Control of cell number in developing retina

描述(申请人提供)：我的实验室的长期科学目标是了解决定视网膜细胞数量的分子机制。以果蝇的复眼为实验模型，我的实验室发现了一种新的信号通路--河马通路，它通过协调调节细胞增殖和细胞死亡来控制视网膜细胞的数量。在上一个项目期间，我的实验室所做的工作使我们能够描绘出一个由Ste20样激酶河马(Hpo)、NDR家族激酶疣(WTS)和转录共激活因子York kie(Yki)组成的河马激酶级联。HPO磷酸化并激活WTS，WTS进而通过在关键残基(S168)将后者磷酸化并将其排除在核之外而使YKI失活，在核中它通常作为TEAD/TEF家族转录因子扇形(SD)的辅助激活因子。河马途径通过转录调控靶基因，如细胞周期调节因子E和细胞死亡抑制因子DIAP1，促进细胞死亡，抑制细胞增殖。哺乳动物的Hpo、Sav、WTS和yki的同源物构成了一个类似的激酶级联，哺乳动物的Hippo途径在器官大小控制中发挥着保守的作用。最近，我们发现Kibra(KBR)是一种新的肿瘤抑制因子，它与Merlin(Mer)和相关的FERM结构域蛋白Expanded(Ex)一起发挥调节河马激酶级联反应的功能。由于HPO上游的信号事件仍不清楚，我们对这一新蛋白复合体的鉴定为研究河马信号通路中这一鲜为人知的方面提供了新的机会。在接下来的项目期间，我们建议在这些发现的基础上，通过以下几个方面的研究进一步阐明河马途径的组成、功能和调控。首先，我们的目标是定义一个完整的河马信号通路，通过对河马通路的其他组成部分进行全基因组RNAi筛选和遗传筛选，将信息从细胞外环境传递到yki磷酸化。其次，我们将确定缺失的调控河马靶基因转录的DNA结合转录因子(S)，因为我们之前对SD和YKI的表征表明，YKI可能与额外的DNA结合转录因子合作来调控河马靶基因的表达。我们将通过进行系统的蛋白质-蛋白质和蛋白质-DNA相互作用筛选来检验这一假设。最后，我们将研究KBR-Ex-Mer复合体在河马途径中发挥作用的分子和细胞机制。除了揭示眼睛发育的基本机制外，拟议中的研究还将对其他组织的发育具有普遍意义。 与公共卫生相关：拟议的研究不仅将使我们能够阐明调控视网膜细胞数量的基本分子机制，而且还将提供关于动物发育过程中其他器官中细胞数量是如何决定的，以及这一过程的异常调控如何导致组织萎缩或肿瘤发生的一般性见解。这种洞察力可能有助于对相关人类疾病的治疗干预，包括视网膜疾病。

DESCRIPTION (provided by applicant): The long-term scientific goal of my laboratory is to understand the molecular mechanisms that specify retina cell number. Using the compound eye of Drosophila as an experimental model, my laboratory has discovered a novel signaling pathway, the Hippo pathway, which controls retina cell number by coordinately regulating cell proliferation and cell death. Work from my laboratory in the last project period has allowed us to delineate a Hippo kinase cascade comprised of the Ste20-like kinase Hippo (Hpo), the NDR family kinase Warts (Wts), and the transcriptional co activator Yorkie (Yki). Hpo phosphorylates and activates Wts, which in turn, inactivates Yki by phosphorylating the latter at a critical residue (S168) and excluding it from the nucleus, where it normally functions as a co activator for the TEAD/TEF family transcription factor Scalloped (Sd). The Hippo pathway promotes cell death and restricts cell proliferation through the transcriptional regulation of target genes such as the cell cycle regulator cyclin E and the cell death inhibitor diap1. The mammalian homologues of Hpo, Sav, Wts and Yki constitute an analogous kinase cascade and that the mammalian Hippo pathway plays a conserved role in organ size control. Most recently, we have discovered Kibra (Kbr) as a novel tumor suppressor that functions together with Merlin (Mer) and the related FERM domain protein Expanded (Ex) to regulate the Hippo kinase cascade. Since signaling events upstream of Hpo still remain poorly defined, our identification of this novel protein complex provides new opportunities to investigate this less understood aspect of the Hippo signaling pathway. In the coming project period, we propose to build on these findings to further elucidate the composition, function and regulation of the Hippo pathway, through the following lines of research. First, we aim to define a complete Hippo signaling pathway that relays information from the extracellular milieu to Yki phosphorylation by conducting a genome-wide RNAi screens and genetic screens for additional components of the Hippo pathway. Second, we will identify the missing DNA-binding transcription factor(s) that regulates Hippo target gene transcription, since our previous characterization of Sd and Yki suggests that Yki may partner with additional DNA-binding transcription factors to regulate the expression of Hippo target genes. We will test this hypothesis by conducting systematic protein-protein and protein-DNA interaction screens. Lastly, we will investigate the molecular and cellular mechanisms by which the Kbr-Ex-Mer complex functions within the Hippo pathway. Besides revealing fundamental mechanisms of eye development, the proposed studies will have general implications for the development of other tissues. PUBLIC HEALTH RELEVANCE: The proposed studies will not only allow us to elucidate the basic molecular mechanism that regulates retina cell number, but also provide general insights into how cell number is determined in other organs during animal development and how aberrant regulation of this process could lead to tissue atrophy or tumorigenesis. Such insights may facilitate the therapeutic interventions of relevant human diseases, including diseases of the retina.