Molecular mechanisms underlying trachea formation and the pathology of tracheomalacia and complete tracheal rings

气管形成的分子机制以及气管软化和完整气管环的病理学

• 批准号: 10543343
• 负责人: Debora Sinner
• 金额: $ 2.06万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-04 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543343
• 关键词: Affect; Biological; Cartilage; Cells; Congenital Abnormality; Cytoskeletal Modeling; Development; Diagnosis; Disease; Dorsal; Endoderm; Epithelial; Etiology; Foundations; Gene Expression; Genes; Genetic Epistasis; Goals; Intervention; Ligands; Light; Mediating; Mesenchymal; Mesenchyme; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Muscle; Myoblasts; Operative Surgical Procedures; Output; Palliative Care; Pathology; Pattern; Publishing; Regulation; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Structural defect; Testing; Therapeutic; Trachea; Tracheobronchomalacia; WNT Signaling Pathway; beta catenin; in vivo; mortality; mouse model; novel; overexpression; receptor; respiratory; transcriptome sequencing

Tracheobronchomalacia (TBM) and Complete Tracheal Rings (CTR), are congenital conditions characterized by abnormal tracheal cartilage and muscle and associated with high morbidity and mortality. The etiologies of TBM and CTR are poorly understood, and current interventions are limited to invasive surgery and palliative care. The overarching goal of the present application is to define underlying molecular mechanisms of congenital diseases affecting trachea development. Our published studies demonstrated a critical role for respiratory endoderm in determining the dorsal-ventral organization of the tracheal mesenchyme where cartilage and smooth muscle developed. Endodermal deletion of Wls (Wntless), a cargo receptor necessary for secretion of Wnt ligands, results in lack of tracheal cartilage that is replaced by abnormally organized smooth muscle, resembling TBM pathology. Our RNA Sequencing analysis identified Notum, and Bmp4, as candidate Wnt target genes in tracheal mesenchyme that regulate cartilage and trachealis smooth muscle formation downstream of epithelial Wls- induced signaling. We hypothesize that Notum acts by suppressing canonical Wnt signaling while simultaneously promoting non-canonical Wnt and BMP signaling required for tracheal mesenchyme differentiation. Thus, Notum is at the core of a novel model of regulation for the output of Wnt signaling wherein epithelial Wnt ligands first promote Wnt/b-catenin signaling and then turn it off to allow for non-canonical Wnt activity. To test the central hypothesis, we will: SA1: To determine the mechanism by which Notum promotes tracheal cartilage formation. Using novel mouse model and ex vivo studies, we will determine the extent to which deletion or overexpression of Notum affects Wnt/b-catenin dependent and independent signaling activity, and the epistasis among Notum, Wnt5a, and Wnt-b-catenin to pattern cartilage. SA2: To define the mechanism by which Bmp4 and Wnt signaling interact to mediate cell fate of tracheal mesenchyme. Performing in vivo and ex vivo studies we will determine whether Wnt/b- catenin synergizes with BMP to promote cartilage by inducing Notum and Wnt5a/Ror2 expression; and whether Notum and Bmp4 cooperate to repress ectopic muscle in ventral tracheal mesenchyme. SA3: To test the hypothesis that epithelial Wnt signaling directs cytoskeletal organization of trachealis smooth muscle. We will examine whether 1) canonical Wnt ligands from the epithelia act directly to promote myoblast cytoskeletal organization by regulating myogenic gene expression; 2) epithelial Wnt ligands activate non-canonical mesenchymal Wnt signaling working via Ror2 to mediate myoblast cytoskeletal organization. The proposed studies will identify critical molecular signaling pathways that mediate tracheal morphogenesis and will shed light on the pathology of TBM and CTR. Thus, these studies will lay the foundation for better diagnosis and therapeutic management of congenital and structural defects of the trachea.

气管支气管软化症（TBM）和完全气管环（CTR）是先天性疾病，其特征在于： 气管软骨和肌肉异常，发病率和死亡率高。TBM的病因 和CTR知之甚少，目前的干预措施仅限于侵入性手术和姑息治疗。的 本申请的首要目标是确定先天性疾病的潜在分子机制 影响气管发育我们已发表的研究表明，呼吸内胚层在 确定气管间充质的背腹组织，其中软骨和平滑肌 开发Wls（Wntless）的内胚层缺失，Wnt配体分泌所必需的货物受体， 导致缺乏气管软骨，被异常组织的平滑肌替代，类似于TBM 病理我们的RNA测序分析确定Notum和Bmp 4为气管上皮细胞中Wnt的候选靶基因。 间充质调节软骨和气管平滑肌形成的上皮Wls下游， 诱导信号。我们假设Notum通过抑制经典Wnt信号传导而起作用， 同时促进气管间充质所需的非经典Wnt和BMP信号传导 分化因此，Notum是Wnt信号传导的输出的新型调节模型的核心，其中 上皮Wnt配体首先促进Wnt/β-连环蛋白信号传导，然后将其关闭以允许非经典Wnt/β-连环蛋白信号传导。 活动为了检验中心假设，我们将：SA 1：为了确定Notum促进 气管软骨形成。使用新的小鼠模型和离体研究，我们将确定 Notum的缺失或过表达影响Wnt/β-连环蛋白依赖性和非依赖性信号传导活性， Notum、Wnt 5a和Wnt-b-连环蛋白之间的上位性以形成软骨。SA 2：通过以下方式定义机制： 其中Bmp 4和Wnt信号相互作用以介导气管间充质的细胞命运。在体内和体外进行 体内研究我们将确定Wnt/B-连环蛋白是否与BMP协同作用，通过诱导 Notum和Wnt 5a/Ror 2的表达;以及Notum和Bmp 4是否合作抑制异位肌， 腹侧气管间充质SA 3：检验上皮Wnt信号传导指导细胞骨架蛋白表达的假设。 气管平滑肌的组织。我们将研究1）来自上皮细胞的典型Wnt配体 通过调节肌源性基因表达直接促进成肌细胞骨架组织; 2）上皮细胞 Wnt配体激活非经典间充质Wnt信号传导，通过Ror 2介导成肌细胞 细胞骨架组织拟议的研究将确定关键的分子信号传导途径， 气管形态发生，并将揭示TBM和CTR的病理学。因此，这些研究将奠定 为更好地诊断和治疗气管先天性和结构性缺陷奠定了基础。