Gene Function Profiling of Neural Crest Cell Diversification

描述(由申请人提供)：确定基因组合如何作为基因调控网络相互作用以产生细胞多样性是理解发育的基础。为了阐明发育过程中细胞多样化的机制，对神经脊(NC)进行了广泛的研究。NC是脊椎动物胚胎前体细胞的一个离散的、看似均一的未分化干细胞样外胚层群体，是多种不同细胞类型的来源，包括周围神经系统的神经元和神经胶质细胞、色素细胞和头面部骨骼的主要元素等。在原肠形成过程中诱导外胚层的NC结构域之后，NC细胞亚群的命运被指定为不同的亚系，最终产生完整的祖细胞群体的细胞派生谱系。 NC亚系的命运是如何在发展过程中被指定的，目前还不完全清楚。在遗传水平上确定NC细胞之间的差异是如何建立的，这对于理解NC是如何产生如此大量的不同细胞类型是至关重要的。对斑马鱼和其他脊椎动物的研究表明，几种转录因子对 NC子系的不同子集和重叠子集的规范，尽管没有一个子集可以单独说明NC细胞的整体多样性。我们在斑马鱼foxd3中发现； TFAP2A双突变体所有NC亚系都未能明确，表明foxd3和TFAP2A是启动NC多样化所需的协同和普遍的基因。此外，我们的 研究表明，foxd3和TFAP2A对NC亚系初始规范的要求部分是由于它们对SoxE家族基因sox9a、sox9b和sox10的NC表达的调节。总之，这些结果确定了一个框架基因调控网络(GRN)，它启动了NC的多样化。然而，关键的是，框架GRN转录因子相互作用启动NC多样化的机制尚不清楚。同样重要的是，已确定的框架GRN不能完全解释NC多样化。因此，我们在已建立的框架GRN的基础上提出了一个研究计划，以回答关于NC亚系命运规范的遗传调控的关键悬而未决的问题，这些基因调控最终产生NC多样性。我们将在分子水平上，使用基于芯片的方法和转基因记者，确定框架之间的GRN转录因子相互作用决定NC细胞命运的机制。此外，我们将利用全基因组微阵列表达谱综合鉴定其他依赖foxd3和tfap2a的基因，根据选择标准，这些基因是GRN控制NC多样化的候选基因。然后，我们将使用转基因报告野生型胚胎和包含框架GRN(foxd3、TFAP2A、sox9a、sox9b和sox10)的基因的单个或双突变的胚胎，结合NC发育的全面表型分析，确定这些候选基因在调节NC多样化方面的功能。我们拟议的研究结果将通过在我们对NC多样化的监管的理解方面取得重大根本性进展来解决该领域的严重缺陷。此外，我们的结果将产生适用的机制范式，以普遍了解细胞的多样性，并为未来全面确定完整的GRN控制NC的发展提供丰富的基础。最后，考虑到NC发展过程中失误导致的临床相关疾病的高发，我们的结果可能为诊断、治疗和预防人类疾病(如神经临界疾病和NC起源的癌症)提供重要的见解。

DESCRIPTION (provided by applicant): Determining how combinations of genes interact as gene regulatory networks to produce cellular diversity is fundamental to understanding development. The neural crest (NC) has been studied extensively to elucidate mechanisms of cell diversification during development. The NC is a discrete and seemingly homogeneous undifferentiated stem cell-like ectodermal population of vertebrate embryonic precursor's cells that is the source of multiple different cell types including neurons and glia of the peripheral nervous system, pigment cells and major elements of the craniofacial skeleton, among others. Subsequent to the induction of the NC domain of the ectoderm during gastrulation, the fates of subsets of NC cells are specified as distinct sublineages that ultimately generate the complete cellular derivative repertoire of the progenitor population. How the fates of NC sublineages are specified during development is incompletely understood. Determining at the genetic level how differences between NC cells are established is essential to understanding how the NC generates such a vast array of different cell types. Studies in zebrafish and other vertebrates have indicated that several transcription factors are essential for the specification of distinct and overlapping subsets of NC sublineages, although none can individually account for NC cell diversification in its entirety. We found that in zebrafish foxd3; tfap2a double mutants all NC sublineages fail to be specified, indicating that foxd3 and tfap2a are synergistically and universally required for the initiation of NC diversification. Further, our studies indicate that the requirement for foxd3 and tfap2a for the initial specification of NC sublineages is due in part to their regulation of the NC expression of the SoxE family genes sox9a, sox9b and sox10. Together, these results have identified a framework gene regulatory network (GRN) that initiates NC diversification. Critically, however, the mechanisms by which framework GRN transcription factor interactions initiate NC diversification are not known. Equally important, the identified framework GRN cannot account for NC diversification in its entirety. Accordingly, we propose a research plan, based on the established framework GRN, to answer critical unresolved questions about the genetic regulation of the specification of NC sublineage fates which ultimately produces NC diversity. We will determine at the molecular level, employing a ChIP-based approach coupled with transgenic reporters, the mechanisms by which interactions between the frameworks GRN transcription factors specify NC cell fates. In addition, we will comprehensively identify additional foxd3- and tfap2adependent genes that, based on selection criteria, are candidates for the GRN controlling NC diversification using whole genome microarray expression profiling. We will then determine the functions of these candidates in regulating NC diversification using loss- and gain-of function approaches employing transgenic reporter wild type embryos and embryos singly or doubly mutant for genes comprising the framework GRN (foxd3, tfap2a, sox9a, sox9b and sox10) coupled with comprehensive phenotypic analysis of NC development. The results of our proposed studies will address critical deficiencies in the field by producing major fundamental advances in our understanding of the regulation of NC diversification. In addition, our results will generate applicable mechanistic paradigms for understanding cell diversification generally and provide a rich foundation for future comprehensive functional determination of the complete GRN controlling NC development. Lastly, given the high prevalence of clinically relevant conditions resulting from miscues during NC development, our results are likely to provide important insights for strategies to diagnose, treat and prevent human diseases such as neurocristopathies and cancers of NC origin.