Figure 1
Genetic models for lineage tracing lymphatic muscle cells in popliteal lymphatic vessels and ex vivo whole mount multicolor fluorescent microscopy. Schematics of the constitutively-active (A.a) and tamoxifen-inducible (A.b) conditional Cre-driver constructs used in this study (Genes of Interest = Pax7, MyoD, Prrx1, and NG2) are presented with the Ai9tdTomato reporter (tdT) construct to illustrate the heterozygous double-transgenic mice that were used in the lineage tracing (red Cre+), and their heterozygous single-transgenic Ai9tdTomato littermates that were used as negative controls (black Cre−). An illustration of the tamoxifen dosing regimen (daily intraperitoneal injections on postnatal days 10–13) is also presented with the standard time of sacrifice at postnatal day 21, while all gene markers that showed PLV-LMC tdT-negativity at any time during neonatal development were additionally sacrificed > 6-weeks to validate earlier observations (A.c). To harvest the popliteal lymphatic vessels (PLVs), mice were anesthetized and injected with 2% Evans Blue dye (Millipore Sigma Cat# E2129) into their footpad, prior to skin removal. A 1.5 × photograph of the exposed lower limb is presented to illustrate how Evans Blue filled PLVs (two PLVs per hindlimb; blue arrows) draining into the popliteal lymph node (white circle) for PLV identification prior to excision (B.a). Magnified photographs of the black boxed regions illustrate incisions on the PLV border opposite the saphenous vein (yellow arrow) to release it from the fat pad (B.b), and then how the PLV was removed +/− the saphenous vein for analysis (B.c). After explant of the PLV (~ 2–3 mm in length), whole mount immunofluorescence was performed using antibodies against Prox1 with a DyLight 650-conjugated secondary antibody to mark lymphatic endothelial cells (purple) (C.a) and directly adjacent Alexa Fluor 488-conjugated antibodies against αSMA to mark lymphatic muscle cells (green) (C.b) with a Hoechst nuclear stain (blue). Direct fluorescent microscopy of the Cre-driven tdT reporter protein (red) in cells of the PLV (example of Prrx1CreER × Ai9tdTomato mouse) identifies positive cells in the lineage trace (C.c). A composite overlay of the three images is shown to illustrate co-expression of αSMA and tdT (yellow) in LMCs of Prox1+ PLVs (C.d).
