Figure 4

PLV-LMCs derive from a unique Prrx1⁺ and NG2⁻ progenitor cell incorporated onto PLVs between P10 and P21. Multicolor fluorescent microscopy was performed on whole mount immunostained PLVs and adjacent blood vessels (BVs) from tamoxifen-inducible Prrx1^CreER (n = 6, P21 depicted), and NG2^CreER (n = 5, P90 depicted) reporter mice as described in Fig. 1. Representative low-magnification overlay images of all channels show the PLVs with a highlighted region of interest (white boxes) (A.a,B.a). High-magnification images of the region of interest show Prox1 immunostain of LECs (A.b,B.b), αSMA immunostain of LMCs (A.c,B.c), Cre-driven tdT expression (A.d,B.d), overlay of all channels (A.e,B.e), and positive control BV VSMCs (A.f,B.f), blood capillary (BC) pericytes, and peripheral nerves (N) all directly adjacent to PLVs (B.f). Lineage tracing of Prrx1^CreER (A.a–f) mice demonstrates tdT expression in PLVs (A.d) that colocalizes with αSMA⁺ LMCs (A.e) along with successful Cre-recombination with tdT expression in positive control VSMCs, as expected (A.f). NG2^CreER (B.a–f) mice show absent tdT expression in PLVs (no red fluorescence in outlined PLV) (B.d) with successful tamoxifen induced NG2-driven Cre-recombination depicted by tdT expression in BV VSMCs, BC pericytes, and peripheral nerves (N), as expected (B.f). Note the few yellow VSMCs on the BV, which indicates that these cells were added to the fully formed BV after tamoxifen treatment.