Dll4 Gene Regulation and Function during Retinogenesis

描述(由申请人提供)：本提案的长期目标是了解导致不同视网膜细胞类型的确定和分化的分子事件。在哺乳动物视网膜发生过程中，在各种内在和外在因素的作用下，有7类细胞是由多能祖细胞特异性产生的。最近涉及功能丧失和功能获得的分子遗传学研究发现，许多转录因子和信号分子是视网膜发生的关键调节因子。这些因子被发现在不同的发育过程中发挥作用，以建立祖细胞的多能性，定义祖细胞的能力，决定细胞的命运，和/或指定细胞类型和亚型。因此，转录因子和信号分子在视网膜形成过程中控制细胞的特化和分化起着至关重要的作用。然而，尽管有这些重要的进展，许多转录因子下游的分子靶点和信号事件仍然知之甚少，这两类分子之间的相互作用在很大程度上仍有待确定。在这一应用中，建议进行实验，重点研究DLL4-Notch信号调节的分子和发育事件，以及DLL4和FOXN4翼状螺旋转录因子之间的遗传关系。我们的初步数据和以前的工作使我们认为DLL4在视网膜细胞类型的指定和分化中可能具有介导Notch信号的特定作用，并且它可能作为FOXN4的直接靶点来调节其在视网膜形成过程中的部分功能。本申请的主要目的是通过追求四个具体目标来检验这一假设：i)测试FOXN4作为DLL4表达的直接上游调控因子。我们的目标是证实FOXN4在调控Notch信号分子中的特异性，并研究FOXN4是否具有通过分子、生物信息学和生物化学相结合的方法直接结合和激活DLL4启动子的能力；ii)通过条件基因打靶研究DLL4在视网膜形成过程中的生物学功能。我们的目标是建立视网膜和阶段特异性的DLL4基因敲除小鼠，并在解剖学、细胞和分子水平上对它们进行功能鉴定，以确定DLL4-Notch信号调节的发育和细胞过程；iii)通过过度表达分析研究DLL4-Notch信号在视网膜形成过程中的作用。我们的目标是使用过表达的方法来确定DLL4及其显性-阴性形式对视网膜细胞指定和分化的可能的细胞自主和非细胞自主的影响；以及iv)绘制表达DLL4的视网膜前体细胞的命运图。我们的目标是使用Cre-loxP命运映射策略确定表达DLL4的视网膜前体细胞的谱系。这些拟议的研究有望为调控基因网络和信号事件提供重要的见解，这些事件是决定和分化不同类型视网膜细胞的基础。 公共卫生相关性：拟议的研究旨在阐明涉及视网膜细胞规范和分化的遗传调控网络和信号事件，从而为更好地理解和治疗致盲的神经视网膜疾病奠定基础。特别是，它们与实现从干细胞中控制再生所需的视网膜细胞类型以达到治疗目的高度相关。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the molecular events that lead to the determination and differentiation of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered numerous transcription factors and signaling molecules as pivotal regulators of retinogenesis. These factors are found to act at various developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, both transcription factors and signaling molecules play essential roles in controlling cell specification and differentiation during retinogenesis. Despite these important advances, however, the molecular targets and signaling events downstream from many transcription factors still remain poorly understood and the interplay between these two classes of molecules remains largely to be determined. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by Dll4-Notch signaling as well as on the genetic relationship between Dll4 and the Foxn4 winged- helix transcription factor. Our preliminary data and previous work have led us to propose that Dll4 may have a specific role in mediating Notch signaling in the specification and differentiation of retinal cell types and that it may act as a direct target of Foxn4 to mediate part of its function during retinogenesis. This application primarily aims to test this hypothesis by pursuing four specific aims: i) to test Foxn4 as a direct upstream regulator of Dll4 expression. Our goal is to confirm the specificity of Foxn4 in regulating Notch signaling molecules and to investigate whether Foxn4 has the ability to directly bind and activate the Dll4 promoter by a combination of molecular, bioinformatic and biochemical approaches; ii) to study the biological function of Dll4 during retinogenesis by conditional gene targeting. We aim to generate retina- and stage-specific Dll4 knockout mice and functionally characterize them at anatomical, cellular and molecular levels to identify the developmental and cellular processes that Dll4-Notch signaling regulates; iii) to investigate Dll4-Notch signaling events during retinogenesis by overexpression analysis. We aim to employ an overexpression approach to determine the presumed cell-autonomous and non-cell-autonomous effects of Dll4 and its dominant-negative form on retinal cell specification and differentiation; and iv) to map the fate of Dll4- expressing retinal progenitors. Our goal is to determine the lineages of Dll4-expressing retinal progenitors using the Cre-loxP fate-mapping strategy. The proposed studies together are expected to provide important insights into the regulatory gene network and signaling events that underlie the determination and differentiation of different retinal cell types. PUBLIC HEALTH RELEVANCE: The proposed studies aim to elucidate the genetic regulatory network and signaling events involved in retinal cell specification and differentiation, and thus will lay the foundation for a better understanding and treatment of blinding neuroretinal diseases. In particular, they are highly relevant to achieving controlled regeneration of desired retinal cell types from stem cells for therapeutic purposes.