Yevgenya Grinblat - US grants

[unreadable] DESCRIPTION (provided by applicant): The staggering functional and anatomical complexity of the vertebrate brain arises gradually during embryonic development from an initially uniform field of cells. We would like to understand the early steps of brain development, in particular, formation of asymmetry along the dorsal/ventral axis of the brain primordium. This study will address the roles of two related transcription factors, zic2 and zic5, during dorsal brain formation. Important functions for both genes have already been demonstrated in several vertebrates, but their mechanisms are not understood. In humans, mutations in zic2 and zic5 have been causally linked to two prevalent birth defects: exencephaly and holoprosencephaly. Expression of zic genes is restricted to the dorsal portion of the neural tube, and this restriction is critical for their correct function. In spite of their obvious importance, the molecular mechanisms of regulation and function of vertebrate zic genes are not well understood. We have obtained exciting preliminary evidence that zic2 and zic5 regulate transcription of wnt1, a gene with essential functions during dorsal brain formation, and that Wnt signaling in turn regulates transcription of zics. These data have led us to propose that wnt and zic genes are involved in a regulatory feedback loop. This novel hypothesis will help explain at least some of the defects observed in zic mutants, and will be tested in a different model system, the zebrafish. Zebrafish embryos are available during all stages of brain development, are easy to observe and manipulate, and have a short generation time. As a result, powerful genetic, genomic and embryological methods have been established for use in this model organism. Strong evolutionary conservation of the zic gene family ensures that this study will uncover shared molecular mechanisms that operate during brain formation in all vertebrates, including humans. Ultimately, this work will contribute to a better understanding of the mechanisms underlying birth defects that affect embryonic development of the vertebrate brain. [unreadable] [unreadable]

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. PUBLIC HEALTH RELEVANCE: 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.

PROJECT SUMMARY/ABSTRACT Embryonic cells divide rapidly while acquiring more restricted fates. There is urgent need to understand how proliferation and fate acquisition controls are coordinated, especially in the context of the developing brain. Embryonic programs that generate neurons, once deciphered, can be stimulated therapeutically in the adult brain, offering new treatments for traumatic brain injury and degenerative disease, health burdens with few available treatment options. Zebrafish is a uniquely powerful model for these studies as it allows individual cells to be observed in living embryos. To harness this potential of the zebrafish, it is necessary first to engineer new transgenic zebrafish strains that encode in vivo cell cycle reporters and conditional mutations in key genes. Zic2, a member of the conserved Zic (zinc finger in the cerebellum) gene family, functions in embryonic stem cells and several embryonic cell lineages, including proliferating neural progenitors in zebrafish. Our previous work supports the hypothesis that zebrafish zic2 function coordinates proliferation and cell fate acquisition in neuronal progenitors. We will test this hypothesis by pursuing two specific aims. Our first Aim is to establish transgenic mitotic cell cycle reporters for interrogating zic2 function in controlling neuronal proliferation. We will engineer transgenic zebrafish to express live cell-cycle reporters encoded by FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) under the control of zic2 transcriptional enhancers. We will use this newly constructed line and confocal imaging of living embryos to analyze cell cycle in individual cells. We will then combine FUCCI transgenics with our established zic2 mutant strains to ask how absence of zic2 function disrupts these normal cell cycle dynamics. Our second Aim is to develop conditional alleles to investigate zic2 function during neuronal specification. We will use the Cre/Lox system together with CRISPR/Cas9 short homology- directed integration to generate (1) floxed alleles of endogenous Zic2 genes and (2) hormone-inducible Cre lines under the control of zic2 transcriptional enhancers. We will use these new zebrafish strains to ask when during development zic2 function is required in neuronal progenitors. This work will generate tools necessary for understanding the role of Zic2 in neurogenesis, enabling future studies that combine single- cell live imaging with single cell transcriptomics and genomics. Importantly, these unique and powerful tools will allow temporally controlled mutagenesis of other genes of interest in cell lineages where zebrafish zic2 genes are expressed, both during development and post-embryonically. In addition to the brain, these include retinal and glial components of the eye and neural-crest cells that generate craniofacial structures of the face. This work will inform disease mechanism investigations broadly and in multiple body systems.