Biophysical and genetic mechanisms underlying diaphragm morphogenesis and Congenital Diaphragmatic Hernias

项目摘要 横隔膜是一种关键的骨骼肌，它将胸腔与腹膜分开，并驱动 吸气期呼吸。横隔膜发育缺陷导致先天性横隔膜疝气 (CDH)，一种常见的结构性出生缺陷(每3,000名新生儿中有1名)，腹部内容物突出进入胸腔， 阻碍肺部发育并导致50%的新生儿死亡。尽管具有重要的功能意义， 横隔膜与先天性巨结肠的患病率、腹股沟形成的生物物理特性以及腹股沟的 细胞外基质(ECM)对横隔膜结构完整性的贡献在很大程度上是未知的。这 该提案将检验ECM组织缺陷导致结缔组织改变的假设 与疝气有关的僵硬(目标1)，确定结缔组织成纤维细胞是否为关键来源 并鉴定受转录因子GATA4调控的ECM组分，GATA4是一个强烈的基因 与CDH相关(目标3)。我最近的工作证明了肌肉关键调节因子的突变是如何 祖细胞迁移导致具有部分肌肉的横隔膜，在那里结缔组织区域缺乏肌肉 但关键的是不要突出。通过直接比较突出的结缔组织(使用先前建立的 CDH模型)到保持其结构完整性的肌肉区域，我将研究生物力学 性能以及与隔膜中的抗拉强度相关的ECM成分和组织。 这项研究将确定关键的细胞外基质成分(K99)的胚胎来源，阐明胶原蛋白和 弹性蛋白交联对横隔膜组织力学的影响(K99，R00)和治疗性 影响横隔膜结缔组织成纤维细胞硬度的候选细胞(R00)。拟议中的实验将 在老鼠遗传学、原子力显微镜和质谱学方面为我提供有价值的培训。在……下面 在加布里埃尔·卡登博士的指导下，我将获得过渡到 独立的学术立场。为了进一步发展我的事业，我将在会议上展示我的研究成果， 指导学生，参加相关课程，并发表我的发现。我召集的K99指导委员会， 由杰夫·韦斯博士、弗拉基米尔·赫莱德博士、柯克·汉森博士、伯努瓦·布鲁诺博士和克里斯汀·关博士组成的 在细胞和体外组织中进行生物物理测量所需的专业知识，包括 对膜片的有限元模型进行测量，表征膜片的ECM轮廓 结缔组织的精确定量，并分析GATA4在发育中的转录网络 横隔膜。独立之路奖将使我能够追求一个雄心勃勃的研究计划 研究ECM对肌肉骨骼发育过程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.