Retinal morphogenesis in zebrafish

描述(申请人提供)：脉络膜裂隙(CF)闭合时的畸形是视网膜形态形成的重要步骤，是先天性视力障碍的重要原因。HH信号在CFs的形态发生过程中起着至关重要的作用；然而，我们对控制这一过程的遗传和细胞机制的理解存在着根本的差距。我们发现斑马鱼Zic2a是一种保守的锌指转录因子，是HH调控的前脑和视网膜基因表达的关键调节因子，并证明了Zic2a在CF形态发生中的重要作用。我们研究的长期目标是阐明控制CF形态发生的基因调控网络。实现这一目标的关键一步是确定Zic2a效应子并详细分析它们在CF形成过程中的功能，这是本提案的目标。我们的研究表明，Zic2a在邻近纤维的视柄中表达，并且非细胞自主地促进纤维的形成。基于这些数据，我们假设Zic2a通过HH信号来控制驱动OS和CF形态发生的差异细胞动力学，并且Zic2a调控OS/视网膜边界形成过程中关键功能基因的转录。这一假说将通过追求两个具体目标来验证：(1)表征OS/视网膜形成边界的细胞动力学，并分析HH信号和Zic功能在调节这些动力学过程中的作用；(2)确定视柄和CF中Zic2a的转录靶点，并测试它们在CF形态发生中的作用。在第一个目标下，将使用高分辨率实时成像来研究CFs形态发生过程中的细胞动力学，无论是在正常发育期间还是在Zic功能和HH信号中断的胚胎中。在第二个目标下，将使用荧光激活分选从Zic2a表达正常或中断的胚胎中分离视柄细胞，并使用高通量测序(RNA-seq)识别需要Zic2a功能才能在这些细胞中正确表达的基因。这些基因在CF形成过程中的功能将通过条件性过表达分析和工程锌指核酸酶的定向突变相结合的方式进行评估。这项拟议的工作意义重大，因为它将识别控制视网膜形态发生并由Hedghog信号协调的非常重要的遗传网络的新组件。这种方法是创新的，因为它在一个实验生物斑马鱼中整合了分子、遗传和细胞生物学方法，以研究Zic2a在CF形态发生过程中的新功能。这种方法将从根本上促进我们对一个重要的悬而未决的问题的理解：HH信号如何在视网膜发育过程中协调图案化和形态发生，并将为分析其他重要但鲜为人知的功能奠定坚实的基础，包括它们在神经管关闭、神经元再生和肿瘤发生中的作用。

DESCRIPTION (provided by applicant): Malformations during choroid fissure (CF) closure, an important step in retinal morphogenesis, are a significant cause of congenital visual impairment. Hh signaling plays a crucial role during CF morphogenesis; however, there is a fundamental gap in our understanding of the genetic and cellular mechanisms that control this process. We have identified a novel role for zebrafish Zic2a, a conserved zinc-finger transcription factor, as a key modulator of Hh-regulated gene expression in the forebrain and retina, and demonstrated an essential role for Zic2a in CF morphogenesis. The long-term goal of our research is to elucidate the gene regulatory network that controls CF morphogenesis. An essential step toward this goal, identification of Zic2a effectors and detailed analysis of their functions during CF formation, is the objective of this proposal. We have shown that Zic2a is expressed in the optic stalk, which is adjacent to the CF, and functions non-cell-autonomously to promote CF formation. Based on these data, we hypothesize that Zic2a functions through Hh signaling to control differential cellular dynamics that drive OS and CF morphogenesis, and that Zic2a regulates transcription of genes with key functions during OS/retinal border formation. This hypothesis will be tested by pursuing two Specific Aims: (1) Characterize cellular dynamics at the forming OS/retinal boundary and analyze the role for Hh signals and Zic function in regulating these dynamics; (2) Identify transcriptional targets of Zic2a in the optic stalk and CF, and test their roles during CF morphogenesis. Under the first aim, cellular dynamics during CF morphogenesis will be examined using high-resolution real-time imaging, both during normal development and in embryos with disrupted Zic function and Hh signaling. Under the second aim, optic stalk cells will be isolated from embryos with normal or disrupted Zic2a expression using fluorescence-activated sorting, and genes that require Zic2a function for their correct expression in these cells will be identified using high-throughput sequencing (RNA-seq). Functions of these genes during CF formation will be assessed through a combination of conditional over expression assays and targeted mutagenesis with engineered zinc finger nucleases. The proposed effort is significant because it will identify new components of a very important genetic network that controls retinal morphogenesis and is coordinated by Hedghog signaling. This approach is innovative because it integrates molecular, genetic and cell biological approaches in one experimental organism, the zebrafish, to examine a novel function for Zic2a during CF morphogenesis. This approach will fundamentally advance our understanding of an important outstanding question: how Hh signaling coordinates patterning and morphogenesis during retinal development, and will build a strong foundation for analyses of other important but poorly understood functions of Zics, including their roles in neural tube closure, neuronal regeneration and tumorigenesis.