Fig. 1

Nr2f1a is expressed in ACs and generation of anr2f1amutant allele. (A,B) IHC for MHC (red), AHMC (purple), and Nr2f1a (green) at 24 and 48 hpf. Nr2f1a⁺ nuclei in cells at the venous pole adjacent to AMHC⁺ cells (yellow arrowheads), which we propose are putatively atrial progenitors. Expression in nuclei of ACs (white arrows). Blue brackets indicate AMHC⁺ region devoid of Nr2f1⁺ nuclei. Images are Z-stacks of confocal sections. V indicates ventricle and A indicates atrium in all figures. Scale bar indicates 10 µm in A,B. ≥ 10 hearts were examined at each stage. (C) Schematic of deletion in nr2f1a exon 1 from the TALENs. (D) Sequence of the 8-bp deletion in the nr2f1a^el512 mutant allele. (E) Schematic indicating the predicted truncation caused by the deletion. Activation domain – AF1 (red), DNA-binding domain – DBD (green), Ligand binding domain – LBD (yellow). Black indicates amino acids after the protein goes out of frame. (F,G) WT and nr2f1a^el512-/- mutant embryos at 48 hpf. (H) Western blot indicating that Nr2f1a protein is lost in nr2f1a mutants. (I,J) Confocal images of whole mount IHC indicating Nr2f1a protein is expressed in the same anatomical structure as shown previously with ISH (Love and Prince, 2012) and is lost in nr2f1a mutants. Images are the dorsolateral with anterior left. e – eyes, p – pharyngeal (arrow in I), c – cerebellum, hb –hindbrain.>20 wt sibling and nr2f1a mutant embryos were examined.