Postnatal Alveolar Formation

摘要：。 该项目的主要长期目标是确定肺泡形成的主要遗传调节因素。 肺泡缺陷是许多新生儿和成人肺部疾病的基础。他们受阻的发展被发现在 婴儿患有BPD(支气管肺发育不良)，而其破坏是肺气肿的特征 成人的肺。尽管有重要的临床相关性，肺泡形成仍然是最不被了解的肺阶段。 发展！ 我们已经开发了两个平行的、基于出生后遗传学的模型来分析 肺泡形成。在转化生长因子βR1/2Gli小鼠中，次级波峰中Alk5和TβR2的条件出生后失活 肌成纤维细胞、SCMF阻断肺泡形成并破坏上皮形态发生。类似的战略在 PDGFRαGLI模型也产生几乎相同的表型。我们推断，由于表型几乎相同， 这两种模型必须共享一些在肺泡形成中不可或缺的遗传因素。 使用RNAseq/生物信息学的初步数据确定了“特定于途径”的差异表达基因 (Deg)转化生长因子β和PDGFA/PDGFRα突变模型。最重要的是，正如预测的那样，我们确定了一个星团 我们提出的“共同”基因中包含一组保守的核心肺泡形成调控基因。这些 初步数据，证明有理由对DEG进行更全面和更广泛的分析，以提高 RNAseq数据(具体目标1)。在具体目标2中，我们将验证并从功能上描述共同的 基因。在最初的簇中，一个值得注意的基因是Igf1，它编码IGF1信号通路的配体。我们 选择IGF1进行进一步的功能分析(具体目标3和4)。 假设：转化生长因子β、PDGFA/PDGFRA和IGF1形成了一个复杂的信号网络的主要分支电路。 SCMF通过一组下游的“肺泡发生调控基因”来控制肺泡的发生。这个集群是 唯一保守的&可以通过使用突变的比较差异基因表达分析来鉴定 三条路径中的每一条&识别共同的DEG。具体目标是： 具体目标1：。通过分析寻找控制肺泡发生的保守的SCMF调控基因 转化生长因子受体αGli1和转化生长因子βR1/2Gli1肺组织差异表达基因的研究 具体目标2：。验证和功能分析来自特定目标1的候选基因。 具体目标3：。开发IGF1信号的条件性、细胞靶向性遗传模型来研究这一点 途径是肺泡组装和上皮形态发生中高度保守的关键调控节点。 具体目标4：。利用重组IGF1确定IGF1调控肺泡生成的候选基因 肺转化生长因子βR1/2Gli1基因的修复 影响：肺泡是呼吸的功能单位。它的正确组装需要协调的功能 在内胚层、中胚层、内皮细胞之间，可能还有免疫细胞。对这些机制的阐明不仅 有影响，但对了解新生儿(如BPD、CDH和肺泡毛细血管发育不良)是必要的 和成人肺部疾病(慢性阻塞性肺病、肺气肿)。这里所描述的研究有很强的理论基础。 和初步数据。虽然这项提议可能看起来过于雄心勃勃，但初步数据的数量已经 在手边，确保在RO1期限内成功完成项目。

ABSTRACT:. The overarching long-term objective of this project is to identify principal genetic regulators of alveogenesis. Alveolar defects underlie many neonatal and adult lung diseases. Their arrested development is found in infants with BPD (BronchoPulmonary Dysplasia), while their destruction is characteristic of emphysematous lungs in adults. Despite significant clinical relevance, alveogenesis remains the least understood phase of lung development! We have developed 2 parallel, postnatal genetically-based models to analyze the molecular basis of alveogenesis. In Tgfβr1/2Gli mice, conditional postnatal inactivation of both Alk5 & Tβr2 in secondary crest myofibroblasts, SCMF blocks alveogenesis & disrupts epithelial morphogenesis. Similar strategy in the PdgfrαGli model also yields a nearly identical phenotype. We reasoned that due to nearly identical phenotypes, the two models must share in common some genetic elements whose role in alveogenesis is indispensable. Preliminary data using RNAseq/bioinformatics identified “pathway-specific” differentially expressed genes (DEG) for TGFβ and PDGFA/PDGFRα mutant models. Most importantly, as predicted, we identified a cluster of “common” genes which we propose contains a cluster of conserved core alveogenesis regulatory genes. These preliminary data, justify a more comprehensive and expanded analysis of DEG to improve the reliability of the RNAseq data (Specific Aim 1). In Specific Aim 2, we will validate and functionally characterize the common genes. A notable gene in the initial cluster is Igf1, which encodes the ligand for the IGF1 signaling pathway. We have selected IGF1 for further functional analyses (Specific Aims 3 and 4). HYPOTHESIS: TGFβ, PDGFA/PDGFRa & IGF1 form major sub-circuits of a complex signaling network in SCMF that controls alveogenesis via a cluster of downstream “alveogenesis regulatory genes”. This cluster is uniquely conserved & can be identified by comparative differential gene expression analysis using mutations in each of the three pathways & identifying the common DEG. The Specific Aims are: Specific Aim 1:. Identify Conserved SCMF Regulatory Genes That Control Alveogenesis Thru Analysis of Differentially Expressed Genes in PdgfrαGli1 & Tgfβr1/2Gli1 Lungs. Specific Aim 2:. Validate and Functionally Analyze Candidate Genes from Specific Aim 1. Specific Aim 3:. Develop Conditional, Cell-Targeted Genetic Model of IGF1 Signaling to Interrogate This Pathway as a Key Highly Conserved Regulatory Node in Alveolar Assembly & Epithelial Morphogenesis. Specific aim 4:. To Use Recombinant IGF1 to Identify Candidate IGF1-Regulated Alveogenesis Regulatory Genes via Rescue of Tgfβr1/2Gli1 Lungs. Impact: The alveolus is the functional unit of respiration. Its proper assembly requires coordinated function amongst endodermal, mesodermal, endothelial & perhaps immune cells. Elucidation of the mechanisms not only has an impact, but is necessary for understanding both neonatal (e.g. BPD, CDH & Alveolar Capillary Dysplasia) and adult pulmonary diseases (COPD, emphysema). The studies described here are grounded in strong rationale and preliminary data. While the proposal may appear over-ambitious, the volume of the preliminary data already at hand, makes certain the successful completion of the project within an RO1 time limit.