The role of Cdx factors in zebrafish neural crest regionalization

项目摘要 这项提议的总体目标是研究CDX蛋白在形成新生神经脊的模式中的作用。 (NC)沿主体轴。NC是一个广泛迁移的多潜能细胞群体，并产生 不同寻常的细胞类型阵列。而大量证据表明，颅骨和躯干NC细胞(NCC) 表现出不同的细胞行为、分化和潜在的转录网络， 建立这些差异的机制仍然没有得到解决。提出的战略是 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 沿前后(AP)轴阵列NC时的系数。CDX功能将在斑马鱼中进行研究 胚胎，因为它们是实时可视化NCC行为的理想选择，并且非常适合基因组方法。 此外，强大的转基因工具和复杂的遗传方法使之成为可能。 标记和修饰特定的细胞类型并评估基因功能。已公布的和初步的结果导致了 我的中心假设是斑马鱼CDX蛋白抑制头颅NC身份，并促进 制定主干数控开发方案。CDX蛋白在AP模式形成中起保守作用 并已被证明可以促进脊髓认同感，抑制后脑发育计划 斑马鱼。鉴于CDX蛋白在形成神经上皮图案化中的作用，我建议将它们作为 建立颅脑和躯干NCC的区别。为了评估这一假设，在目标1中，我将 确定CDX是否调整特定于轴的NC特征。特别是，我会确定损失和 CDX功能的获得利用单个平面影响轴向特异性细胞行为和分化潜能 照明显微镜及相关方法。在目标2中，我将全面描述CDX表达的细节 在NC开发期间使用荧光报告器，并利用一种新的方法来描述基因组基因座 由NCC谱系中的CDX4结合。从这一数据中，我希望确定CDX4和CDX4直接监管的目标 确定CDX如何建立躯干NC基因调控网络。该项目在使用小说方面具有创新性。 转基因方法、尖端显微镜和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 潜在的转录网络。这项拟议的研究具有重要意义，因为它解决了一个长期存在的 以及该领域的根本性问题。重要的是，这个项目将提供一个理想的培训机会 支持我的长期职业目标，即成为一名专注于 研究发育过程中的图案化和细胞命运调节，并开发创新的方法和 使科学探索成为可能的技术。 好了！

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.! !