retina.

In the developing Drosophila retina, the proneural gene for photoreceptor neurons is atonal, a basic helix-loop-helix transcription factor. Using atonal as a marker for proneural maturation, we examine the stepwise resolution of proneural clusters during the initiation of ommatidial differentiation in the developing eye disc. In addition, evidence is provided that atonal is negatively regulated by rough, a homeobox-containing transcription factor expressed exclusively in the retina. This interaction leads to the refinement of proneural clusters to specify R8, the first neuron to emerge in the retinal neuroepithelium. Ectopic expression of atonal or removal of rough results in the transformation of a discrete 'equivalence group' of cells into R8s. In addition, ectopic expression of rough blocks atonal expression and proneural cluster formation within the morphogenetic furrow. Thus, rough provides retina-specific regulation to the more general atonal-mediated proneural differentiation pathway. The opposing roles of atonal and rough are not mediated through the Notch pathway, as their expression remains complementary when Notch activity is reduced. These observations suggest that homeobox-containing genes can provide tissue-specific regulation to bHLH factors.

DaughterlessIs Required forDrosophilaPhotoreceptor Cell Determination, Eye Morphogenesis, and Cell Cycle Progression

Initiation ofDrosophilaperipheral nervous system (PNS) development requires theachaete–scutecomplex (AS-C) and theatonal(ato) genes. The AS-C andatoencode basic helix-loop-helix (bHLH) transcription factors that dimerizein vitrowith another bHLH protein,daughterless(da).dahas many functions duringDrosophilaembryonic development, as it is required for proper sex determination, oogenesis, and neurogenesis. Here, we examine the expression and function ofdawithin the developingDrosophilaeye. The use of a monoclonal antibody to the Da protein revealed that Da levels are modulated across the developing eye disc. Within the morphogenetic furrow (MF) and photoreceptor cell R8, there is a cell-by-cell correspondence between high levels of Da protein expression and Ato protein expression. Mosaic analysis of adult tissue demonstrates thatdafunction is cell autonomous and required within R2, R3, R4, R5, and R8. Examination of gene expression inda−imaginal disc clones reveals thatdaregulates Ato expression in the MF, affects the progression of the MF, and is necessary for the reestablishment of the G2and M phases of the synchronized cell cycle posterior to the MF.

The role of yan in mediating the choice between cell division and differentiation.

An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Drosophila epidermal growth factor receptor (Egfr) gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis revealed that aop is allelic to yan and genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones showed that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. Our studies suggest that the role of yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation. The relationship of the Egfr and Notch pathways to this developmental role of yan is discussed.

Early decisions in Drosophila eye morphogenesis

Recent analyses have shed light on the roles of genes involved in early events of eye cell determination and the spatiotemporal control of differentiation within the eye field. These genes function at sequential steps in the programming, initiation, or progression of differentiation, highlighting an elegant orchestration of gene activities to achieve this striking developmental event. Progress has been made in the study of the coordination between cell cycle control and cell differentiation, as well as in the genetic control of morphogenetic movements within the developing eye disc.

Hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates

The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginal disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.

The effect of eye reduction on white position-effect variegation in Drosophila melanogaster.

Position-effect variegation for the white locus was studied in normally shaped eyes and in reduced eyes of Bar (B) and Drop (Dr) flies. The average number of spots per eye is successively lower in +, B, and Dr eyes; moreover, B eyes show a relatively strong pigmentation. No simple relation seems to be present between the degree of pigmentation and the number of facets, either between +, B, and Dr eyes or within classes of Dr eyes that have been analysed. The chance that ommatidia will become pigmented follows a gradient across mottled eyes of wild-type shape that seems fixed early in development. The gradient is less clear or absent in B eyes. The results are best interpreted on the basis of the cell-lineage theory and an early one-sided action of B on the developing eye disc after fixation of the gradient.