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如何整合来自多个信号通路的指令并以适当的方式作出反应的机制。这些信号通路的不受控制或失调导致人类肿瘤发生和/或发育缺陷，反映了精确编排的信号在所有生物过程中发挥的关键作用。我们的方法是利用遗传易处理的果蝇系统来揭示新的策略，通过保守的信号转导级联聚集在核转录因子网络上，指导视网膜发育过程中的细胞增殖、命运规范、分化、形态发生和存活。由于发育信号机制在进化过程中一直高度保守，对果蝇分子电路的了解将从根本上推进我们对细胞命运如何被指定和维持，以及为什么调控不当导致哺乳动物癌症和疾病的理解。本提案的目标是探索一种新的信号整合机制，该机制是由我们正在进行的协调视网膜规范和发育的转录调节网络研究提出的。特别是Eyes absent (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，有可能通过指导下游基因表达模式来响应上游信号输入，并通过其磷酸酶功能影响这些相同途径的活性。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介导的转录调节的关系仍知之甚少。该提案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Eya的两个基本功能（一个作为核转录因子，一个作为磷酸酪氨酸信号网络的组成部分）的适当协调和调节对视网膜发育至关重要的假设。该研究计划的成功完成将为Eya的PTP功能所需的细胞和发育背景提供深入的见解，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。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Eyes absent （Eya）参与的磷酸酪氨酸信号通路，并研究在体外表达筛选中发现的新结合伙伴对Eyes absent的转录因子和磷酸酶两种功能的贡献。并验证在细胞核和细胞质之间缺乏功能的眼睛的空间划分有助于视网膜正常发育的假设。因为我们正在研究的蛋白质和途径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。公共卫生相关性：基因功能的不适当调控是广泛的人类发育异常和疾病（包括癌症）的基础。这项研究的目的是揭示在眼睛正常发育过程中调节关键决定的细胞机制，作为理解当这些关键控制丧失时出现的疾病和缺陷并最终开发有效治疗干预措施的重要的第一步。

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.