Multiple preexisting SMCs give rise to excess smooth muscle in elastin mutants. Transverse descending aortic sections of SMA-CreER^T2 mice also carrying ROSA26R^(mTmG/+) (A) or ROSA26R^(Rb/+) (B) and of the indicated elastin genotype induced with tamoxifen at E12.5. (A) Sections at P0.5 were stained for SMA, GFP (lineage marker), CD31, and nuclei (DAPI), and GFP⁺ SMCs are included in extra aortic wall layers in Eln^(+/−) (outer layer; open arrowheads) and in Eln^(−/−) (inner layer adjacent to endothelium). The boxed regions are shown as close-ups with closed arrowheads indicating increased SMC projections in elastin mutants. (B) Section at E18.5 was analyzed with direct fluorescence of Rb colors and staining for DAPI. Excess inner layer SMCs of Eln^(−/−) aorta include cells of multiple colors (asterisks) indicating polyclonality. For each genotype, n = 4 pups in A and n = 3 embryos in B. Lu, aortic lumen; E, endothelial layer; 1–7, smooth muscle layers. Bars, 10 µm.