Gene Function Profiling of Neural Crest Cell Diversification

神经嵴细胞多样化的基因功能分析

• 批准号: 8513967
• 负责人: CHRISTINE E BEATTIE
• 金额: $ 18.3万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-19 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513967
• 关键词: Accounting; Address; Biological Models; Candidate Disease Gene; Cells; Complementary DNA; Coupled; Data; Development; Developmental Process; Diagnosis; Diagnostic; Disease; Ectoderm; Elements; Embryo; Embryonic Development; Fluorescence; Foundations; Future; Gene Combinations; Gene Components; Gene Expression; Gene Expression Profile; Gene Family; Genes; Genetic; Genetic Programming; Genome; High Prevalence; Human; Human Genetics; Learning; Malignant Neoplasms; Mediating; Messenger RNA; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Neoplasm Metastasis; Neural Crest; Neural Crest Cell; Neuroglia; Neurons; Nucleic Acid Regulatory Sequences; Oligonucleotide Microarrays; Organism; Pattern; Peripheral Nervous System; Phenotype; Pigments; Play; Population; Prevention; Process; Regulation; Regulator Genes; Reporter; Research; Research Design; Role; Sampling; Selection Criteria; Skeleton; Source; Specific qualifier value; Staging; Stem cells; System; Tissues; Transgenic Organisms; Undifferentiated; Vertebrates; Whole Organism; Work; Zebrafish; base; blastomere structure; body system; cell type; clinically relevant; craniofacial; gain of function; gastrulation; gene function; human disease; in vivo; insight; member; molecular marker; mutant; novel; precursor cell; prevent; progenitor; transcription factor

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.

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。神经嵴（NC）已被广泛研究，以阐明发育过程中细胞多样化的机制。 NC 是脊椎动物胚胎前体细胞的离散且看似同质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、色素细胞和颅面骨骼的主要元素，其中其他的。原肠胚形成过程中外胚层 NC 结构域的诱导后，NC 细胞亚群的命运被指定为不同的亚系，最终产生祖细胞群的完整细胞衍生谱系。 NC 亚系的命运在发育过程中是如何确定的尚不完全清楚。在基因水平上确定 NC 细胞之间的差异是如何建立的，对于理解 NC 如何产生如此大量的不同细胞类型至关重要。对斑马鱼和其他脊椎动物的研究表明，几种转录因子对于 NC 亚系的不同和重叠子集的规范，尽管没有一个可以单独解释 NC 细胞的整体多样性。我们在斑马鱼foxd3中发现了这一点； tfap2a 双突变体所有 NC 亚系均未能被指定，表明foxd3 和 tfap2a 对于 NC 多样化的启动具有协同作用且普遍需要。此外，我们的 研究表明，NC 亚系的初始规范需要 Foxd3 和 tfap2a，部分原因在于它们对 SoxE 家族基因 sox9a、sox9b 和 sox10 的 NC 表达的调节。总之，这些结果确定了启动 NC 多样化的基因调控网络 (GRN) 框架。然而，重要的是，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 GRN 框架无法全面解释 NC 多样化。因此，我们基于已建立的GRN框架提出了一项研究计划，以回答有关NC亚谱系命运规范的遗传调控的关键未解决问题，最终产生NC多样性。我们将采用基于 ChIP 的方法与转基因报告基因相结合，在分子水平上确定 GRN 转录因子框架之间的相互作用指定 NC 细胞命运的机制。此外，我们将根据选择标准，全面鉴定额外的 Foxd3 和 tfap2 依赖性基因，这些基因是使用全基因组微阵列表达谱控制 NC 多样化的 GRN 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）的基因的单突变或双突变胚胎，使用功能丧失和获得功能方法来确定这些候选者在调节NC多样化中的功能。 ) 结合 NC 发育的综合表型分析。我们提出的研究结果将通过在我们对数控多样化监管的理解方面产生重大的根本性进展来解决该领域的关键缺陷。此外，我们的结果将产生适用的机制范式，以普遍理解细胞多样化，并为未来控制完整 GRN 的 NC 开发的全面功能确定提供丰富的基础。最后，鉴于 NC 开发过程中的失误导致临床相关疾病的发生率很高，我们的结果可能为诊断、治疗和预防人类疾病（例如神经脆病和 NC 起源的癌症）的策略提供重要见解。