Project-2: Modeling TE birth defects in animals

计划2|项目摘要 未能将胎儿前肠分离成不同的气管和食管（TE）可能导致生命谱- 威胁气管食管缺损（TEDs）。TED的遗传病因学知之甚少， 只有12%的TED患者存在变异。即使在已知致病突变的情况下， 就像发育转录因子（TF）一样，它们如何导致TED还不清楚，因为直到最近， TE形态发生的机制尚不清楚。我们在上一个奖项中取得了重大进展。使用 结合爪蟾和小鼠胚胎学，我们阐明了保守的细胞事件驱动TE 形态发生，并表明破坏任何步骤都可能导致TED样表型。我们用非洲爪蟾 CRISPR筛选以验证来自患者基因组序列的潜在TED致病变体。一个主要 一项新发现是，在Hedgehog（HH）信号传导和发育TF的下游，内体介导的 需要膜重塑以将前肠分离成不同的TE管。与此相一致，基因组 对项目1中185名TED患者的测序显示， 膜/囊泡运输基因，包括内吞衔接ITSN 1，在初步数据中， 证实了非洲爪蟾TE形态发生所必需的。CLEAR联合体项目2的目标是 在动物模型中用细胞分辨率定义TED的发展基础。我们将：确定 内体运输调节TE形态发生的机制（Aim 1）。测试的假设 发育TF控制效应蛋白的细胞特异性表达， 机械或货物（目标2，与项目3合作）。评估患者变体在动物中的致病性 验证内体病可能是TEDs的主要原因这一挑衅性假设（Aim 3）。

PROJECT 2 | PROJECT SUMMARY Failure to separate the fetal foregut into distinct trachea and esophagus (TE) can result in a spectrum of life- threatening tracheoesophageal defects (TEDs). The genetic etiology of TEDs is poorly understood, and risk variants are known in only 12% of TED patients. Even in cases where the causative mutations are known such as in developmental transcription factors (TFs), how they result in TEDs is unclear because up until recently the mechanisms of TE morphogenesis were ill defined. We made significant progress in the previous award. Using a combination of Xenopus and mouse embryology, we elucidated the conserved cellular events driving TE morphogenesis and showed that disrupting any step can result in TED-like phenotypes. We used Xenopus CRISPR screens to validate potential TED-causing variants from patient genome sequences. One major discovery was that downstream of Hedgehog (HH) signaling and developmental TFs, endosome-mediated membrane remodeling is required to separate the foregut into distinct TE tubes. Consistent with this, genome sequencing of 185 TED patients from Project 1 revealed an enrichment of damaging variants in membrane/vesicular trafficking genes including the endocytic adaptor ITSN1, which in preliminary data we demonstrated is required for Xenopus TE morphogenesis. The goal of the CLEAR consortium Project 2 is to define the developmental basis of TED with cellular resolution in animal models. We will: Determine the mechanism by which endosome trafficking regulates TE morphogenesis (Aim1). Test the hypothesis that developmental TFs control the cell-specific expression of effector proteins such as endosome trafficking machinery or cargo (Aim2, in collaboration with Project 3). Assess the pathogenicity of patient variants in animals testing the provocative hypothesis that endosomeopathies might be a major cause of TEDs (Aim3).