Spatiotemporal Profiling Defines the Epithelial and Mesenchymal Transition Window in Embryonic Lung Morphogenesis

Lung organogenesis is orchestrated by dynamic epithelial–mesenchymal interactions during embryogenesis, yet the gene regulatory programs and signaling dynamics governing these processes in the pseudoglandular stage remain incompletely understood. In this study, we integrated spatial and single-cell transcriptomic data across embryonic developmental stages to systematically characterize epithelial and mesenchymal dynamics during lung development. To achieve more refined cell types at single-cell resolution in spatial transcriptomic data, we developed a bin-based deconvolution strategy that enabled high-precision cell-type assignment. We subsequently constructed a 3D spatiotemporal landscape of lung development and elucidated the molecular regulatory mechanisms underlying epithelial–mesenchymal maturation during lung morphogenesis. In addition, we analyzed transcription factor module activity, intercellular communication signaling, and predicted downstream target genes, while integrating public GWAS metadata to link developmental programs with lung cancer-related features. We observed pronounced stage-specific functional heterogeneity between the pseudoglandular and late embryonic stages. Notably, E13.5 emerged as a critical transition window, during which progenitor states shifted toward more mature cellular phenotypes. We reconstructed epithelial–mesenchymal interactions and uncovered coordinated rewiring of ligand–receptor signaling and transcriptional networks across developmental stages. Regulatory network analysis further identified temporally coordinated transcription factor modules centered on Tbx3, Tbx5, Gli1, Gata4/5, Foxa1/2, and Cebpa, which collectively orchestrated branching morphogenesis, epithelial patterning, and tissue stabilization. Integration with lung cancer genome-wide association data demonstrated that embryonic lung progenitor states exhibit strong associations with lung cancer-related transcriptional programs, particularly involving epithelial–mesenchymal plasticity and RNA-splicing pathways. Furthermore, TP53/HNRNP-mutant lung adenocarcinomas displayed embryonic-like molecular features associated with cytoskeletal remodeling and progenitor-state reactivation. Together, our study provided a spatiotemporally resolved framework of embryonic lung development and identifies a critical transition window linking lung morphogenesis, regulatory network remodeling, and cancer-associated epithelial plasticity.