Genetic control of neuronal progenitor proliferation in zebrafish

项目总结/摘要 胚胎细胞分裂迅速，同时获得更多的限制命运。迫切需要了解如何 增殖和命运获得控制是协调的，特别是在发育中的大脑的情况下。 胚胎时期产生神经元的程序一旦被破译，就可以在成年人身上进行治疗性刺激。 大脑，为创伤性脑损伤和退行性疾病提供新的治疗方法， 可用的治疗方案。斑马鱼是这些研究的一个独特的强大模型，因为它允许个体 在活胚胎中观察到的细胞。为了利用斑马鱼的这种潜力，首先需要 设计新的转基因斑马鱼品系，其编码体内细胞周期报告基因和条件突变 在关键基因中。Zic 2是小脑锌指基因家族的一员，其功能是在小脑中表达锌指蛋白。 胚胎干细胞和几种胚胎细胞谱系，包括增殖的神经祖细胞， 斑马鱼我们先前的工作支持了斑马鱼zic 2功能协调的假设， 神经元祖细胞的增殖和细胞命运获得。我们将通过以下方式来验证这一假设： 两个具体目标。我们的第一个目标是建立转基因有丝分裂细胞周期报告基因， ZIC 2在控制神经元增殖中的功能。我们将设计转基因斑马鱼， 由FUCCI（基于荧光泛素化的细胞周期指示剂）编码的细胞周期报告基因， ZIC 2转录增强子的控制。我们将使用这个新构建的线和共焦成像， 活胚胎来分析单个细胞的细胞周期。然后我们将联合收割机FUCCI转基因与我们的 建立了zic 2突变株，以研究zic 2功能的缺失是如何破坏这些正常细胞周期的 动力学我们的第二个目标是开发条件等位基因，以研究zic 2在 神经元特化我们将使用Cre/Lox系统与CRISPR/Cas9短同源- 定向整合以产生（1）内源性Zic 2基因的floxed等位基因和（2）可诱导的Cre 在ZIC 2转录增强子控制下的细胞系。我们将用这些新的斑马鱼品系来询问 在发育过程中，神经元祖细胞需要ZIC 2功能。这项工作将产生工具， 这对于理解Zic 2在神经发生中的作用是必要的，使得未来的研究能够将联合收割机与单一的 单细胞转录组学和基因组学的细胞活成像。重要的是，这些独特而强大的工具 将允许在斑马鱼ZIC 2 基因在发育期间和胚胎后都表达。除了大脑， 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嵴细胞， 脸上这项工作将广泛地为疾病机制研究提供信息，并在多个身体系统中进行。

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.