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Abstract

Development of an adult organism from a fertilized embryo requires coordinated cell proliferation, specification, differentiation and morphogenesis. These processes are controlled by a limited collection of conserved signaling pathways that integrate with tissue specific gene regulatory networks. The best understood mechanism for integration involves transcriptional interactions, in which combinatorial control of gene expression by signaling effectors together with transcription factors dictates the appropriate developmental program. Additional opportunities for interaction present when one considers factors that can act both in the nucleus and cytoplasm. The Drosophila retinal determination gene eyes absent (eya) provides an excellent model for studying how such a protein might interact with signaling pathways in multiple locations within a cell. In this dissertation, I explore Eya’s interactions with the Jak/Stat pathway in the nucleus and the cytoplasm, and how these interactions differ between developmental contexts. Eya, though initially discovered in Drosophila, is conserved across metazoans and encodes a dual-function transcriptional cofactor and phosphatase that shuttles between the nucleus and cytoplasm. Eya’s nuclear role in transactivation has been extensively studied, but understanding of its functions in the cytoplasm remain limited. In Drosophila, tyrosine phosphorylation of Eya promotes its cytoplasmic accumulation, leading to the hypothesis that interactions with phosphotyrosine-based signaling networks will direct its cytoplasmic functions. In this study, I show that eya synergizes with components of the Jak/Stat pathway in photoreceptor axon targeting. This discovery both identifies a phosphotyrosine-based signaling circuit with which Eya may interact and defines a novel developmental context for Jak/Stat signaling. Further characterization of the eya-Jak/Stat genetic synergy using subcellularly restricted eya transgenes suggests that the interactions involve cytoplasmic Eya. Expanding on this, biochemical analyses in cell culture show that Eya can form protein complexes with three Jak/Stat pathway members, Hop, Socs36E and Socs44A. Structure-function experiments conclude that the Eya-Socs44A complex is mediated by direct interaction between the Src Homology 2 domain of Socs44A and tyrosine phosphorylated Eya. Subcellularly, Eya becomes redistributed into cytoplasmic punctate structures, suggesting interactions with Socs44A contribute to the dynamics of Eya localization and function within a cell. To investigate whether Eya interacts with Jak/Stat members in contexts outside the eye and if the interactions have effects on pathway signaling, genetic interactions were explored in two additional tissues, the egg chamber and wing imaginal disc. I find that eya has a novel role in regulating border cell migration during egg chamber maturation, a process which also requires Jak/Stat signaling. Interestingly, eya and effectors of the pathway no longer genetically synergize as they do in axon targeting, suggesting context-specific interactions. In the wing, I find that ectopic eya increases expression of a Jak/Stat pathway reporter, indicating that Eya can provide positive input to signaling. Characterization of this effect with subcellularly restricted eya transgenes reveals involvement of Eya’s role in the nucleus as a transcriptional coactivator. This suggests that Eya, together with its transcriptional cofactor Sine oculis (So), may regulate activity of the pathway by altering gene expression, for example members of the Jak/Stat signaling pathway. Transcriptional interactions appear mutual in which the Jak/Stat pathway may also augment Eya-So transcriptional output, as effectors of the pathway synergize with ectopic eya to induce expression of the Eya-So target dachshund. Taken all together, my work identifies novel interactions between Eya and the Jak/Stat signaling pathway, and suggests that the mechanism and consequence of these interactions differ depending on the tissue. This work sets forth a model in which gene networks integrate with signaling pathways both during cytoplasmic signal transduction and downstream transcriptional regulation to generate context-dependent cellular behaviors.

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