BRD4 promotes endodermal cell fate during mammalian lung development

Lung development relies on diverse cell intrinsic and extrinsic mechanisms to ensure proper cellular differentiation and compartmentalization. In addition, it requires precise integration of multiple signaling pathways to temporally regulate morphogenesis and appropriate cell specification. To accomplish this, organogenesis relies on epigenetic and transcriptional regulators to promote cell fate and inhibit alternative cell fates. Using genetic mouse and human embryonic stem cell (hESC) differentiation models, tissue explants, and single-cell transcriptomic analysis, we demonstrated that Bromodomain Containing Protein 4 (BRD4) is required for mammalian lung morphogenesis and cell fate. Endodermal deletion of BRD4 impaired epithelial-mesenchymal crosstalk, leading to disrupted proximal-distal patterning and branching morphogenesis. Moreover, temporal deletion of BRD4 revealed developmental stage-specific defects in airway and alveolar epithelial cell specification with a predominant role in proximal airway cell fate. Similarly, BRD4 promoted lung endodermal cell differentiation into airway lineages in a hESC-derived lung organoid model. Together, these data demonstrated that BRD4 orchestrates early lung morphogenesis and separately regulates cell specification, indicating a multifunctional and evolutionarily conserved role for BRD4 in mammalian lung development. Overall design: Approximately 16500 EPCAM+ cells were sorted by FACS from the lungs of multiple Shh-Cre/+, Brd4-fl/+ (HET control) or Shh-Cre/+, Brd4-fl/fl (KO) mice sacrificed at E17.5 and loaded into independent lanes of a 10X Chromium GEX 3' v3 microfluidics chip for 3'-biased scRNA-seq in order to compare Brd4 HET vs. Brd4 KO in the Shh+ lung epithelium.