Control of Cell Proliferation and Differentiation in the Developing Retina

描述(申请人提供)：这项资助的长期目标是阐明细胞内在转录因子和发育信号通路在视网膜发育过程中控制细胞命运、细胞增殖和分化的机制。这项研究将以果蝇作为模型系统进行，因为果蝇和哺乳动物系统之间控制视网膜发育的基因和途径在功能上高度保守，而且很容易在苍蝇系统中结合生化、遗传、细胞和发育方法进行活体研究。在果蝇视网膜发育过程中，原神经基因无性系(ATO)的表达是视网膜前体细胞启动神经元分化的关键。ATO的表达最初由ATO 3‘增强子上调，由ATO 5’增强子提纯。对ATO 3‘增强子的分析表明，ATO受视网膜决定(RD)网络蛋白的直接调控，包括无眼(Ey)、无眼(Eya)和正弦(SO)。我们的初步研究表明，ATO 3‘增强子也受bHLH蛋白的正向和负向调节。在形态发生沟之后，Notch信号通过抑制ATO的表达负向调节光感受器的分化，同时促进第二有丝分裂波的S期。我们发现，在SMW的S期，Da也是必需的，而且Da和Notch信号都是通过CycE光盘增强子来调节SMW中CycE的表达。根据我们的初步结果，我们推测不同的bHLH二聚体参与了ATO‘增强子的激活或抑制，这是基于它们与RD蛋白的相互作用。同样，特定的Da二聚体也参与激活SMW中的CycE光盘增强子，这是基于它们与Notch信号的相互作用。此外，ATO 3‘增强子的激活也为研究RD因子的功能和调节提供了一个良好的体内系统。为了验证这些假说，并利用果蝇系统来研究RD网络蛋白，我们有三个具体的目的：1.研究Da调节ATO 3‘增强子的机制。2.利用果蝇模型研究Ey/Pax6的功能和调控。3.确定Da和Notch信号协同调控SMW细胞增殖和光感受器分化的机制。从果蝇身上获得的知识可以应用到哺乳动物的系统中，并将为控制正常视网膜发育过程中的细胞增殖和分化提供新的机械性见解。了解对正常视网膜发育的控制对于最终开发预防、诊断和治疗视网膜疾病以及其他涉及细胞增殖或分化缺陷的人类疾病的新方法至关重要。 与公共健康相关：这项研究将为正常视网膜发育过程中细胞命运规范、细胞增殖和分化的控制提供新的机械性见解。了解正常视网膜发育的控制对于最终开发预防、诊断和治疗视网膜疾病的新方法至关重要。

DESCRIPTION (provided by applicant): The long term objective of this grant is to elucidate the mechanisms by which cell intrinsic transcription factors and developmental signaling pathways interact in the control of cell fate specification, cell proliferation, and differentiation during retinal development. This investigation will be carried out using Drosophila as a model system because of the high functional conservations of the genes and pathways controlling retinal development between the Drosophila and the mammalian systems and because of the ease of combining biochemical, genetic, cellular, and developmental approaches in the fly system to carry out in vivo studies. During Drosophila retinal development, expression of the proneural gene Atonal (ato) is critical for retinal progenitor cells to initiate neuronal differentiation. Ato expression is initially upregulated by the Ato 3' enhancer and is refined by the Ato 5' enhancer. Dissection of the Ato 3' enhancer revealed that Ato is directly regulated by retinal determination (RD) network proteins Eyeless (Ey), Eyes absent (Eya), and Sine oculis (So). Our preliminary studies revealed that Ato 3' enhancer is also regulated by bHLH proteins both positively and negatively. Posterior to the morphogenetic furrow (MF), Notch signaling negatively regulates photoreceptor differentiation by inhibiting Ato expression and simultaneously promotes S phase in the second mitotic wave (SMW). We show that Da is also required for S phase in the SMW and that both Da and Notch signaling regulate CycE expression in the SMW through the CycEdisc enhancer. We hypothesize based on our preliminary results that different bHLH dimers are involved in the activation or inhibition of the Ato 3' enhancer based on their interactions with the RD proteins. Similarly specific Da dimers are also involved in the activation of the CycEdisc enhancer in the SMW based on their interactions with the Notch signaling. In addition, the activation of Ato 3' enhancer also provides a nice in vivo system to investigate the function and regulation of RD factors. To test these hypotheses and to use the Drosophila system to characterize the RD network proteins, we have three Specific Aims: 1. Characterize the mechanisms by which Da regulates Ato 3' enhancer. 2. Characterize the function and regulation of Ey/Pax6 using the Drosophila model. 3. Determine the mechanisms by which Da and Notch signaling coordinate the control of cell proliferation and photoreceptor differentiation in the SMW. Knowledge obtained from this study in Drosophila can be applied to the mammalian systems and will provide new mechanistic insights into the control of cell proliferation and differentiation during normal retinal development. Understanding the control of normal retinal development is critical for the eventual development of new approaches for the prevention, diagnosis, and treatment of retinal diseases as well as other human diseases involving cell proliferation or differentiation defect. PUBLIC HEALTH RELEVANCE: This research will provide new mechanistic insights into the control of cell fate specification, cell proliferation and differentiation during normal retinal development. Understanding the control of normal retinal development is critical for the eventual development of new approaches for the prevention, diagnosis, and treatment of retinal diseases.