Genetic control of neuronal progenitor proliferation in zebrafish

项目摘要/摘要 胚胎细胞快速分裂，同时获得更多受限的命运。我们迫切需要了解如何 增殖控制和命运获取控制是协调的，特别是在大脑发育的背景下。 一旦破译了产生神经元的胚胎程序，就可以在成人身上进行治疗刺激 Brain，为创伤性脑损伤和退行性疾病提供新的治疗方法，健康负担很少 可用的治疗方案。斑马鱼是这些研究的一个独特而强大的模型，因为它允许个体 活体胚胎中观察到的细胞。为了利用斑马鱼的这种潜力，首先有必要 设计新的转基因斑马鱼品系，编码体内细胞周期报告和条件突变 在关键基因中。Zic2是Zic(小脑中的锌指)基因家族中保守的成员，在 胚胎干细胞和几种胚胎细胞谱系，包括增殖的神经前体细胞 斑马鱼。我们之前的工作支持斑马鱼zic2功能坐标的假设 神经前体细胞的增殖和细胞命运的获得。我们将通过以下方式来验证这一假设 有两个明确的目标。我们的第一个目标是建立转基因有丝分裂细胞周期报告程序 ZIC2在控制神经元增殖中发挥作用。我们将改造转基因斑马鱼来表达活的 Fucci(基于荧光泛素化的细胞周期指示器)编码的细胞周期报告 ZIC2转录增强子的调控。我们将使用这条新构建的直线和共聚焦成像 活胚胎来分析单个细胞的细胞周期。然后我们将把Fucci转基因技术与我们的 已建立的zic2突变株询问zic2功能的缺失如何扰乱这些正常的细胞周期 动力学。我们的第二个目标是开发条件等位基因来研究zic2在 神经元规格。我们将使用CRE/Lox系统和CRISPR/Cas9短同源- 定向整合产生(1)内源Zic2基因的花等位基因和(2)激素诱导的Cre 在zic2转录增强子控制下的品系。我们将利用这些新的斑马鱼品种来询问 在发育过程中，神经前体细胞需要zic2功能。这项工作将生成工具 了解Zic2在神经发生中的作用是必要的，使未来的研究能够结合单一的- 单细胞转录组学和基因组学的细胞活体成像。重要的是，这些独特而强大的工具 将允许对斑马鱼zic2细胞谱系中其他感兴趣的基因进行时间控制的突变 基因在发育过程中和胚胎发育后都有表达。除了大脑，这些 包括眼睛的视网膜和神经胶质成分，以及产生颅面结构的神经脊细胞 那张脸。这项工作将为疾病机制研究提供广泛和多个身体系统的信息。

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.