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.
The proposed research is relevant to human health because it targets a conserved but poorly understood genetic mechanism that controls an aspect of retinal morphogenesis (choroid fissure formation), particularly vulnerable to disruption during human development. This work will identify novel candidate genes for human coloboma and will set the stage for small-molecule screens to identify modulators of CF closure, with the ultimate goal of designing pharmacological interventions to alleviate coloboma-associated visual impairment.