Biophysical and genetic mechanisms underlying diaphragm morphogenesis and Congenital Diaphragmatic Hernias

项目摘要 横膈膜是一种关键的骨骼肌，它将胸腔和腹腔分开， 吸气相呼吸。先天性膈疝是由膈肌发育缺陷引起的 (CDH)，一种常见的结构性出生缺陷（每3,000名新生儿中有1名），即腹部内容物疝入胸腔， 阻碍肺部发育并导致50%的新生儿死亡。尽管功能的重要性， 隔膜和CDH的患病率，疝形成的生物物理特性和 细胞外基质（ECM）对横膈膜的结构完整性的贡献在很大程度上未被探索。这 一项提案将检验ECM组织缺陷导致结缔组织改变的假设 与疝相关的僵硬（目的1），确定结缔组织成纤维细胞是否是一个关键来源 的ECM（目的2），并确定ECM成分的转录因子GATA 4，一个基因强烈调控 与CDH相关（Aim 3）。我最近的工作证明了肌肉关键调节因子的突变 祖细胞迁移导致隔膜部分肌肉，其中结缔组织区域缺乏肌肉 但关键是不要疝气。通过直接比较疝出的结缔组织（使用先前建立的 CDH模型）的肌肉区域，保持其结构完整性，我将研究生物力学 特性以及ECM成分和与隔膜中的拉伸强度相关的组织。 这项研究将确定关键ECM成分（K99）的胚胎来源，阐明胶原蛋白和 弹性蛋白交联影响隔膜中的组织力学（K99，R 00）并表征治疗 影响隔膜结缔组织成纤维细胞硬度的候选物（R 00）。拟议的实验将 在老鼠遗传学、原子力显微镜和质谱学方面给我提供了宝贵的培训。下 加布里埃尔·卡顿博士的指导，我将获得必要的经验和培训，过渡到一个 独立的学术地位。为了促进我的职业发展，我将在会议上展示我的研究， 指导学生，参加相关课程，并发表我的研究结果。我召集的K99导师委员会 由Jeff韦斯博士、弗拉基米尔Hlady博士、Kirk汉森博士、Benoit Bruneau博士和Kristen Kwan博士组成， 在细胞和离体组织中进行生物物理测量所需的专业知识，包括这些 测量到隔膜的有限元模型中，表征隔膜的ECM轮廓 结缔组织的定量精度，并分析GATA 4转录网络的发展 隔膜独立之路奖将使我能够从事一项雄心勃勃的研究计划 研究ECM对肌肉骨骼发育中结缔组织结构完整性的调节。

Project Summary The diaphragm is a critical skeletal muscle that separates the thoracic from the abdominal cavity and drives the inspiration phase respiration. Defects in diaphragm development cause congenital diaphragmatic hernias (CDH), a common structural birth defect (1 in 3,000 births) where abdominal contents herniate into the thorax, obstructing lung development and leading to 50% neonatal mortality. Despite the functional significance of the diaphragm and the prevalence of CDH, the biophysical properties underlying hernia formation and the contribution of extracellular matrix (ECM) to the structural integrity of the diaphragm is largely unexplored. This proposal will test the hypothesis that defects in ECM organization lead to alterations in connective tissue stiffness associated with hernias (Aim 1), determine whether connective tissue fibroblasts are a critical source of ECM (Aim 2), and identify ECM components regulated by the transcription factor GATA4, a gene strongly associated with CDH (Aim 3). My recent work has demonstrated how mutations in key regulators of muscle progenitor migration lead to a diaphragm with partial muscle, where regions of connective tissue lack muscle but critically do not herniate. By directly comparing herniated connective tissue (using a previously established model of CDH) to amuscular regions that maintain their structural integrity, I will investigate biomechanical properties as well as ECM composition and organization associated with tensile strength in the diaphragm. This research will identify the embryonic source of key ECM components (K99), clarify how collagen and elastin crosslinking impacts tissue mechanics in the diaphragm (K99, R00) and characterize therapeutic candidates affecting stiffness of diaphragm connective tissue fibroblasts (R00). The proposed experiments will provide me with valuable training in mouse genetics, atomic force microscopy, and mass spectrometry. Under the mentorship of Dr. Gabrielle Kardon, I will gain the experience and training necessary to transition to an independent academic position. To further my career development, I will present my research at conferences, mentor students, attend relevant courses, and publish my findings. My assembled K99 mentorship committee, composed of Drs. Jeff Weiss, Vladimir Hlady, Kirk Hansen, Benoit Bruneau and Kristen Kwan, will provide the necessary expertise to perform biophysical measurements in cells and ex vivo tissues, incorporate these measurements into finite element models of the diaphragm, characterize the ECM profile of diaphragm connective tissue with quantitative precision, and analyze the GATA4 transcriptional network in the developing diaphragm. The Pathway to Independence Award will enable me to pursue an ambitious research program investigating ECM regulation of connective tissue structural integrity in musculoskeletal development.