Figure 1. Marking cell clones in developing lung mesenchyme.

A, Diagram of embryonic day E11.5 mouse lung (ventral view) showing airway epithelium (AE; white), mesenchyme (Me; light gray), mesothelium (Mt; dark gray), airway (ASM) and vascular (VSM) smooth muscle (red), and pulmonary arteries (PA) and vascular plexus (Pl) in blue. B, Labeled mesenchymal progenitor cell (green) in early lung (left), which at E11.5 (right) has generated a mixed lineage clone (green, dashed circle) with labeled ASM, mesothelial cells, and undifferentiated fibroblasts. C-F, Close-up of clones (pseudo-colored green) in indicated tissues of lungs of indicated ages. Clones were marked with MADM (C, E, F) or Rainbow (D) strategies and immunostained for E-cadherin (airway epithelium; blue), PECAM (vascular endothelium; blue in F) and smooth muscle α-actin (ASM and VSM; red). Cells in each clone are numbered. G, Structure of lung mesenchyme specific Cre transgene using lung mesenchyme-specific enhancer (LME) from Tbx4 locus and P_hsp68 minimal promoter to drive Cre recombinase expression. H-J, Cre mRNA in situ hybridization of lungs from Tbx4^LME-Cre transgenic mice of indicated ages. mRNA is robustly detected by E10.5 exclusively in lung mesenchyme (H), remains strong at E13.5 (I), and begins to downregulate by E14.5 (J). Similar results were obtained for Tbx4^LME-CreER (Figure S1). K, Tiled image of transverse section through E13.5 Tbx4^LME-Cre; R26R-EYFP lung immunostained for YFP lineage trace marker (white). Lineage-traced cells are confined to lung. NT, neural tube; Li, liver; St, stomach. L, Section through E13.5 Tbx4^LME-Cre; R26R-EYFP lung immunostained for YFP (green) and E-cadherin (magenta). Nearly all mesenchyme cells are lineage labeled (green). Lineage label is not detected in epithelium (magenta). M-O, Tbx4^LME-CreER; Rainbow embryos given limiting doses of tamoxifen (0.6 mg/dam) at E9.5 (M) or E10.5 (N, O) and analyzed at E13.5 by immunostaining for smooth muscle α-actin (SMA) and clone markers indicated. Clones are labeled by a single recombination reporter, either mCherry (M), mOrange (N) or Cerulean (O). Cells in each clone are numbered. Bars, 50 μm (C-F; H-J), 100 μm (L-O).