Fig. 2

Atrial endocardial cell proliferation is increased upon Wnt8a overexpression or in nkx2.5^vu179 mutants. a Quantifications of endocardial cell numbers at 30 hpf reveals that there is no increase of atrial endocardial cells in Tg(hsp70l:wnt8a-GFP)^w34 (n = 9 hearts), or nkx2.5^vu179 mutant embryos (n = 10 hearts) compared with WT (kdrl:GFP n = 10 hearts, fli1a:nEGFP n = 9 hearts) at this stage. At the later heart ballooning stage (52 hpf), a significant increase of endocardial atrial cell numbers has occurred upon Wnt8a overexpression [Tg(hsp70l:wnt8a-GFP)^w34 (n = 8 hearts)] or in nkx2.5^vu179 mutants (n = 27 hearts) compared to WT (kdrl:GFP n = 11 hearts, fli1a:nEGFP n = 27 hearts). Mean values ± SD are shown. Two-way ANOVA was used to compare each condition with its WT control in each individual chamber (ns: not significant,****p < 0.0001). b Schematic model of the EdU assay used. WT and Tg(hsp70l:wnt8a-GFP)^w34 transgenic embryos were heat shocked at 24 hpf. Embryos of all genotypes were injected with EdU into the circulatory system at 30 hpf and analyzed at 52 hpf. c, g, k, o Reconstructions of confocal z-stacks showing representative hearts at 52 hpf of (c) WT, (g) upon Wnt8a overexpression, (k) in nkx2.5^vu179 mutants, or (o) in nkx2.5/nkx2.7 double morphants. Endocardial tissue is marked by Tg(kdrl:EGFP)^s843 or Tg(fli1a:nEGFP)^y7 reporters (white) and proliferative cells are marked by EdU incorporation (red). A, atrium; V, ventricle. Scale bars, 30 μm. d–f, h–j, l–n, p–r Shown are magnifications of insets (yellow boxes) with kdrl:GFP or fli1a:nEGFP/EdU double positive cells (yellow arrowhead). Single channel with kdrl:GFP or fli1a:nEGFP (d, h, l, p), EdU incorporation (e, i, m, q) and the merge of both channels (f, j, n, r). Scale bars, 10 μm. s Quantifications of the share of EdU + atrial or ventricular endocardial cells relative to the total number of endocardial cells within the respective cardiac chamber in WT (kdrl:GFP n = 8 hearts, fli1a:nEGFP n = 13 hearts), upon Wnt8a overexpression (n = 9 hearts), nkx2.5^vu179 mutants (n = 8 hearts), or nkx2.5/nkx2.7 double morphants (n = 8 hearts). Upon Wnt8a overexpression or loss of Nkx2.5 or Nkx2.5/Nkx2.7, proliferation significantly increases in the developing atrial endocardium. Mean values ± SD are shown. Two-way ANOVA was used to compare each condition with its WT control in each individual chamber (ns: not significant; ***p < 0.001, ****p < 0.0001)