Figure 1. The IFT is expanded in smo mutants by 24 hpf.

(A-J) Wild-type (wt; A,C,E,G,I) and smo mutant (B,D,F,H,J) hearts at 48 hpf are shown after in situ hybridization (A-H) or after immunofluorescence (I,J). Frontal views; arrowheads indicate the IFT. (A,B) bmp4 expression is expanded in the venous pole of smo mutants relative to wt siblings (n=8 wt, 12 smo). (C-H) hcn4, tbx18, and shox2 are similarly expanded in the venous pole of smo (n=10 wt, 12 smo for hcn4; n=12 wt, 12 smo for tbx18; n=26 wt, 13 smo for shox2). (I,J) Isl1 (white) is present in the nuclei of IFT cardiomyocytes; myocardium is labeled with MF20 (green). More Isl1+ cardiomyocytes are observed in the IFT of smo mutants than in wt siblings (n=15 wt, 11 smo). Scale bars: 50 μm.

(K-P) In situ hybridization depicts bmp4 expression in wt (K,M,O) and smo (L,N,P). Frontal views; arrowheads indicate the IFT. Expression of bmp4 is already expanded in the IFT of smo mutants, relative to wt siblings, at 32 hpf (K,L; n=6 wt, 8 smo). This expansion is maintained at 40 (M,N; n=8 wt, 10 smo) and 48 hpf (O,P; n=8 wt, 12 smo).

(Q) Graph indicates the number of Isl1+ cardiomyocytes in the IFT. See Materials and Methods for cell counting technique. The number of Isl1+ cardiomyocytes in smo mutants, relative to wt siblings, is increased as early as 24 hpf. This increase is maintained at 32 and 48 hpf. The number of Isl1+ cardiomyocytes in wt is also steadily maintained throughout this timeframe. **p<0.01; ****p<0.0001.