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Fig. 6 Llgl1 is required for timely epicardial emergence and ventricular colonisation. (A-F′) Confocal z-slices through the ventricle of Tg(myl7:LifeAct-GFP) transgenic embryos visualising the myocardium (green) and anti-Cav1a antibody (magenta), highlighting the epicardium at 76 hpf (A-D) and 96 hpf (E,F). Wild-type embryos show full epicardial coverage at 76 hpf (A, n=12/12), whereas only a small subset of llgl1 mutants have full epicardial coverage (C, n=3/15), or only a few epicardial cells attached (D′, blue arrow, n=3/15) and the majority have partial epicardial coverage (B′, yellow arrows, n=9/15). By 96 hpf Cav1a+ epicardium surrounds the ventricle in both wild type (E, n=4/4) and the majority of llgl1 mutant embryos (F, n=5/6). A′-F′ show Cav1a staining only. Scale bars: 25 µm. (G-J) mRNA in situ hybridisation analysis of wt1a expression in wild-type and llgl1 mutant embryos at 55 hpf. G and I show the ventral view, dashed line outlines the heart; H and J show the lateral view. Scale bars: 50 µm. Both wild-type and llgl1 mutant embryos express wt1a in proepicardial cells in the dorsal pericardium; however; whereas wild-type embryos have wt1a+ cells attached to the ventral ventricular wall (red arrow, H), these cells are reduced or absent in llgl1 mutants (red arrow, J). (K) Quantification of the number of epicardial cells attached to the ventral ventricular wall in wild type (n=28) and llgl1 mutants (n=21). Comparative analysis performed using unpaired two-tailed t-test (**P<0.01). Data are mean±s.d. (L) Schematic model of epicardium, laminin and Crb2a dynamics between 55 hpf and 84 hpf.