Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.

Respiratory organs are composed of branched tubular epithelial networks that optimize gas flow and shield against airborne microbes and noxious chemicals. A systematic account of lung epithelial heterogeneity is expected to elucidate epithelial cell functions and facilitate new repair strategies upon infection. Here, we combined mouse genetics, single-cell mRNA sequencing and multiplex in situ mapping to generate a topological lineage map of adult airway epithelial cells. Apart from ciliated cells, we identified and focused on 4 major adult secretory cell states with distinct localizations along the proximo-distal axis of intrapulmonary airways. Positional differences in cell type occurrence reveal a gradient of epithelial heterogeneity, where proximal and distal epithelial cells highly express characteristic gene programs, whereas cells in intermediate positions of the airway stalks are characterized by low levels of both proximal and distal gene programs. Lineage-tracing experiment combined with single cell sequencing and spatial analysis suggest the hierarchical activation of genetic programs establishing mature secretory and ciliated epithelial cell types from embryonic progenitors. We tested if the distinct transcriptomic states of secretory cells reflect functional differences in vitro, by comparing differentiation potentials of secretory cells isolated from distinct locations. Distally located secretory cells showed an increased potency compared to proximal cells and Scgb1a1-Sftpc double positive cells (DPs). To identify distal determinant signals that generate this diversity, we investigated the significance of postnatal upregulation of FGF signaling components in secretory cells. Conditional inactivation of Fgfr2 suggests that FGF signaling selectively controls the identity of distal secretory cells by upregulating surfactant and lipid metabolism genes and repressing alveolar type I genes, without affecting the expression of proximal-distal graded programs. Our work elucidates the spatial heterogeneity of the airway secretory epithelium and reveals its developmental origin.