Kevin Moses

Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway

Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.

Genetics of epithelial polarity and pattern in the Drosophila retina.

This review is focused on recent advances in our understanding of the development of coordinated cell polarity, through experiments on the Drosophila compound eye. Each eye facet (or “ommatidium”) contains a set of eight photoreceptor cells, placed so that their rhabdomeres form an asymmetric trapezoid. The array of ommatidia is organized so that these trapezoids are aligned in two mirror‐image fields, dorsal and ventral to the eye midline (or “equator”). The development of this pattern depends on two systems of positional information that inform the cluster of cells that will form an ommatidium of anterior/posterior (a/p) and dorsal/ventral (d/v) direction. The former (a/p) is encoded by a progressive wave of development (the morphogenetic furrow). The latter (d/v) involves molecules known to act in tissue polarity in other organs and organisms. Our understanding of the function of these molecules rests not only on their mutant phenotypes, biochemistry, and expression patterns, but also on the spatial effects when mutant patches of cells are made (genetic mosaics). BioEssays 21:275–285, 1999.

Drosophila eye development

Retinal Specification and Determination in Drosophila * Regulators of the Morphogenetic Furrow * NOTCH and the Patterning of Ommatidial Founder Cells in the Developing Drosophila Eye * The Epidermal Growth Factor Receptor in Drosophila Eye Development * Cell Fate Specification in the Drosophila Eye * Tissue Polarity in the Retina * Regulation of Growth and Cell Proliferation During Eye Development * Evolution of Color Vision * Developmental Regulation Through Protein Stability * Programmed Death in Eye Development * Drosophila Compound Eye Morphogenesis: Blind Mechanical Engineers? The Establishment of Retinal Connectivity * Homologies Between Vertebrate and Invertebrate Eyes * Applications of the Drosophila Retina to Human Disease Modeling.

MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing.

Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK.

Expression of evolutionarily conserved eye specification genes during Drosophila embryogenesis

Abstract. Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sineoculis (so), eyesabsent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field.

Pointed regulates an eye-specific transcriptional enhancer in the Drosophila hedgehog gene, which is required for the movement of the morphogenetic furrow.

Drosophila development depends on stable boundaries between cellular territories, such as the embryonic parasegment boundaries and the compartment boundaries in the imaginal discs. Patterning in the compound eye is fundamentally different: the boundary is not stable, but moves (the morphogenetic furrow). Paradoxically, Hedgehog signaling is essential to both: Hedgehog is expressed in the posterior compartments in the embryo and in imaginal discs, and posterior to the morphogenetic furrow in the eye. Therefore, uniquely in the eye, cells receiving a Hedgehog signal will eventually produce the same protein. We report that the mechanism that underlies this difference is the special regulation of hedgehog (hh) transcription through the dual regulation of an eye specific enhancer. We show that this enhancer requires the Egfr/Ras pathway transcription factor Pointed. Recently, others have shown that this same enhancer also requires the eye determining transcription factor Sine oculis (So). We discuss these data in terms of a model for a combinatorial code of furrow movement.

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.

smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

The Drosophila Mitogen Activated Protein Kinase (MAPK) Rolled is a key regulator of developmental signaling, relaying information from the cytoplasm into the nucleus. Cytoplasmic MEK phosphorylates MAPK (pMAPK), which then dimerizes and translocates to the nucleus where it regulates transcription factors. In cell culture, MAPK nuclear translocation directly follows phosphorylation, but in developing tissues pMAPK can be held in the cytoplasm for extended periods (hours). Here, we show that Moleskin antigen (Drosophila Importin 7/Msk), a MAPK transport factor, is sequestered apically at a time when lateral inhibition is required for patterning in the developing eye. We suggest that this apical restriction of Msk limits MAPK nuclear translocation and blocks Ras pathway nuclear signaling. Ectopic expression of Msk overcomes this block and disrupts patterning. Additionally, the MAPK cytoplasmic hold is genetically dependent on the presence of Decapentaplegic (Dpp) and Hedgehog receptors.

Genetic and biochemical analysis of the role of Egfr in the morphogenetic furrow of the developing Drosophila eye

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 (Egfrtsla) 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 Egfrtsla is a serine to phenylalanine change in a conserved extracellular ligand-binding domain. We show by biochemical and genetic approaches that the Egfrtsla 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.

Function of the Drosophila TGF-α homolog Spitz is controlled by Star and interacts directly with Star

Drosophila Spitz is a homolog of transforming growth factor alpha (TGF-alpha) and is an activating ligand for the EGF receptor (Egfr). It has been shown that Star is required for Spitz activity. Here we show that Star is quantitatively limiting for Spitz production during eye development. We also show that Star and Spitz proteins colocalize in Spitz sending cells and that this association is not coincident with the site of translation--consistent with a function for Star in Spitz processing or transmission. Finally, we have defined minimal sequences within both Spitz and Star that mediate a direct interaction and show that this binding can occur in vivo.