Figure 5. Effect of Wnt bead implants and transgenic activation of Wnt pathway show that Wnt signaling primes ASM progenitors.

A-C, Stereoscope images of stalk-to-tip mesenchyme grafts performed as in Figure 4D, except also implanted with control (A) or ligand-soaked (B, C) agarose beads as indicated. Asterisk, position of bead. A'-C' show single confocal slices of graft region after immunostaining for GFP (graft marker, green), E-cadherin (epithelium, blue), and SMA (ASM, red). Graft cells exposed to PBS- or FGF10-soaked beads showed little or no migration along airways (A', B'), whereas those exposed to WNT1-soaked beads (C') migrated along airways where some (arrowheads) differentiated to ASM (separate channels shown in Figure S5E). D, Quantification of graft cell invasion in untreated grafts (reproduced from Figure 4B), and following exposure to ligand-soaked beads and Wnt inhibitors, as indicated. Note that WNT1 beads (column 5) induce stalk mesenchyme invasion at levels comparable to that seen in tip-to-tip grafts (column 1), and the effect is reduced in the presence of 100 ng/ml Wnt inhibitors DKK1 or SFRP1 (column 6). E-H, Right middle lobes of E12.5 control (E) and Tbx4^LME-Cre;βcat^ex3/+ lungs that express stabilized β-catenin to activate canonical Wnt pathway throughout mesenchyme (F-H) immunostained for E-cadherin (epithelium, green), SMA (smooth muscle, red), and PECAM (endothelium, blue). Wnt pathway activation results in disorganized smooth muscle around airways (F) and ectopic wrapping of vascular plexus by cells continuous with ASM (arrowheads in G). (H) Single confocal slice of branch as in G showing that the ectopic smooth muscle wrapping the plexus (arrowhead) expresses vascular smooth muscle marker NG2 (magenta). Bars, 50 μm (A-E), 25 μm (F, G).