Neural Crest and Cardiac Outflow Tract Formation

(改编自申请者摘要)应聘者是一名医学博士，具有广泛的 在儿科、新生儿-围产期医学和 儿科心脏病学。他在分子和生物科学领域拥有丰富的研究经验 他目前正在寻求密集的基础研究 培训以过渡到具有最终目标的独立调查员 成为一名学术内科科学家。在颁奖时， 候选人将是费城儿童医院的教职员工 还是宾夕法尼亚大学的儿科学讲师。 这项建议集中在胚胎心脏发生的一个关键时刻 流出道的分离和形成是一个过程 涉及到神经脊的贡献。发育缺陷发生在 这一时期导致了大量的先天性心脏缺陷。 阐明心脏流出道形成的机制将有助于 提高我们对这些疾病的认识。初步实验 由申请人执行的目的是识别已知的和 新基因在流出道间隔时在心脏中被激活 开始(E11.5)。其中一个克隆(Clone 35)编码一种未知功能的蛋白质 这是通过进化来保守的。它具体表达在 流出道内和神经沟来源的间充质内 主-肺间隔。这里提出了对此进行深入的分析。 新基因及其在神经脊中的潜在作用 介导的心脏形态发生。此外，还创造了一种新的转基因小鼠 在赞助商的实验室里将被用来分析神经脊的命运 几个患有先天性心脏病的突变小鼠的细胞。特定的 目的是：1)深入分析《克隆人35》。这将包括隔离 全长cdna，广泛的表达分析，制备抗体 允许亚细胞定位、基因组染色体作图和生成 该基因靶向缺失的转基因小鼠。2)细胞命运 先天缺陷小鼠突变系神经脊细胞的检测 心脏病(斑点、补丁、NFL和崩溃3个突变胚胎)通过使用 表达Pax3启动子近端Cre重组酶的转基因小鼠 在神经脊细胞中直接表达，以及适当的Cre-Report 老鼠。

(Adapted from applicant's abstract) The candidate is an MD with extensive medical training in the fields of pediatrics, neonatal-perinatal medicine and pediatric cardiology. He has acquired research experience in molecular and developmental biology and he is currently seeking intensive basic research training to transition to an independent investigator with the ultimate goal to become an academic physician scientist. At the time of the award the candidate will be a faculty member of the Children's Hospital of Philadelphia and Instructor in Pediatrics at the University of Pennsylvania. This proposal focuses on a critical time during embryonic cardiogenesis in which septation and formation of the outflow tract take place, a process involving neural crest contribution. Developmental defects occurring during this period account for a large number of congenital heart defects. Elucidation of the mechanisms involved in cardiac outflow tract formation will enhance our understanding of these diseases. Preliminary experiments performed by the applicant were aimed towards the identification of known and novel genes activated in the heart at the time when outflow tract septation begins (E11.5). One of them (Clone 35) encodes a protein of unknown function that is conserved through evolution. It is expressed specifically in the outflow tract and in neural crest derived mesenchyme at the time of aortopulmonary septation. Here, it is proposed an in depth analysis of this novel gene with particular attention to its potential role in neural crest mediated cardiac morphogenesis. In addition, a novel transgenic mouse created in the sponsor's laboratory will be used to analyze the fate of neural crest cells in several mutant mouse lines with congenital heart disease. Specific aims are: 1) In depth analysis of Clone 35. This will include isolation of full length cDNA, extensive expression analysis, preparation of antibodies to allow subcellular localization, genomic chromosomal mapping, and generation of transgenic mice with targeted deficiency of this gene. 2) Cell fate determination of neural crest cells in mouse mutant lines with congenital heart disease (Splotch, Patch, Nfl and collapsing 3 mutant embryos) by using transgenic mice expressing Cre-recombinase from the proximal Pax 3 promoter, that directs expression in neural crest cells, and appropriate Cre-reporter mice.