The role of connective tissue in regulating muscularization and innervation of the diaphragm

项目摘要 横隔膜是一种重要的骨骼肌，在呼吸过程中起着至关重要的作用，起着屏障的作用。 将胸腔和腹腔分开。膜片的发展需要集成 来自不同胚胎来源的肌肉、结缔组织、肌腱、骨骼和神经。中的缺陷 横隔肌肌化可导致先天性横隔膜疝气(CDH)，这是一种常见的出生缺陷(1/2 3,000名新生儿)，肝脏突出进入胸腔，阻碍肺部发育，导致50% 新生儿死亡率。横隔膜的发育需要肌肉前体从体节迁移 以及将膈神经轴突(神经支配的唯一来源)延伸至结缔组织前体细胞。 然而，尽管CDH的发生率很高，而且隔膜的功能很重要，但细胞 相互作用和分子信号指导肌肉迁移和横隔膜所需的轴突引导 肌化和神经支配在很大程度上是未知的，也是这项提议的主题。我们的实验室最近 证明肌肉和结缔组织之间的相互作用对正常发育至关重要。 横隔肌和这种相互作用中的缺陷是CDH的一个来源。肌肉结缔组织 起源于暂时性的胚胎结构，称为胸膜腹膜皱褶(PPFS)。这个项目的重点是 PPFS，尤其是PPFS分泌的肝细胞生长因子(HGF)在导向中的作用 肌祖细胞和膈神经连接到发育中的横隔膜。初步数据显示 Met在膈肌祖细胞中的表达和HGF在PPF成纤维细胞中的表达提示PPF来源 HGF在将MET+肌祖细胞招募到发育中的横隔膜中起重要作用。然而，更早的一次 符合从体节分离祖细胞的要求排除了对这一点的明确测试 假设。目标1将确定哪些体节是横隔肌的来源并检验这一假设 HGF是一种重要的PPF来源的分泌信号，它关键地调节肌肉向 横跨横隔膜。在肌肉祖细胞迁移后通过有条件地删除PPF中的HGF 从体节，我将测试HGF/MET信号在肌肉祖细胞迁移到横隔膜中的作用。 初步数据表明，PPF来源的HGF的丢失扰乱了正常的肌化和膈神经的引导 神经轴突进入横隔膜。目标2将检验PPF和肌肉衍生信号是 引导膈神经轴突进入和穿过横隔膜所必需的。我会决定是否 胸膜腹膜皱褶在培养中具有趋化特性，并表征了细胞的耗竭 包括肝细胞生长因子在内的分泌信号影响膈神经轴突的导向。总而言之，这些研究 利用小鼠强大的遗传学来确定结缔组织前体细胞在肌肉迁移和 轴突对发育中的横隔膜的引导，并为了解结缔组织如何 缺陷可导致先天性横隔膜疝气。

Project Summary The diaphragm is an essential skeletal muscle, playing a critical role in respiration and serving as a barrier that separates the thoracic and abdominal cavities. Development of the diaphragm requires the integration of muscle, connective tissue, tendon, bone, and nerves that arise from different embryonic sources. Defects in muscularization of the diaphragm cause congenital diaphragmatic hernias (CDH), a common birth defect (1 in 3,000 births) where the liver herniates into the thorax, impeding lung development and resulting in a 50% neonatal mortality rate. Diaphragm development entails the migration of muscle precursors from the somites and the extension phrenic nerve axons (the sole source of innervation) to connective tissue progenitors. However, in spite of the high incidence of CDH and functional importance of the diaphragm, the cellular interactions and molecular signals directing muscle migration and axon guidance required for diaphragm muscularization and innervation are largely unknown and the subject of this proposal. Our lab has recently demonstrated that interactions between muscle and connective tissue are crucial for normal development of the diaphragm muscle and defects in this interaction are a source of CDH. The muscle connective tissue originates from transient embryonic structures, termed the pleuroperitoneal folds (PPFs). This project focuses on the role of PPFs and, in particular, Hepatocyte Growth Factor (HGF) secreted by the PPFs, in guiding muscle progenitors and the phrenic nerve to the developing diaphragm. Preliminary data showing the expression of Met in the diaphragm's muscle progenitors and Hgf in PPF fibroblasts suggests that PPF-derived HGF is important for recruiting MET+ muscle progenitors into the developing diaphragm. However, an earlier MET requirement for delamination of progenitors from the somites has precluded an explicit test of this hypothesis. Aim 1 will determine which somites are the source of diaphragm muscle and test the hypothesis that HGF is an important PPF-derived secreted signal that critically regulates migration of muscle into and throughout the diaphragm. By conditional deletion of Hgf in PPFs after muscle progenitors have emigrated from the somite, I will test the role of HGF/MET signaling in muscle progenitor migration into the diaphragm. Preliminary data suggest that loss of PPF-derived Hgf disrupts normal muscularization and guidance of phrenic nerve axons into the diaphragm. Aim 2 will test the hypothesis that PPF and muscle derived signals are required for guiding phrenic nerve axons to and throughout the diaphragm. I will determine whether the pleuroperitoneal folds have chemoattractant properties in culture and characterize how the depletion of secreted signals, including Hgf, affect the guidance of phrenic nerve axons. Taken together, these studies exploit the powerful genetics of mice to define the role of connective tissue progenitors in muscle migration and axon guidance to the developing diaphragm and establish a basis for understanding how connective tissue defects can contribute to congenital diaphragmatic hernias.