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，以阐明在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均质的未分化干细胞样外胚层群体，其是多种不同细胞类型的来源，包括外周神经系统的神经元和神经胶质、色素细胞和颅面骨骼的主要元素等。在原肠胚形成期间诱导外胚层的NC结构域之后，NC细胞亚群的命运被指定为最终产生祖细胞群体的完整细胞衍生物库的不同亚系。 如何在发展过程中指定的NC亚系的命运是不完全理解。在遗传水平上确定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。在斑马鱼和其他脊椎动物中的研究表明，几种转录因子是必需的， NC亚系的不同和重叠子集的规范，虽然没有一个可以单独占NC细胞多样化的整体。我们发现，在斑马鱼foxd 3; tfap 2a双突变体所有NC亚系未能被指定，表明foxd 3和tfap 2a是协同作用的，普遍需要的NC多样化的起始。此外，我们的 研究表明，NC亚系的初始特化需要foxd 3和tfap 2a部分是由于它们调节SoxE家族基因sox 9a、sox 9 b和sox 10的NC表达。总之，这些结果已经确定了一个框架基因调控网络（GRN），启动NC多样化。然而，至关重要的是，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，确定的框架GRN不能占NC多样化的全部。因此，我们提出了一个研究计划，基于已建立的框架GRN，回答关键的未解决的问题，NC亚系命运的规范，最终产生NC多样性的遗传调控。我们将确定在分子水平上，采用基于ChIP的方法加上转基因报告，GRN转录因子的框架之间的相互作用指定NC细胞命运的机制。此外，我们将全面确定额外的foxd 3和tfap 2a依赖基因，根据选择标准，是候选人的GRN控制NC多样化使用全基因组微阵列表达谱。然后，我们将确定这些候选人的功能，在调节NC多样化使用损失和增益的功能的方法，采用转基因报告野生型胚胎和胚胎单或双突变体的基因，包括框架GRN（foxd 3，tfap 2a，sox 9a，sox 9 b和sox 10）加上全面的表型分析NC发展。我们提出的研究结果将解决该领域的关键缺陷，产生重大的根本性进展，我们的理解NC多样化的监管。此外，我们的研究结果将产生适用的机制范式，了解细胞多样化一般，并提供了一个丰富的基础，为未来的全面功能确定的完整的GRN控制数控开发。最后，鉴于NC发展过程中的失误导致的临床相关疾病的高患病率，我们的研究结果可能为诊断，治疗和预防人类疾病（如NC起源的神经损伤和癌症）的策略提供重要的见解。