Cell-Cell Signaling in Embryonic and Retinal Development

描述(由申请人提供)：这项工作的长期目标是阐明细胞整合来自多个信号通路的指令并以适合背景的方式做出反应的机制。这些信号通路的不受控制或失调的活动会导致人类肿瘤的发生和/或发育缺陷，这反映了精确编排的信号在所有生物过程中所起的关键作用。我们的方法是利用遗传上易驯化的果蝇系统来发现新的策略，在视网膜发育过程中，保守的信号转导级联聚合在核转录因子网络上，指导细胞增殖、命运指定、分化、形态发生和生存。由于发育信号机制在进化中高度保守，了解果蝇使用的分子电路将从根本上提高我们对细胞命运是如何指定和维持的，以及为什么调控不当会导致哺乳动物癌症和疾病的理解。这项建议的目标是探索一种新的信号整合机制，这是我们正在进行的对编排视网膜规范和发育的转录调控网络的研究提出的。尤其是EYA，通过其作为转录因子和蛋白酪氨酸磷酸酶(PTP)的不寻常的双重功能，既可以通过引导下游的基因表达模式来响应上游信号输入，又可以通过其磷酸酶功能来影响这些相同通路的活性。尽管Eya PTP功能受损与包括眼部缺陷在内的人类疾病有关，但Eya PTP活性的正常生物学功能及其与Eya介导的转录调控的关系仍不清楚。这项建议描述了一种结合遗传、生化和细胞生物学分析的多方面方法，以解决以下假设：适当协调和调节Eya的两个基本功能，一个是作为核转录因子，另一个是作为磷酸酪氨酸信号网络的组成部分，对视网膜发育至关重要。拟议研究计划的成功完成将提供对Eya PTP功能所需的细胞和发育背景的洞察，并将揭示Eya、视网膜决定基因网络和其他对眼睛发育至关重要的信号模块之间的串扰新机制。其具体目的是通过探索与Abelson(Abl)和Src64酪氨酸激酶的遗传和生化相互作用，阐明EYA参与的磷酸酪氨酸信号通路，研究体外表达筛选中发现的新的结合伙伴对EYA转录因子和磷酸酶两项功能的贡献，并验证EYA在细胞核和细胞质之间的空间分割有助于视网膜的正常发育。因为我们正在研究的蛋白质和通路在哺乳动物中具有保守的功能，我们的工作揭示的新的信号策略将促进对人类发育和疾病的理解。与公共卫生相关：对基因功能的不适当调控是包括癌症在内的一系列人类发育异常和疾病的基础。这项研究的目标是揭示在眼睛正常发育过程中调节关键决定的细胞机制，作为了解当这些关键控制丢失时出现的疾病和缺陷，并最终开发有效的治疗干预措施的必要的第一步。

DESCRIPTION (provided by applicant): The long-term objectives of this work are to elucidate the mechanisms whereby cells integrate instructions from multiple signaling pathways and respond in a context-appropriate manner. Reflecting the key role that precisely orchestrated signaling plays in all biological processes, uncontrolled or dysregulated activity of these pathways leads to tumorigenesis and/or developmental defects in humans. Our approach is to exploit the genetically tractable Drosophila system to uncover novel strategies whereby conserved signal transduction cascades converge on nuclear transcription factor networks to direct cell proliferation, fate specification, differentiation, morphogenesis and survival during retinal development. Because developmental signaling mechanisms have been highly conserved in evolution, knowledge of the molecular circuitries used in Drosophila will fundamentally advance our understanding of how cell fates are designated and maintained, and why misregulation results in cancer and disease in mammals. The goal of this proposal is to explore a novel mechanism of signal integration suggested by our ongoing study of transcriptional regulatory networks that orchestrate retinal specification and development. Specifically Eyes absent (Eya), through its unusual dual functionality as transcription factor and protein tyrosine phosphatase (PTP), has the potential both to respond to upstream signaling inputs by directing downstream patterns of gene expression and to influence the activity of these same pathways through its phosphatase function. Although impaired Eya PTP function has been implicated in human disease, including ocular defects, the normal biological function of Eya's PTP activity, and its relationship to Eya-mediated transcriptional regulation, remains poorly understood. This proposal describes a multi-faceted approach combining genetic, biochemical, and cell biological assays to address the hypothesis that proper coordination and regulation of Eya's two essential functions, one as a nuclear transcription factor and one as a component of phosphotyrosine signaling networks, is critical for retinal development. Successful completion of the proposed research plan will provide insight into the cellular and developmental contexts in which Eya's PTP function is required and should uncover novel mechanisms of cross-talk between Eya, the retinal determination gene network and other signaling modules critical for eye development. The specific aims are to elucidate the phosphotyrosine signaling pathways in which Eyes absent (Eya) participates by exploring genetic and biochemical interactions with the Abelson (Abl) and Src64 tyrosine kinases, to investigate the contribution of new binding partners identified in an in vitro expression screen with respect to Eyes absent's two functions as transcription factor and phosphatase, and to test the hypothesis that spatial partitioning of Eyes absent function between nucleus and cytoplasm contributes to proper retinal development. Because the proteins and pathways we are studying have conserved functions in mammals, the new signaling strategies revealed by our work will advance understanding of human development and disease. PUBLIC HEALTH RELEVANCE: Inappropriate regulation of gene function underlies a broad spectrum of human developmental abnormalities and diseases, including cancer. The goal of this research is to reveal the cellular mechanisms that regulate critical decisions during normal development of the eye, as an essential first step toward understanding the diseases and defects that arise when these key controls are lost and eventually toward developing effective therapeutic interventions.