Fig. 2

Llgl1 is required for timely apical redistribution of Crb2a. (A-D) Confocal z-slices of the ventricle of Tg(myl7:LifeAct-GFP) transgenic embryos visualising the myocardium (green) and anti-Crb2a antibody (magenta). (A′-D′) Crb2a staining. Scale bars: 25 µm. C and D show higher magnification of yellow boxes in A and B, respectively. Scale bars: 10 µm. In wild-type embryos at 72 hpf low levels of Crb2a are distributed across the apical myocardial membrane (A,C), whereas in llgl1 mutants bright Crb2a puncta are observed at apical CM junctions (B,D). (E-F’) Example measurements of Crb2a in wild-type and llgl1 mutant embryos. (G) Schematic showing quantification of Crb2a across the apical CM surface. AM, apical membrane; Ap, apical; Ba, basal; J, junction. (H,I) Example quantifications of Crb2a intensity across the standardised apical membrane of individual cardiomyocytes in wild type (H) and llgl1 mutants (I) at 72 hpf. Crb2 is elevated at the cell boundaries in llgl1 mutants (I) compared with wild type (H). Individual traces represent individual embryos. Grey boxes indicate apicobasal positions used to bin data into junctional domains. (J) Quantification of junctional/apical Crb2a intensity in wild-type siblings and llgl1 mutants at 55 hpf (wt, n=139; llgl1−/−, n=113), 72 hpf (wt, n=117; llgl1−/−, n=106) and 80 hpf (wt, n=112; llgl1−/−, n=159). At 72 hpf wild-type embryos have slightly elevated Crb2a at CM junctions compared with across the apical membrane, but llgl1 mutants have significantly more junctional Crb2a. By 80 hpf, overall levels of Crb2a in llgl1 mutants is reduced compared with wild-types. One-way ANOVA with multiple comparisons (****P<0.0001, **P<0.01, *P<0.05). ns, non significant. Box plots show median values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges; dots indicate data points. (K) Schematic depicting dynamics of Crb2a distribution in wild type and llgl1 mutants at 72 hpf and 80 hpf.