Molecular Interactions During Neural Crest Formation

描述（由申请人提供）：神经嵴干细胞参与人类疾病，包括肿瘤和颅面和心脏畸形。它们产生周围神经系统的大部分细胞、颅面骨和软骨、心脏流出道的细胞和黑素细胞以及其他衍生物。研究神经嵴的基本发育生物学是更好地理解，诊断和治疗这些人类疾病的关键一步。这项建议的长期目标是研究在神经嵴细胞形成过程中发挥作用的分子相互作用在尽可能早的阶段。因此，必须确定诱导的时间、地点和细胞或组织。最近已经表明，上胚层的一个区域被指定在原肠胚形成期间形成神经嵴细胞，这些相同的区域将在不久之后表达Pax 7，并且Pax 7是神经嵴形成所需的。鉴于这些发现，建议调查何时，何地以及如何神经嵴细胞的诱导和指定，虽然经典的胚胎学和分子生物学方法相结合。具体目标是：1）鉴定参与神经嵴诱导和特化的时间、位置和组织; 2）表征BMP、FGF和WNT分子在神经嵴形成中的参与;和3）表征神经嵴形成期间Pax 7分子相互作用。这些目标是由一组可检验的假设，分析神经嵴细胞的发育在囊胚/原肠胚阶段，挑战他们的诱导通过神经和非神经/中胚层相互作用的公认大炮，并分析在分子水平上的参与三个信号通路和Pax 7的作用模式。体内和体外实验将记录神经嵴发育的进展与一组分子标记物。为此，将通过从待测试的上胚层组织中将胶原外植体和移植物移植到幼稚胚胎环境中来获得特化和诱导图谱（具体目标1）;不同的BMP、FGF和WNT信号传导条件（包括组合的激活和抑制方法，沿着特定的细胞自主和非细胞自主试剂），将在诱导前后应用于预期和非预期神经嵴组织（具体目标2）;最后，我们将描述Pax 7和Ubc 9之间的相互作用，以及它们在神经嵴发育过程中的作用（具体目标3）。

DESCRIPTION (provided by applicant): Neural crest stem cells are involved in human illnesses including tumors and craniofacial and heart malformations. They generate most of the cells of the peripheral nervous system, craniofacial bone and cartilage, cells of the outflow tract of the heart and melanocytes amongst other derivatives. Studying the basic developmental biology of the neural crest is a critical step to better understand, diagnose, and treat these human conditions. The long term objective of this proposal is to study the molecular interactions at play during neural crest cell formation at the earliest possible stages. Therefore it is imperative to identify the time, place and cells or tissues involved in their induction. It has been recently shown that a region of the epiblast is specified to form neural crest cells during gastrulation, these same region will express Pax7 soon after, and that Pax7 is required for neural crest formation. In light of these findings it is proposed to investigate when, where and how neural crest cells are induced and specified though a combination of classic embryology and molecular biology approaches. The specific aims are: 1) identify time, location and tissues involved in the induction and specification of the neural crest; 2) characterize the participation of BMP, FGF and WNT molecules in neural crest formation; and 3) Characterize Pax7 molecular interactions during neural crest formation. These aims are guided by a set of testable hypotheses that analyze neural crest cell development at the blastula/gastrula stage, challenge the accepted cannon of their induction through neural and non-neural/mesodermal interactions, and analyze at the molecular level the participation of three signaling pathways and the mode of action of Pax7. In vivo and in vitro experiments will record the progression of neural crest developments with a battery of molecular markers. To this end, specification and induction maps will be obtained through collagen explants and grafts to naive embryonic environments from epiblast tissues to be tested (specific aim 1); distinct BMP, FGF and WNT signaling conditions (including combinatorial activation and inhibition approaches, along with specific cell autonomous and non-cell autonomous reagents), will be applied to prospective and non-prospective neural crest tissues before and after induction (specific aim 2); and finally we will characterize the interaction between Pax7 and Ubc9, and their role during neural crest development, (specific aim 3).