Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation.

The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.     

The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis.

Ultraspiracle (usp) encodes the Drosophila cognate of RXR, the human retinoid X receptor. To examine how RXR subfamily members function in development, we have undertaken a phenotypic analysis of usp mutants. usp is required at multiple stages of development for functions that occur in a wide variety of tissues. usp is required in the eye-antennal imaginal disc for normal eye morphogenesis and in the somatic and germline tissues of adult females for fertilization, eggshell morphogenesis and embryonic development. An unusual sunken eye phenotype with marked ventral-dorsal polarity appears to be caused by a lack of usp function in the imaginal disc cells that reside between the eye and antennal anlage. The usp functions include cell autonomous and non-cell autonomous components, suggesting that usp controls the production of factors important for both cell-cell communication and cellular differentiation. These usp signalling pathways have mechanistic parallels to steroid and retinoid action in developing vertebrate tissues.     

Surviving Drosophila eye development

During eye development, cell death interplays dynamically with events of differentiation to achieve the remarkably patterned structure of the fly compound eye. Mutations in genes that affect the normal developmental process can lead to excessive death of progenitor cells, or, alternatively, to the differentiation of supernumerary neurons, pigment and cone cells due to survival of cells that would normally be eliminated. These data reveal that eye development contains cell selection processes: only certain cells are selected to undergo differentiation, and supernumerary cells are actively eliminated by cell death pathways to achieve the highly ordered lattice of the eye. The final number of cells that comprise the eye is controlled through a balance of cell proliferation with proper cell differentiation and removal by cell death.     

Migration and function of glia in the developing Drosophila eye.

Although glial cells have been implicated widely in the formation of axon tracts in both insects and vertebrates, their specific function appears to be context-dependent, ranging from providing essential guidance cues to playing a merely facilitory role. Here we examine the role of the retinal basal glia (RBG) in photoreceptor axon guidance in Drosophila. The RBG originate in the optic stalk and have been thought to migrate into the eye disc along photoreceptor axons, thus precluding any role in axon guidance. Here we show the following. (1) The RBG can, in fact, migrate into the eye disc even in the absence of photoreceptor axons in the optic stalk; they also migrate to ectopic patches of differentiating photoreceptors without axons providing a continuous physical substratum. This suggests that glial cells are attracted into the eye disc not through haptotaxis along established axons, but through another mechanism, possibly chemotaxis. (2) If no glial cells are present in the eye disc, photoreceptor axons are able to grow and direct their growth posteriorly as in wild type, but are unable to enter the optic stalk. This indicates that the RBG have a crucial role in axon guidance, but not in axonal outgrowth per se. (3) A few glia close to the entry of the optic stalk suffice to guide the axons into the stalk, suggesting that glia instruct axons by local interaction.     

Cytosolic Ras supports eye development in Drosophila

Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development.     

Ommatidial development in Drosophila eye disc fragments

We have tested the hypothesis that the leading edge of the growing Drosophila compound eye acts as a template that organizes unpatterned cells of the retinal epithelium into the accurate cellular mosaic of the eye. Unpatterned fragments of the epithelium, not containing the leading edge of the growing field, were transplanted into larval hosts. After hosts pupated, the implants were recovered; most contained ommatidia, demonstrating that the leading edge of the growing eye pattern is not required for its propagation. In a second set of experiments, implants were recovered before hosts pupated and examined for ommatidia using a monoclonal antibody. These implants likewise differentiated ommatidia and the temporal progress of retinal development in the implants mirrored that of normal development. A schedule of ommatidial development thus appears to be mapped onto the retinal epithelium in advance of the leading edge.     

Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer

The steroid hormone 20-hydroxyecdysone coordinates the stages of Drosophila development by activating a nuclear receptor heterodimer consisting of the ecdysone receptor, EcR, and the Drosophila RXR receptor, USP. We show that EcR/USP DNA binding activity requires activation by a chaperone heterocomplex like that required for activation of the vertebrate steroid receptors, but not previously shown to be required for activation of RXR heterodimers. Six proteins normally present in the chaperone complex were individually purified and shown to be sufficient for this activation. We also show that two of the six (Hsp90 and Hsc70) are required in vivo for ecdysone receptor activity, and that EcR is the primary target of the chaperone complex.     

Membrane trafficking in Drosophila wing and eye development

It is clear that membrane transport is essential to the proper sorting and delivery of membrane bound receptors and ligands, and secreted signaling molecules. Molecular genetic studies in Drosophila are particularly well suited to studies of membrane transport in development. The conservation of cell signaling pathways and membrane transport molecules between Drosophila and other species makes the results obtained in these studies of general interest. In addition, the ability to generate gain- and loss-of-function genetic mutations of various strengths, and the ability to generate transgenic flies that direct protein expression to tissues during development are of particular advantage. Several recent papers suggest that interesting and novel roles for membrane transport processes will be uncovered by studying classically defined membrane transport proteins in developmental contexts. Together these studies suggest that regulation of membrane transport may represent an additional mechanism to regulate the strength of cell-cell signaling during development.     

Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.     

Polarity determination in the Drosophila eye.

In the Drosophila eye, photoreceptor clusters are arranged with mirror-image symmetry about the dorso-ventral midline. Recent work indicates critical r?les for Wingless and JAK/STAT signalling in setting up long-range gradients of positional information that determine this symmetry. Furthermore, important functions have been elucidated for Notch signalling in specifying the position of the axis of symmetry and then later in development in interpreting gradient information.     

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.     

PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development.

Mutations in the tumor suppressor gene PTEN (MMAC1/TEP1) are associated with a large number of human cancers and several autosomal-dominant disorders. Mice mutant for PTEN die at early embryonic stages and the mutant embryonic fibroblasts display decreased sensitivity to cell death. Overexpression of PTEN in different mammalian tissue culture cells affects various processes including cell proliferation, cell death and cell migration. We have characterized the Drosophila PTEN gene and present evidence that both inactivation and overexpression of PTEN affect cell size, while overexpression of PTEN also inhibits cell cycle progression at early mitosis and promotes cell death during eye development in a context-dependent manner. Furthermore, we have shown that PTEN acts in the insulin signaling pathway and all signals from the insulin receptor can be antagonized by either Drosophila or human PTEN, suggesting a potential means for alleviating symptoms associated with altered insulin signaling.     

Cell proliferation, survival, and death in the Drosophila eye.

The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a "Second Mitotic Wave" cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival.