Abstract Rationale Alveolar septation and repair depend on distinct fibroblast populations that coordinate extracellular matrix (ECM) assembly and epithelial differentiation. Matrix fibroblasts provide mechanical stability, while lipofibroblasts (LipoFBs) support AT2 function through lipid transfer and paracrine signaling. GATA6 has emerged as a transcriptional regulator of matrix fibroblast identity. Its loss increases expression of the lipofibroblast-associated factor TCF21, suggesting reciprocal regulation of matrix and lipogenic fibroblast states. We investigated how GATA6 deletion and TCF21 overexpression influence fibroblast fate and alveolar structure in vivo and in vitro. Methods GATA6 was deleted (PDGFRαCreER^;Gata6ᶠl/fl^) and TCF21 overexpressed (PDGFRαCreER^;RosaTCF21^) at postnatal day (PN) 1. Lungs were analyzed at PN7 and PN28 by histology, morphometry, immunofluorescence, and electron microscopy (EM). CRTAP and fibronectin served as matrix markers, and lipid droplets were quantified by ADRP/Plin2 staining. Gene and single-cell data defined fibroblast subtype transitions. Complementary studies in IMR90 fibroblasts examined GATA6 knockdown and TCF21 overexpression on lipid accumulation and lipogenic gene expression. Results GATA6 deletion caused alveolar simplification, septal thickening, and loss of CRTAP and fibronectin, indicating loss of matrix fibroblast function. PDGFRα⁺ fibroblasts accumulated lipid droplets and upregulated Tcf21, Plin2, and Pparγ without induction of myogenic genes. Single-cell analysis revealed a shift from adventitial-like to lipogenic fibroblast states. In contrast, TCF21 overexpression in vivo reproduced alveolar simplification and loss of CRTAP but did not increase lipid content or fully induce lipogenic markers. EM demonstrated AT1 cell membrane blebbing consistent with impaired basement membrane support, linking TCF21-driven matrix defects to epithelial distortion. In IMR90 fibroblasts, both GATA6 loss and TCF21 overexpression increased Plin2, Pparγ, and lipid accumulation, confirming an intrinsic GATA6-TCF21-PPARγ regulatory axis controlling fibroblast lipogenic potential. Conclusions GATA6 preserves matrix fibroblast identity and ECM organization, whereas TCF21 promotes a lipogenic program that is incomplete in vivo but robust in vitro. Loss of GATA6 reprograms fibroblasts toward a lipogenic state, disrupting alveolar architecture, while TCF21 alone induces matrix instability reflected by AT1 blebbing. Together, these findings define a GATA6-TCF21 “yin-yang” axis governing fibroblast plasticity, matrix integrity, and epithelial-mesenchymal coordination during alveolar development and repair. This abstract is funded by: R01HL154720 and R01HL164552 (A. K. T. P. ) and the NHLBI Training Grant T32HL152465 (C. A. S. )
Perl et al. (Fri,) studied this question.