Direct regulatory interaction of the eyeless protein with an eye-specific enhancer in the sine oculis gene during eye induction in Drosophila.
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The Pax-6 gene encodes a transcription factor with two DNA-binding domains, a paired and a homeodomain, and is expressed during eye morphogenesis and development of the nervous system. Pax-6 homologs have been isolated from a wide variety of organisms ranging from flatworms to humans. Since loss-of-function mutants in insects and mammals lead to an eyeless phenotype and Pax-6 orthologs from distantly related species are capable of inducing ectopic eyes in Drosophila, we have proposed that Pax-6 is a universal master control gene for eye morphogenesis. To determine the extent of evolutionary conservation of the eye morphogenetic pathway, we have begun to identify subordinate target genes of Pax-6. Previously we have shown that expression of two genes, sine oculis (so) and eyes absent (eya), is induced by eyeless (ey), the Pax-6 homolog of Drosophila. Here we present evidence from ectopic expression studies in transgenic flies, from transcription activation studies in yeast, and from gel shift assays in vitro that the EY protein activates transcription of sine oculis by direct interaction with an eye-specific enhancer in the long intron of the so gene. (+info)
Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development.
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The small GTP-binding protein Ral is activated by RalGDS, one of the effector molecules for Ras. Active Ral binds to a GTPase activating protein for CDC42 and Rac. Although previous studies suggest a role for Ral in the regulation of CDC42 and Rac, which are involved in arranging the cytoskeleton, its in vivo function is largely unknown. To examine the effect of overexpressing Ral on development, transgenic Drosophila were generated that overexpress wild-type or mutated Ral during eye development. While wild-type Ral caused no developmental defects, expression of a constitutively activated protein resulted in a rough eye phenotype. Activated Ral did not affect cell fate determination in the larval eye discs but caused severe disruption of the ommatidial organization later in pupal development. Phalloidin staining showed that activated Ral perturbed the cytoskeletal structure and cell shape changes during pupal development. This phenotype is similar to that caused by RhoA overexpression. In addition, the phenotype was synergistically enhanced by the coexpression of RhoA. These results suggest that Ral functions to control the cytoskeletal structure required for cell shape changes during Drosophila development. (+info)
ebi regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development. (+info)
A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors.
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1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids. (+info)
Dual role for Hedgehog in the regulation of the proneural gene atonal during ommatidia development.
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The differentiation of cells in the Drosophila eye is precisely coordinated in time and space. Each ommatidium is founded by a photoreceptor (R)8 cell and these founder cells are added in consecutive rows. Within a row, the nascent R8 cells appear in precise locations that lie out of register with the R8 cells in the previous row. The bHLH protein Atonal determines the development of the R8 cells. The expression of atonal is induced shortly before the selection of a new row of R8 cells and is initially detected in a stripe. Subsequently atonal expression resolves into regularly spaced clusters (proneural clusters) that prefigure the positions of the future R8 cells. The serial induction of atonal expression, and hence the increase in the number of rows of R8 cells, requires Hedgehog function. Here it is shown that, in addition to this role, Hedgehog signalling is also required to repress atonal expression between the nascent proneural clusters. This repression has not been previously described and appears to be critical for the positioning of Atonal proneural clusters and, therefore, the R8 cells. The two temporal responses to Hedgehog are due to direct stimulation of the responding cells by Hedgehog itself. (+info)
Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster.
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During animal development, growth of the various tissues and organs that make up the body must be coordinated. Despite recent progress in understanding growth control within the cell unit, the mechanisms that coordinate growth at the organismal level are still poorly understood. To study this problem, we performed a genetic screen for larval growth-defective mutants in Drosophila melanogaster. Characterization of these mutants revealed distinct types of larval growth defects. An allelic series for the translation initiation factor, Eif4A, showed different growth rates and suggests that Eif4A could be used as a dose-dependent growth regulator. Two mutants that fail to exit cellular quiescence at larval hatching (milou and eif4(1006)) have a DNA replication block that can be bypassed by overexpression of the E2F transcription factor. A mutation (bonsai) in a homolog of the prokaryotic ribosomal protein, RPS15, causes a growth defect that is non-cell-autonomous. Our results emphasize the importance of translational regulation for the exit from quiescence. They suggest that the level of protein synthesis required for cell cycle progression varies according to tissue type. The isolation of non-cell-autonomous larval growth-defective mutants suggests that specialized organs coordinate growth throughout the animal and provides new tools for studies of organismal growth regulation. (+info)
Role of Xrx1 in Xenopus eye and anterior brain development.
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The anteriormost part of the neural plate is fated to give rise to the retina and anterior brain regions. In Xenopus, this territory is initially included within the expression domain of the bicoid-class homeobox gene Xotx2 but very soon, at the beginning of neurulation, it becomes devoid of Xotx2 transcripts in spatiotemporal concomitance with the transcriptional activation of the paired-like homeobox gene Xrx1. By use of gain- and loss-of-function approaches, we have studied the role played by Xrx1 in the anterior neural plate and its interactions with other anterior homeobox genes. We find that, at early neurula stage Xrx1 is able to repress Xotx2 expression, thus first defining the retina-diencephalon territory in the anterior neural plate. Overexpression studies indicate that Xrx1 possesses a proliferative activity that is coupled with the specification of anterior fate. Expression of a Xrx1 dominant repressor construct (Xrx1-EnR) results in a severe impairment of eye and anterior brain development. Analysis of several brain markers in early Xrx1-EnR-injected embryos reveals that anterior deletions are preceded by a reduction of anterior gene expression domains in the neural plate. Accordingly, expression of anterior markers is abolished or decreased in animal caps coinjected with the neural inducer chordin and the Xrx1-EnR construct. The lack of expansion of mid-hindbrain markers, and the increase of apoptosis in the anterior neural plate after Xrx1-EnR injection, indicate that anterior deletions result from an early loss of anterior neural plate territories rather than posteriorization of the neuroectoderm. Altogether, these data suggest that Xrx1 plays a role in assigning anterior and proliferative properties to the rostralmost part of the neural plate, thus being required for eye and anterior brain development. (+info)
Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila.
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Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal. (+info)