(A) A summary of cell numbers, gating of fluorescence-activated cell sorting (FACS, performed for E15 and P4 samples), median number of unique molecular identifiers (nUMI), and median number of genes (nGene) for each sample in the wild-type mouse trachea atlas presented in Figure 2. (B) Vertical bar graphs showing the proportion of cell types and states from each time point for each tracheal cell type and states colored by developmental stages. The overall representation of stromal cells from our atlas is consistent with those previously published lung atlas (Xie et al., 2018; Guo et al., 2019; Zepp et al., 2017) and reflects the diversity of cell types and states present in the developing airway. (C) The cellular composition of all cell types identified at E15 and P4. FACS was performed for cells from ShhCre/R26mTmG mice before RNA-sequencing to distinguish cells of the Shh-expressing lung endoderm lineage (green) from the rest non-endoderm lineages (red). In agreement with the established lineage relationships of the airway endoderm, GFP+ cells derived from Shh+ endoderm consistently expressed the epithelial marker Epcam. These Epcam+ cells were annotated as 1) basal cells, 2) ciliated cells, 3) secretory cells, and 4) cilia-secretory hybrid cells. RFP+ cells derived from non-Shh expressing lineages consisted of a large collection of mesenchymal cells, muscle cells, endothelial cells, immune cells, Schwann cells, and neuronal cells. Legends for cell types and cell states are shared in B and C D Doublet scores for all cells. Each dot represents a cell. Colors indicate cell clusters. (E) Expressions of thyroid markers Tg and Pax8 projected onto tSNE shown in Figure 2B. (F) Expressions of erythrocyte markers Alas2 and Hba-a2 projected onto tSNE shown in Figure 2B. Our dataset comprises a collection of diverse mesenchymal cell types, many of which have not been characterized at single-cell resolution. Vascular smooth muscle cells and pericytes are identified based on the expression of Notch3 and Rgs5, whereas airway smooth muscle cells express higher levels of Myh11 and Acta2. Endothelial cells express Pecam1 and can be further grouped into lymphatic endothelial cells based on the expression of Lyve1 and Thy1, and vascular endothelial cells based on the expression of Cd34. We identified two immune cell clusters, including a population of Fcerig+/Cd3g+ T cells and a population of Cx3cr1+/C1qa+ monocytes. Our dataset includes a cluster of Wnt2+ mesenchymal cells which persist across all time points included in this study. These cells are marked by Pi16, Cd34, and Ly6a (Sca-1), similar to the molecular signatures of adipose progenitor-like cells. Because Wnt2+ lineages can serve as cardiopulmonary progenitors and define a mesenchymal alveolar niche important for self-renew and repair in the lung, we annotated this Wnt2+/Cd34+/ Ly6a+ cluster as mesenchymal progenitors, which may generate the reservoirs of mesenchymal cell types during development and tissue repair.