The role of Cdx factors in zebrafish neural crest regionalization

项目摘要 本研究的总体目标是研究Cdx蛋白在新生神经嵴形成中的作用 (NC)沿着身体轴线。NC是一种多能细胞群，其广泛迁移并产生一种免疫应答。 一系列的细胞类型虽然大量证据表明，颅和躯干NC细胞（NCC） 在细胞行为、分化和潜在的转录网络方面表现出差异， 建立这些差异的机制仍然没有得到解决。该战略旨在 研究这一关键的发育问题将集中在Cdx的作用上，Cdx是一种同源结构域转录， 因素，在沿前后（AP）轴对NC进行沿着排列时。Cdx功能将在斑马鱼中进行研究 胚胎，因为这些是理想的可视化NCC行为在真实的时间和基因组方法非常适合。 此外，强大的转基因工具和复杂的遗传方法的可用性使其成为可能 来标记和修饰特定的细胞类型并评估基因功能。公布的和初步的结果导致了 我的中心假设，斑马鱼Cdx蛋白抑制颅NC身份，并促进 主干数控开发程序的建立。Cdx蛋白在AP模式中起保守作用 并已被证明可以促进脊髓的特性，抑制后脑发育程序， 斑马鱼考虑到Cdx蛋白在神经上皮细胞模式化中的作用，我建议将它们作为候选蛋白， 确定颅侧和躯干NCC之间的差异。为了评估这一假设，在目标1中，我将 确定Cdx是否调节轴向特定NC特征。特别是，我将确定是否损失和 Cdx功能的获得影响轴特异性细胞行为和分化潜能，使用单平面 照明显微镜和相关方法。在目标2中，我将充分表征Cdx表达的细节 在NC开发过程中使用荧光报告，并利用一种新的方法来分析基因组位点 在NCC谱系中被Cdx 4结合。从这些数据中，我希望确定Cdx 4的直接调节目标， 确定Cdx如何建立主干NC基因调控网络。这个项目是创新的使用新颖的 转基因方法，尖端显微镜，以及新开发的转录分析方法 因子-DNA相互作用，以研究Cdx在建立躯干NCC身份，行为和 底层的转录网络拟议的研究是重要的，因为它解决了一个长期存在的问题。 也是最基本的问题重要的是，该项目将提供理想的培训机会， 支持我的长期职业目标，成为一名独立的学术研究者，专注于 研究发育过程中的模式和细胞命运调节，开发创新方法， 这些技术将使科学探索成为可能。 !

Project Summary The overall goal of this proposal is to investigate the role of Cdx proteins in patterning the nascent neural crest (NC) along the body axis. The NC is a multipotent cell population that migrates extensively and gives rise to a remarkable array of cell types. While substantial evidence suggests that cranial and trunk NC cells (NCCs) exhibit differences in cellular behaviors, differentiation, and the underlying transcriptional network, the mechanism by which these differences are established remains unaddressed. The strategy proposed to investigate this key developmental question will be to focus on the role of Cdx, a homeodomain transcription factor, in patterning the NC along the anteroposterior (AP) axis. Cdx function will be studied in zebrafish embryos, as these are ideal for visualizing NCC behaviors in real time and well suited for genomic approaches. Moreover, the availability of powerful transgenic tools and sophisticated genetic approaches make it possible to label and modify specific cell types and evaluate gene function. Published and preliminary results have led to my central hypothesis that zebrafish Cdx proteins repress cranial NC identity, and promote the establishment of the trunk NC developmental program. Cdx proteins play a conserved role in AP patterning and have been shown to promote spinal cord identity and repress the hindbrain developmental program in the zebrafish. Given the role of Cdx proteins in patterning the neurepithelium, I propose them as candidates for establishing the differences between cranial and trunk NCCs. To evaluate this hypothesis, in Aim 1 I will determine whether Cdx regulates axial-specific NC features. In particular, I will determine whether loss and gain of Cdx function impacts axial-specific cellular behaviors and differentiation potential using Single Plane Illumination Microscopy and related approaches. In Aim 2 I will fully characterize details of Cdx expression during NC development using fluorescent reporters, and utilize a novel approach to profile the genomic loci bound by Cdx4 in the NCC lineage. From this data, I expect to identify targets of direct regulation by Cdx4 and determine how Cdx establishes the trunk NC gene regulatory network. This project is innovative in using novel transgenic approaches, cutting-edge microscopy, and newly developed methods for profiling transcription factor-DNA interactions in order to investigate the role of Cdx in establishing trunk NCC identity, behaviors, and the underlying transcriptional network. The proposed research is significant as it addresses a long-standing and fundamental question in the field. Importantly, this project will provide an ideal training opportunity to support my long-term professional goal of becoming an independent academic investigator focused on investigating patterning and cell-fate regulation during development and developing innovative approaches and technologies that will enable scientific exploration.! !