. 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 Tgf?r1/2Gli mice, conditional postnatal inactivation of both Alk5 & T?r2 in secondary crest myofibroblasts, SCMF blocks alveogenesis & disrupts epithelial morphogenesis. Similar strategy in the Pdgfr?Gli 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 TGF? and PDGFA/PDGFR? 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: TGF?, 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 Pdgfr?Gli1 & Tgf?r1/2Gli1 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 Tgf?r1/2Gli1 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.
Alveogenesis is the most critical and the least understood phase of lung development. A detailed understanding of alveogenesis is undoubtedly important to unraveling the mechanisms of many lung diseases that involve alveolar defects. These include both neonatal (e.g. BPD, CDH & Alveolar Capillary Dysplasia) and adult pulmonary diseases (COPD). For example, in Bronchopulmonary dysplasia or BPD, a chronic lung disease in infants born prematurely, alveogenesis is impaired and the children with this disease develop not only long term lung problems, but also neurodevelopmental problems & difficulties with school performance. The combined effects of this terrible lung disorder are extremely costly to society and also expensive to the health care system. Unfortunately, much of the studies on the lungs have and remain focused on early period of development which are not directly relevant to BPD or other alveolar diseases. Due to technical difficulties and absence of tools, the process of alveogenesis, so important and relevant to BPD and adult alveolar diseases such as COPD has not received adequate attention. The present project proposes to do just that. The proposal is based on some very strong foundations from experimental studies on mice. But, it also incorporates information from human studies that are not amenable to experimentation. Successful completion of this project will add significantly and robustly to understanding alveolar formation and may indeed pave the way towards a treatment for BPD in children and COPD in adults.
