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Fig. 3 Loss of Grem2 leads to cardiac jogging and looping defects, aberrant expression ofpitx2and abnormal development of cardiac chambers. (A) In situ hybridization analysis using cmlc2 riboprobe shows that in wild-type (WT) embryos the heart jogs leftward, whereas cardiac morphogenesis is randomized in grem2 morphants (grem2 MO). The cardiac tube, consisting of a single atrium (A, white arrowheads) and a single ventricle (V), is thinner, shorter and fails to loop in grem2 morphants. Left/Right (L/R) axis orientation is indicated. (B) In situ hybridization analysis using vmhc and amhc riboprobes shows that loss of Grem2 leads to lower expression levels of both vmhc and amhc, with amhc being essentially absent at 19 hpf (arrowheads). Pitx2 expression (black arrowheads) at 19 hpf is enhanced (white arrowheads), whereas lefty2 expression at 24 hpf is abolished (black arrowheads). Quantification of expression levels in wild-type and morphant embryos is shown below the corresponding images (expression is relative to wild type, which was set as arbitrary value 1). Immunofluorescence analysis using MF20 (red, labels both ventricular and atrial cardiomyocytes) and S46 (green, labels only atrial cardiomyocytes, which appear yellow) antibodies shows abnormal development of both chambers, with the atrium being more deformed and reduced in size than the ventricle. Arrowheads mark the position of the atrio-ventricular boundary. Scale bars: 50 μm. *P<0.05.