Semester

Fall

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Ashok Bidwai

Abstract

Notch signaling is an evolutionary conserved pathway that mediates binary cell-fate specification throughout animal development. Through a process termed lateral inhibition, Notch signaling drives two equipotent cells to adopt distinct fates. Binary cell-fate determination has been exceptionally well studied during Drosophila neurogenesis, particularly, in eye and bristle development. In the eye, Notch mediates the selection of R8 photoreceptors from clusters of R8 precursors by antagonizing the activity of the proneural activator Atonal (Ato). In the bristle, Notch drives the selection of the sensory organ precursors (SOP's) from a group of equipotential cells that expressed the proneural activators encoded by the achaete scute complex (ASC). In either case, the conserved basic-Helix-Loop-Helix (bHLH) repressors encoded by the Enhancer of split Complex (E(spl)C) mediate lateral inhibition by antagonizing Ato or ASC. Accumulating evidence indicates that phosphorylation of the E(spl) member M8 by protein kinase CK2 is required for antagonism of Ato/ASC. This modification appears to convert M8 from an autoinhibited state to one that is competent for binding to, and antagonism of Ato or ASC. The work described in this dissertation aims to extend these findings and provide a more detailed understanding of the mechanisms by which M8 mediates neural repression. The studies described in Chapter-2 provide a fundamental reinterpretation of the mechanism by which the m8 allele E(spl)D ablates Ato expression and eye development. Our work indicates that the eye defects of E(spl)D reflect the unique biphasic requirements of Notch during R8 specification, where Notch elicits Ato expression and later elicits E(spl) expression. Specifically, we show that the product of E(spl)D, a truncated protein called M8*, lacks the autoinhibitory domain thereby allowing it to interfere with the first phase of Notch signaling, itself. As a result, M8* impairs expression of Ato to a level that is insufficient to confer the R8 fate. The work of Chapter-3 provides in vivo evidence in support of the autoinhibition model. Using assays for impaired Notch signaling, we show that the C-terminal domain (CtD) of M8 mediates autoinhibition even when expressed as a free peptide. This ability is abolished when the CtD contains a phosphomimetic Asp substitution at the CK2 consensus site, indicating that the 56-residue CtD peptide is sufficient to mediate autoinhibition. Chapter-4 provides genetic evidence that implicates multisite/hierarchical phosphorylation in the regulation of M8 activity. Our studies suggest that CK2 may act as the primary gatekeeper of this cascade of events, which later involve modifications of M8 by MAPK, CK1 and GSK3. Evidence is presented that the MAPK site in M8 is important for neural repression, and that this site is responsive to alteration in EGFR signaling. Multisite phosphorylation may act as a 'timer' controlling the onset of repression, a regulation that is bypassed by the E(spl)D mutation. The studies in Chapter-5 demonstrate direct genetic interactions between alleles of CK2, Notch and E(spl). The eye, bristle and wing margin defects provide strong evidence that CK2 is a participant in Notch signaling. In Chapter-6, we extend our findings to the bHLH protein Hairy, a member of the HES family. Using in vitro and in vivo assays, we demonstrate that CK2 is required for repression by Hairy as well. Together, the studies described in this dissertation provide novel insights into neural repression, and indicate that posttranslational regulation imposes control over inhibitory Notch signaling.

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