Biophysical and genetic mechanisms underlying diaphragm morphogenesis and Congenital Diaphragmatic Hernias

膈肌形态发生和先天性膈疝的生物物理和遗传机制

基本信息

批准号：
10224292
负责人：
Elizabeth Marie Sefton
金额：
$ 13.39万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224292
关键词：
Abdomen Abdominal Cavity Affect Atomic Force Microscopy Award Binding Biological Models Biomechanics Biophysical Process Biophysics Birth Breathing Cell Culture Techniques Cells ChIP-seq Chest Collagen Congenital Abnormality Congenital diaphragmatic hernia Connective Tissue Data Defect Development Down-Regulation Elastic Fiber Elasticity Elastin Elements Embryo Embryology Embryonic Structures Enzymes Etiology Exhibits Extracellular Matrix Extracellular Matrix Proteins Fibroblasts GATA4 gene Gene Expression Genes Genetic Genetic Transcription Genomic Segment Hernia Human In Vitro Knockout Mice LOX gene Lead Liver Mass Spectrum Analysis Measurement Mechanics Mentors Mentorship Modeling Morphogenesis Mus Muscle Musculoskeletal Development Musculoskeletal Diseases Mutagenesis Mutation Neonatal Mortality Nerve Pathway interactions Phase Positioning Attribute Prevalence Production Property Publishing Regulation Repression Research Research Support Respiration Respiratory Diaphragm Role Skeletal Muscle Source Structural Congenital Anomalies Structure Surface Tensile Strength Testing Thoracic cavity structure Tissues Training Transcriptional Regulation Work base biophysical analysis biophysical properties career development confocal imaging crosslink experience experimental study fibulin-4 liquid chromatography mass spectrometry lung development migration mortality mouse genetics overexpression progenitor programs recruit skills student mentoring symposium therapeutic candidate transcriptome sequencing

项目摘要

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.

项目概要膈肌是重要的骨骼肌，将胸腔与腹腔分开并驱动吸气阶段呼吸。膈肌发育缺陷导致先天性膈疝 (CDH)，一种常见的结构性出生缺陷（每 3,000 名新生儿中就有 1 例），腹部内容物疝入胸腔，阻碍肺部发育并导致 50% 的新生儿死亡率。尽管具有功能意义膈肌和 CDH 的患病率、疝气形成的生物物理特性以及细胞外基质（ECM）对隔膜结构完整性的贡献在很大程度上尚未被探索。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相关的僵硬（目标 1），确定结缔组织成纤维细胞是否是关键来源 ECM（目标 2），并鉴定受转录因子 GATA4（一种强烈的基因）调节的 ECM 成分与 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如何祖细胞迁移导致隔膜具有部分肌肉，其中结缔组织区域缺乏肌肉但关键不要疝出。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，我将研究生物力学特性以及与隔膜拉伸强度相关的 ECM 成分和组织。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、R00）并表征治疗影响隔膜结缔组织成纤维细胞硬度（R00）的候选者。拟议的实验将为我提供了小鼠遗传学、原子力显微镜和质谱方面的宝贵培训。在下面在 Gabrielle Kardon 博士的指导下，我将获得过渡到独立的学术地位。为了进一步发展我的职业发展，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发现。我组建的K99导师委员会，由博士组成。 Jeff Weiss、Vladimir Hlady、Kirk Hansen、Benoit Bruneau 和 Kristen Kwan 将提供在细胞和离体组织中进行生物物理测量所需的专业知识，将这些结合起来测量隔膜的有限元模型，表征隔膜的 ECM 轮廓定量精确的结缔组织，并分析发育中的 GATA4 转录网络隔膜。独立之路奖将使我能够追求雄心勃勃的研究计划研究肌肉骨骼发育中结缔组织结构完整性的 ECM 调节。