Cellular and Molecular Analysis of Body Wall Closure

哺乳动物腹侧体壁关闭缺陷，包括腹裂、胸腹裂和 脐膨出与腹侧体壁、羊膜和脐环的形态发生有关。 腹侧体壁畸形是人类出生缺陷的主要类别之一， 大约每2000个活产婴儿中就有一个。虽然这些缺陷的发生是相对的 常见的是，人们对腹体的发育和关闭缺乏了解，这一点令人惊讶。 无论是老鼠还是人的墙。这一领域的复杂性被一系列关于心力衰竭发病机制的理论所复杂化 未经实验测试的人体体壁缺陷。这项提议旨在产生一种范例 我们对哺乳动物腹侧体闭合的理解的转变，可以提供一个机械框架和 全面的资源，用于未来了解这一过程是如何因遗传和/或 环境原因。转录因子AP-2由TFAP2a基因编码，在 小鼠体壁关闭和以前的研究表明，它调节外胚层，间充质， 外周神经系统，以及驱动腹侧体壁发育的中胚层相互作用。 TFAP2A基因缺失的小鼠有一种严重的腹壁闭合缺陷，即胸腹裂，其中腹壁 胸部和腹部的覆盖物不能形成，从而使心脏、肺、肝脏和肠道暴露在外。关键是， 这是为数不多的具有完全穿透性且一致的体壁闭合缺陷的简单老鼠模型之一。 这可以用来理解这一重要的发展过程是如何失败的。在第一个目标中，相关 将在特定的胚胎时间从对照组和TFAP2A缺失的小鼠身上收集组织，并对其进行单次 细胞RNA序列(ScRNAseq)分析。这将确定小鼠体壁的正常基因表达模式 关闭以及受TFAP2A丢失影响的细胞类型和基因。其他几个基因是 已知会影响体壁关闭，我们将能够将这些基因归因于相关的组织群体 了解他们可能如何影响这一关键的发展过程。在第二个目标中，我们将 对所有控制时间点以及与突变体最相关的时间点执行scATACseq分析。 然后，我们将整合所有scRNAseq和scATACseq数据，以确定组织、细胞类型和基因 通常与体壁闭合相关的表情签名。此外，我们将确定如何 TFAP2A的缺失改变了染色质在体壁中的可及性。将执行验证分析以 确定与正常和异常相关的细胞群体和基因表达谱的关键变化 体壁闭合。随后，来自这些研究的信息将被综合成一部小说和 强大的正常体壁关闭模型，可作为未来理解的重要框架 这类重要的人类先天缺陷。

Mammalian ventral body wall closure defects, including gastroschisis, thoracoabdominoschisis, and omphalocele are linked with the morphogenesis of the ventral body wall, the amnion, and the umbilical ring. Malformations of the ventral body wall comprise one of the leading categories of human birth defects and are present in about one out of every 2000 live births. Although the occurrence of these defects is relatively common, there is a surprising lack of knowldege concerning the development and closure of the ventral body wall in mouse or human. This field is further complicated by the array of theories on the pathogenesis of human body wall defects that have not been experimentally tested. This proposal aims to produce a paradigm shift in our understanding of mammalian ventral body closure that can provide a mechanistic framework and comprehensive resource for future understanding of how this process goes awry because of genetic and/or environmental causes. The transcription factor AP-2, encoded by the gene Tfap2a, has an essential role in mouse body wall closure and previous studies have shown that it regulates ectodermal, mesenchymal, peripheral nervous system, and mesodermal interactions that drive development of the ventral body wall. Tfap2a null mice have a severe form of ventral closure defect, a thoracoabdominoschisis, in which the ventral covering of the chest and abdomen fails to form so that the heart, lungs, liver, and gut are exposed. Critically, this is one of the few simple mouse models that gives a fully penetrant and consistent body wall closure defect that can be used to understand how this important developmental process can fail. In the first Aim, relevant tissue will be collected from control and Tfap2a null mice at specific embryonic times and subjected to single cell RNA seq (scRNAseq) analysis. This will identify the normal gene expression patterns of mouse body wall closure as well as cell types and genes that are impacted by loss of Tfap2a. Several additional genes are known to affect body wall closure and we will be able to attribute these genes to relevant tissue populations to understand how they may be influencing this critical developmental process. In the second Aim, we will perform scATACseq analysis on all control time points as well as the most relevant time points for the mutant. We will then integrate all the scRNAseq and scATACseq data to identify the tissues, cell types, and gene expression signatures that are normally associated with body wall closure. Further, we will identify how chromatin accessibility in the body wall is altered by loss of Tfap2a. Verification analyses will be performed to identify critical changes in cell populations and gene expression profiles associated with normal and abnormal body wall closure. Subsequently, the information from these studies will be synthesised into a novel and powerful model for normal body wall closure that can serve as an important framework for future understanding of this important class of human birth defect.