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

• 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
• 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

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.

项目摘要 隔膜是一种关键的骨骼肌，将胸腔与腹腔分开并驱动 灵感阶段呼吸。隔膜发育的缺陷导致先天性diaphragmatragmatic Hernias （CDH），一种常见的结构性先天缺陷（3,000个出生），腹部含有腹部丘脑的胸腔， 阻塞肺发育并导致50％的新生儿死亡率。尽管功能意义 diaphragm和CDH的患病率，疝气形成的生物物理特性和 细胞外基质（ECM）对隔膜结构完整性的贡献在很大程度上没有探索。这 提案将检验以下假设：ECM组织中的缺陷导致结缔组织的改变 与疝气相关的刚度（AIM 1），确定结缔组织成纤维细胞是否是关键来源 ECM的（AIM 2），并识别由转录因子GATA4调节的ECM组件，一种基因强烈 与CDH相关（AIM 3）。我最近的工作证明了主要肌肉关键调节器中的突变是如何的 祖细胞迁移导致隔膜具有部分肌肉，结缔组织的区域缺乏肌肉 但是批判性地不疝。通过直接比较椎间盘突出的结缔组织（使用先前建立的 CDH的模型）到保持其结构完整性的娱乐区域，我将研究生物力学 diaphragm中与拉伸强度相关的特性以及ECM组成和组织。 这项研究将确定关键ECM组件的胚胎来源（K99），阐明胶原蛋白和如何 弹性蛋白交联会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（R00）刚度的候选者。提出的实验将 为我提供小鼠遗传学，原子力显微镜和质谱法的宝贵训练。在下面 加布里埃尔·卡尔登（Gabrielle Kardon）博士的指导，我将获得过渡到过渡到的经验和培训 独立的学术职位。为了进一步发展我的职业发展，我将在会议上介绍我的研究， 指导学生，参加相关课程并发布我的发现。我组装的K99指导委员会， 由Drs组成。杰夫·魏斯（Jeff Weiss），弗拉基米尔·赫拉迪（Vladimir Hlady），柯克·汉森（Kirk Hansen），贝努瓦·布鲁诺（Benoit Bruneau）和克里斯汀·夸（Kristen Kwan）将提供 在细胞和离体组织中执行生物物理测量的必要专业知识，将其结合在一起 测量隔膜的有限元模型，表征了隔膜的ECM曲线 结缔组织具有定量精度，并分析发育中的GATA4转录网络 膜片。获得独立奖的途径将使我能够追求雄心勃勃的研究计划 研究结缔组织结构完整性在肌肉骨骼发育中的结缔组织结构完整性。