Stc1-expressing myofibroblasts are a developmentally distinct lineage cleared through apoptosis in the neonatal lung

Lung myofibroblasts are necessary for early postnatal alveolar growth. The unique contributions of individual myofibroblast lineages to alveolar development is unresolved by existing genetic tools. We generated a Stc1CreERT2 mouse line that labels the developmentally transient secondary crest myofibroblasts (SCMFs), distinguishing them from alveolar duct myofibroblasts (DMF) and smooth muscle. SCMFs expand through clonal proliferation of Stc1-expressing progenitors and are cleared by apoptosis. Deleting the apoptosis effectors Bax and Bak1 in the Stc1-lineage prevented SCMF clearance during alveologenesis. Single-cell RNA sequencing showed that surviving Stc1-lineage cells lose myofibroblast identity while coexpressing SCMF and DMF markers. Embryonic lineage tracing identified distinct progenitors for SCMFs and DMFs, and genetic activation of Hedgehog (Hh) or Wnt signaling pathways failed to interconvert these lineages. These findings establish Stc1-lineage SCMFs as a discrete, developmentally divergent population, and define their life cycle independent of other myofibroblast lineages. Overall design: Stc1-CreERT2 conditional apoptosis knockout (Stc1-BB) mice were injected with tamoxifen at P1 and sacrificed at P30 for lung cell harvest and scRNA-seq. Cells from each animal were labelled with cell multiplexing oligos (CMOs) prior to FACS in order to assign single cells to animals (biological replicates) within the joined scRNA-seq data. Cell suspensions were not combined across experimental conditions (i.e. HET/KO libraries were generated separately).