ABSTRACT: An in-depth understanding of the mechanisms that regulate alveolar septation and septal wall maturation will be required to develop therapies for premature infants with Bronchopulmonary Dysplasia (BPD) and for developing potential therapies for adult lung regeneration. We and others have begun to define changes in PDGFRa myo, lipo and matrix fibroblast (FB) function during alveolarization, homeostasis, regeneration, and fibrosis. Our long-term goal is elucidation of molecular regulators of FB functions and how diverse FBs support epithelial cells. Our objective herein is to identify molecular mech- anisms that result in functional changes in alveolar fibroblasts that regulate ECM organization and epithelial differentia- tion. Integration of transcriptomic datasets predicted GATA6 as a regulator of PDGFRa-FB differentiation. Our preliminary data show that conditional inactivation of GATA6 in perinatal PDGFRa+ FBs resulted in loss of matrix and gain in lipo fibro- blast function comparable to findings in BPD and animal models of hyperoxia. GATA6 inactivation in PDGFRa+ FBs also resulted in fragmented collagen and detachment of AT1 cells from the basal lamina demonstrating a thus far unidentified role of alveolar FBs in ECM organization. Inactivation of GATA6 resulted in significant increase of the lipo FB transcription factor TCF21 expression and preliminary in vitro data suggest suppression of TCF21 by GATA6. The central hypothesis is that GATA6 and transcriptional cofactors regulate FB function in alveolar FBs by suppressing lipo FB differentiation. The rationale for this research is a new understanding of the regulation of matrix FB function and how these functionally different stages modify extracellular matrix and the FB-epithelial crosstalk. Aim 1 will test the hypothesis that GATA6 suppresses lipo FB function and promotes ECM deposition in the alveolar FBs. This aim will use lineage tracing to deter- mine trans-differentiation of the alveolar FB to a lipo FB. And use in vitro cell derived matrices to identify ECM modifica- tions by lipo and matrix FBs. Aim 2 will test the hypothesis that GATA6 is in a transcriptional network that regulates lipo and matrix FB function. In this aim we will use in vitro gain and loss of function of GATA6 and TCF21 and identify and validate transcriptional networks and partnering transcription factors. Aim 3 will test the hypothesis that GATA6 regulates ECM synthesis and paracrine signaling in PDGFRa+ FBs that direct AT2 to AT1 differentiation. The proposed studies in this application will identify the transcriptional regulation of matrix FB function and their role in extracellular matrix deposition and cell-cell signaling in the alveolar niche. The proposed research is conceptually innovative, because we ask questions regarding the nature of fibroblast plasticity and functional switches. The proposed research is scientifically innovative, because the contractile function of alveolar fibroblasts, has b...