Zygotic rnf2 mutant zebrafish embryos show a pleiotropic phenotype. (a) Lateral view of wildtype embryos (left panel) and rnf2 mutant embryos (right panel) at 3 dpf. The rnf2 mutants show a pleiotropic phenotype, including motility problems, craniofacial defects (arrowheads), lack of pectoral fins, and a pronounced heart edema (arrowheads). Not all phenotypes are visible in the pictures. Scale bar = 1 mm. (b) Expression of tissue-specific markers was assessed by WISH at 3 dpf in wildtype and rnf2 mutant embryos. fabp2: intestinal marker, fabp10: liver marker, try: exocrine pancreas marker (arrowhead indicates pancreatic lobe), and myl7: cardiomyocyte marker. Scale bar is 200 µm.

Zebrafish rnf2 mutant embryos show cardiac looping defects. (a) Fluorescent images of wildtype and rnf2 mutant sibling embryos in a Tg(myl7::GFP) background at 3 dpf. Scale bar is 500 µm. (b) Stills of live-imaging by light sheet microscopy of wildtype and rnf2 mutant hearts in a Tg(myl7::GFP) background starting at 1 dpf. Scale bar is 100 µm.

Cardiac chamber identity is disrupted in rnf2 mutants at 3 dpf. (a) GSEA using the list of cardiac genes reported by Hill et al.³⁸ indicates that the group of transcription factors is significantly enriched in being upregulated in rnf2 mutant hearts at 3 dpf as detected by Single HeartsRNA-seq (FDR q-value = 0.001; NES = 1.96). The leading transcription factors (n = 9) are listed in the zoom of the GSEA plot. (b) GSEA indicates the annotation ‘Structural Group’ genes (n = 23) to be enriched for downregulation upon the rnf2 mutation in hearts at 3 dpf. The leading genes (n = 8) are listed in the zoom of the GSEA plot. FDR q-value = 0.054; NES = −1.67. (c) Normalized counts as found by Single HeartsRNA-seq for nppa, myl7, myh6, and vmhc at 3 dpf in wildtype and rnf2 mutant hearts with their accompanying whole mount in situ hybridizations. Scale bar is 200 µm.