Extended Data Figure 1. Molecular characterization of the murine cardiac lymphatic vasculature.

Whole-mount DAB staining of hearts (n= 3 per time point) with the lymphatic marker VEGFR-3 revealed cardiac lymphatics vessels first sprout from the region of the sinus venosus, on the dorsal side of the heart at E14.5 (a, white box, enlarged in b). At E16.5, ventrally the first small vessels arose between the atria (c), while the main dorsal vessels spread inferiorly from the sinus venosus at the inflow region of the heart (d). At E18.5 the network appears similar with little expansion (e, f). From birth, post-natal day (P) 0, lymphatic vessels branch and expand onto the ventral epicardial surface of the heart such that by P10 the network has expanded significantly coincident with cardiac growth (g, h). Consistent with the systemic lymphatic vasculature, cardiac lymphatic vessels are fully developed by P15 (i, j), with no difference in vessel density at later stages (data not shown). Whole-mount DAB staining of E17.5 hearts with the lymphatic marker Prox1 (n=4) further confirmed extensive spread of the sprouting lymphatics inferiorly from the outflow tract region (k) and sinus venosus, at the inflow region of the heart (l). White inset box in (k, l) is shown in (m, n; respectively) highlighting the punctate nuclear expression of Prox1 in coronary lymphatics. Whole-mount confocal imaging of E17.5 hearts (n=4) stained with VEGFR-3, Prox1 and LYVE-1 confirmed co-labelling of coronary lymphatic vessels (o-v). Note that whilst at this developmental stage VEGFR-3 is restricted to LECs (p, t), Prox1 is also expressed in the underlying myocardium (o, s) and LYVE-1 labels tissue-resident macrophages (q, u). Scale bars: a 750μm; b 300μm; c, d 750μm; e, f 1mm; g, h 2mm; i, j 2.5mm; k, l 400μm; m, n 200μm; o, s 100μm.