During retinal patterning a diverse array of cell fates are specified with remarkable precision through a complex interplay of signaling pathways, molecular factors, and mechanisms that are highly conserved. An example is the process by which the 'founding' neural cell type, the R8-precursor, is specified in Drosophila. Notch signaling mediates this specification in a wave of differentiation, the morphogenetic furrow that sweeps across the eye disc. In an early phase, Notch elicits a broad expression of the basic-helix-loop-helix (bHLH) transcription factor Atonal. Later, Notch elicits expression of bHLH repressors encoded by the Enhancer of Split, E(spl) complex that antagonize Atonal in all but the 'founding' R8 cells. This restriction of Atonal is critical for refining the number of R8 cells, and its perturbation interferes with proper eye development. E(spl)m8 mediates this 'refinement', because its over expression and mutation in the E(spl)D allele (encoding M8*) severely antagonizes Atonal and results in reduced eyes. We have shown that the conserved protein kinase CK2 phosphorylates E(spl)M8 at Ser159. We now demonstrate that replacement of Ser159 with Asp (M8SD) elicits a reduced eye phenotype that mimics the eye phenotype and mechanism of E(spl)D. We hypothesize that phosphorylation by CK2 permits E(spl)M8 to antagonize Atonal and is, therefore, critical for proper retinal patterning. We propose three specific aims to test this hypothesis. (1) Analyze the residual ommatidia of flies expressing M8SD, define the mechanism of its dominant behavior via over expression of Atonal, and define the role of CK2 via analysis of CK2 mutants, RNA-interference/mosaics, and over expression of CK2 in R8-precursors. (2) Test the hypothesis that phosphorylation stabilizes M8 and/or mediates hierarchical phosphorylation. (3) Test the hypothesis that CK2 phosphorylation switches M8 into an active repressor of Atonal, and identify phosphatases that counteract CK2. Our studies are likely to be relevant to humans, because specification of fly R8 photoreceptors and mammalian retinal ganglion cells involve similar mechanisms, and, as in the E(spl)M8-Atonal interaction, CK2 activity mediates interaction of Hes6-Hesl, two vertebrate bHLH proteins involved with rod-photoreceptor specification. Our studies will provide a better understanding of the roles of CK2 in this process, and may provide insights into how misregulated cell fate specification contributes to eye defects.

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National Eye Institute (NEI)
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Neurogenesis and Cell Fate Study Section (NCF)
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Mariani, Andrew P
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West Virginia University
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Bandyopadhyay, Mohna; Arbet, Scott; Bishop, Clifton P et al. (2016) Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development. Pharmaceuticals (Basel) 10:
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Eastwood, Karen; Yin, Chong; Bandyopadhyay, Mohna et al. (2011) New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment. Mol Cell Biochem 356:217-25
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Kahali, Bhaskar; Bose, Anasua; Karandikar, Umesh et al. (2009) On the mechanism underlying the divergent retinal and bristle defects of M8* (E(spl)D) in Drosophila. Genesis 47:456-68
Kahali, Bhaskar; Trott, Regina; Paroush, Ze'ev et al. (2008) Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo. Biochem Biophys Res Commun 373:637-42
Bose, Anasua; Kahali, Bhaskar; Zhang, Sophia et al. (2006) Drosophila CK2 regulates lateral-inhibition during eye and bristle development. Mech Dev 123:649-64
Shaffer, Jonathan M; Karandikar, Umesh C; Bidwai, Ashok P (2006) An activity-based two-hybrid system for the selective identification of substrates of protein kinases. Anal Biochem 355:154-6

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