Molecular basis of dorsal-ventral patterning of the conducting airways

项目摘要 这项提议的目标是研究传导呼吸道的形成机制。 背腹轴产生软骨和气管肌肉是适当和有效的必要的 通风运动。虽然呼吸道软化症(呼吸道缺乏适当的软骨)的发生率很高， 这些疾病的病因在很大程度上仍不清楚。我们生成了一个小鼠模型 气管支气管软化症，其中介导Wnt配体分泌的货物受体Wls在 气管上皮。明确上皮细胞Wnt信号促进气管软骨形成的机制 我们对对照组软骨形成前期气管组织中的基因表达进行了无偏倚分析 和Wlsf/f；ShhCre胚胎。我们发现了在呼吸道中从未被描述过的基因，包括 Lgi3和Notom，呈现明显的背腹表达模式。在Wlsf/f；ShhCre气管组织中， 促进软骨形成的基因，包括BMP配体，下调了表达，而调节基因 平滑肌分化上调。这一提议将检验Wnt配体的假设 由肺上皮产生的物质诱导促进软骨形成的基因表达， 同时，诱导抑制气管腹侧平滑肌分化的基因 间充质的我。这一提议的具体目的是：1)检验差异空间基因的假设 上皮性Wnt配体介导的表达模式是气管软骨腹侧分化所必需的。 这一目标将确定新的Wnt靶基因在气管组织中的空间表达模式，以及 WNT信号在体外检测中提供位置信息。使用体外试验，这一目标也将 确定新的靶基因是否促进Sox9和软骨形成。2)来检验这样的假设 促进气管平滑肌发育受到腹侧上皮Wnt信号的抑制 对气管的影响。在这一目标中，我们将通过体内和体外研究来确定促进平滑的基因 WLS上皮缺失后肌肉发育和上调，包括MYOCD、MYH11、Acta2和 Myo5c受Wnt信号调节。它还将测试靶基因在上皮细胞死亡后是否下调 WLS的缺失能够调节促进平滑肌的基因的表达。这项建议是 创新，因为它将定义在传导呼吸道中表达的新基因在促进气管生长中的作用 软骨或肌肉分化。从长远来看，这一知识将促进新细胞的发展 有效地治疗影响正常气管构型的疾病并修复受损或 畸形的呼吸道。 这项建议还将允许申请人，K01职业发展奖获得者，开发新的假设 关于呼吸道的模式，这将是未来R01提案的基础。因此，该项目将促进 申请人的科学成长和独立性。

Project Summary The goal of this proposal is to investigate mechanisms by which the conducting airways are patterned across the dorsal ventral axis given rise to cartilage and trachealis muscle necessary for appropriate and efficient ventilation movements. While the incidence of airway malacias (airways lacking appropriate cartilage) is high, the etiology of these conditions remains largely unknown. We generated a mouse model of tracheobronchomalacia wherein Wls, a cargo receptor mediating Wnt ligand secretion, was deleted in the epithelium of the trachea. To define mechanisms by which epithelial Wnt signaling promotes tracheal cartilage formation we performed an unbiased analysis of gene expression in prechondrogenic tracheal tissue of control and Wlsf/f;ShhCre embryos. We identified genes that have never been described in respiratory tract, including Lgi3 and Notum, presenting a distinct dorsal-ventral pattern of expression. In Wlsf/f;ShhCre tracheal tissue, genes promoting chondrogenesis, including Bmp ligands, were down regulated, while genes mediating smooth muscle differentiation were upregulated. This proposal will test the hypothesis that Wnt ligands produced by the pulmonary epithelium induce expression of genes promoting chondrogenesis and, simultaneously, induce genes that repress smooth muscle differentiation in the ventral tracheal mesenchyme. The specific aims of this proposal are: 1) To test the hypothesis that a differential spatial gene expression pattern mediated by epithelial Wnt ligands is required for ventral differentiation of tracheal cartilage. This aim will define the spatial expression pattern of novel Wnt target genes in the tracheal tissue and whether Wnt signaling provides positional information in ex vivo assays. Using in vitro assays, this aim will also determine whether novel target genes promote Sox9 and chondrogenesis. 2) To test the hypothesis that genes promoting tracheal smooth muscle development are repressed by epithelial Wnt signaling in the ventral aspect of the trachea. In this aim we will determine via in vivo and in vitro studies whether genes promoting smooth muscle development and upregulated after epithelial deletion of Wls, including Myocd, MyH11, Acta2, and Myo5c, are modulated by Wnt signaling. It will also test whether target genes downregulated after epithelial deletion of Wls are capable to modulate expression of genes promoting smooth muscle. This proposal is innovative, as it will define the role of novel genes expressed in conducting airways in promoting tracheal cartilage or muscle differentiation. In the long term this knowledge will facilitate the development of novel cell based therapies to efficiently treat conditions affecting the normal tracheal patterning and to repair damaged or malformed airways. This proposal will also allow the applicant, a K01 career development awardee, to develop novel hypothesis about patterning of respiratory tract that will be the basis of future R01 proposal. Thus, this project will promote the scientific growth and independence of the applicant.