Molecular Interactions During Neural Crest Formation

描述（由申请人提供）：神经嵴干细胞与人类疾病有关，包括肿瘤、颅面和心脏畸形。它们产生了周围神经系统的大部分细胞，颅面骨和软骨，心脏流出道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起作用的分子相互作用在尽可能早的阶段。因此，有必要确定时间、地点和参与诱导的细胞或组织。最近的研究表明，在原肠胚形成过程中，外胚层的一个区域被指定为形成神经嵴细胞，这些区域很快就会表达Pax7，并且Pax7是神经嵴形成所必需的。鉴于这些发现，建议研究何时，何地以及如何通过经典胚胎学和分子生物学方法的结合来诱导和指定神经嵴细胞。具体目的是：1)确定神经嵴诱导和规范的时间、部位和组织；2)表征BMP、FGF和WNT分子在神经嵴形成中的作用；3)表征Pax7分子在神经嵴形成过程中的相互作用。这些目标是在一系列可测试的假设的指导下进行的，这些假设分析了神经嵴细胞在囊胚/原胚阶段的发育，挑战了通过神经和非神经/中胚层相互作用诱导它们的公认理论，并在分子水平上分析了三种信号通路的参与和Pax7的作用模式。体内和体外实验将用一系列分子标记记录神经嵴发育的进展。为此，将从待测试的外胚层组织中通过胶原外植体和移植物到原始胚胎环境中获得规范和诱导图（具体目的1）；不同的BMP、FGF和WNT信号条件（包括组合激活和抑制方法，以及特定的细胞自主和非细胞自主试剂）将应用于诱导前后的前瞻性和非前瞻性神经嵴组织（特异性目标2）；最后，我们将描述Pax7和Ubc9之间的相互作用，以及它们在神经嵴发育中的作用（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).