Fig. 1

Zygotic and Maternal Zygotic aoh Mutants Display Laterality Phenotypes and Defects in the Nodal Signaling Pathway

(A) Lateral view of WT and Zaoh mutant embryos at 48 hpf.

(B) Characterization of the Zaoh mutant heart and gut phenotype. ISH at 28 hpf for myl7 and at 55 hpf for Foxa3 (dorsal view; anterior top).

(C) At 26 hpf, MZaoh mutant embryos displayed slight developmental delay and a mild dorsalization phenotype in the tailfin (arrowhead). At 55 hpf, incidence of reversed gut looping and organ position was observed (arrowhead pointing at liver).

(D) ISH for spaw (arrowhead indicates anterior-ward expansion) in WT, Zaoh, and MZaoh embryos.

(E) FurinA protein and functional domains. Star, aoh mutation.

(F) Agarose beads preincubated in recombinant BMP and Nodal protein were implanted in MZaoh mutant embryos at 15 somites. Analysis of heart position was determined by myl7 ISH.

(G and H) Spaw-EGFP fusion protein (G). Furin recognition sites 1 and 2 are underlined. Changes in amino acids in the noncleavable forms are detailed (H) western blot with anti-GFP. Incubation of Spaw with Furin results in cleavage and appearance of the mature 41 kDa Spaw-EGFP (asterisk). Incubation of the different Spaw-EGFP variants with Furin demonstrates that cleavage happens only at the single (optimal) predicted Furin cleavage site 1.

(I) Dorsalization phenotypes induced by injection of Spaw-EGFP variants mRNA (1 picogram [pg]) at 24 hpf. Abbreviations, liver (l), pancreas (p), and gut (g).