Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for ﬁbroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ ﬁbroblasts during sacculation and alveolarization. Confocal microscopy identiﬁed spatial association of PDGFRα expressing ﬁbroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ ﬁbroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myoﬁbroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ ﬁbroblasts identiﬁed diﬀerentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression proﬁle patterns. The presence of myoﬁbroblast and smooth muscle precursors at E16.5 and PN7 was reﬂected by at wo-peak gene expression proﬁle on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional diﬀerences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myoﬁbroblasts at PN7 were suggested by a single peakingeneexpressionatPN7 with functional enrichment in cell projection and muscle cell diﬀerentiation. Immunophenotyping of subsets of PDGFRα+ ﬁbroblasts by ﬂowcytometry conﬁrmed the predicted increase in proliferation at E16.5 and PN7, and identiﬁed subsets of CD29+ myoﬁbroblasts and CD34+ lipoﬁbroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization.
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