Genetic control of neuronal progenitor proliferation in zebrafish

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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.