Retinal morphogenesis in zebrafish

描述（由申请人提供）：脉络膜裂隙闭合过程中的畸形是视网膜形态发生的重要步骤，是先天性视力障碍的重要原因。Hh信号在CF的形态发生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根本性的差距。我们发现Zic2a是一种保守的锌指转录因子，是斑马鱼前脑和视网膜中hh调控基因表达的关键调节剂，并证明Zic2a在CF形态发生中起重要作用。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效应物并详细分析其在CF形成过程中的功能，这是本提案的目标。我们已经证明Zic2a在靠近CF的视柄中表达，并非细胞自主地促进CF的形成。基于这些数据，我们假设Zic2a通过Hh信号控制驱动OS和CF形态发生的差异细胞动力学，并且Zic2a调节OS/视网膜边界形成过程中具有关键功能的基因的转录。这一假设将通过追求两个特定目标来验证：(1)表征形成OS/视网膜边界的细胞动力学，并分析Hh信号和Zic功能在调节这些动力学中的作用；(2)确定Zic2a在视柄和CF中的转录靶点，并测试其在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检查正常发育期间和Zic功能和Hh信号中断的胚胎中CF形态发生期间的细胞动力学。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轴细胞，并使用高通量测序（RNA-seq）鉴定需要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过表达试验和工程锌指核酸酶的靶向诱变相结合来评估。这项工作意义重大，因为它将确定一个非常重要的遗传网络的新组成部分，该网络控制视网膜形态发生，并由hedgehog信号协调。这种方法是创新的，因为它整合了分子，遗传和细胞生物学的方法，在一个实验生物体，斑马鱼，来检查一个新的功能，Zic2a在CF形态发生。这种方法将从根本上推进我们对一个重要的突出问题的理解：在视网膜发育过程中Hh信号如何协调模式和形态发生，并将为分析Zics的其他重要但知之甚少的功能，包括它们在神经管闭合，神经元再生和肿瘤发生中的作用奠定坚实的基础。

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.