Adam T. Majot

Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Ashok P Bidwai

Committee Co-Chair

Clifton P Bishop

Committee Member

Philip Keeting

Committee Member

Peter H Mathers

Committee Member

Shuo Wei

Abstract

Drosophila retinogenesis has long served as a paradigm of tissue patterning. Notch, an anciently conserved signaling pathway, is critical to both the formation of and proper spacing of founding R8 photoreceptors. Notch is intimately interwoven into the activity of Atonal (Ato), a proneural basic helix-loop-helix (bHLH) that is required for R8 specification. Ato is expressed through dual enhancers: ato-3' and 5'-ato. ato-3' is activated in response to Notch-independent signaling cues whereas 5'-ato exhibits complex dependence on Notch signaling. First, Notch is required for 5'-ato activation, eliciting formation of Ato intermediate groups (IGs, evenly spaced clusters of 10--20 cells). Subsequently, Notch is required to repress ato, to resolve its expression from IGs to isolated R8s. This is accomplished through the induction of E(spl)bHLH repressors to disrupt proneural function. The former process is independent of the Notch pathway transcription factor Suppressor of Hairless (SuH) and the latter requires Su(H). Current views regarding this process suggest that either 1) the Su(H)-dependent response may require a greater threshold of Notch signal activity before engaging, or 2) E(spl) repressors are initially expressed but lack proper posttranslational activation to repress ato. The work of Chapter 2 refutes both scenarios. We demonstrate that E(spl)bHLHs are expressed prior to the perceived activation of 5'-ato. E(spl) repressors are active at this time, as their removal elicits precocious de-repression of 5'-ato. Thus, both the Su(H)-independent and -dependent processes initiate simultaneously. These findings suggest that ato-3' activity is crucial during IG formation. Analysis of the hypermorphic allele E( spl)D delineates a role for posttranslational activation of E(spl)M8 during the repressive phase of Ato patterning. The work of Chapter 3 further probes the importance of ato-3' in the formation of IGs. Anterior open (Aop) has previously been characterized as an effector of MAPK and is an ortholog of tumor suppressor Tel/ETV6. Aop coexpresses with Ato only during early IG formation, prior to the onset of autoregulation via 5'-ato. We demonstrate that Aop is required cell-autonomously during early IG formation to promote continued expression from ato-3'. Furthermore, Aop is induced by Notch activity via Su(H), indicating that Su(H) co-opts Aop to instill a delay in Notch-mediated repression. The work of Chapter 4 assesses the role of ato in a broader context. The region of the developing eye that expresses Ato is referred to as the morphogenetic furrow (MF). The MF relies upon sophisticated signaling between Notch, Hedgehog (Hh) and Decapentaplegic (Dpp). Our work indicates that, in addition to Ato's role in facilitating neurogenesis, it is proactive in the production of neural progenitors through its elicitation of Dpp, as assessed by a lacZ reporter. In total, these studies emphasize the complexity of the relationship between Notch and Ato and suggest interesting new studies into possible nodes of signaling crosstalk with Notch.

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