Fig. 3

NO Signaling Regulates Liver Size in a cGMP-Independent Manner Involving Modulation of S-Nitrosothiol Homeostasis

(A) Effect of chemical modulators of NO signaling on liver formation in cloche mutants that lack a vasculature. Cloche mutants and siblings (sib) were exposed to chemicals (5 μM) from 24 to 72 hpf and subjected to in situ hybridization for the hepatocyte gene lfabp. Representative photomicrographs were taken at 10× magnification.

(B and C) Phenotype analysis of liver size in WT and cloche mutants exposed to the cGMP analog, 8BrGMP (10 μM), or the S-nitrosothiol GSNO (10 μM) from 24 to 72 hpf as determined by in situ hybridization for lfabp (n > 25 embryos per treatment).

(D) Effect of chemical inhibition or morpholino-mediated knockdown of GSNOR on liver size as determined by in situ hybridization for the hepatocyte gene lfabp at 72 hpf. The translation blocking ATG morpholino against GSNOR was injected at a concentration of 50 ¼M. Representative photomicrographs were taken at 10× magnification.

(E) Phenotype analysis of liver size in GSNOR-deficient embryos at 72 hpf as determined by in situ hybridization for lfabp (n > 25 embryos per treatment).

(F) Loss of GSNOR function alters the percentage of hepatocytes specified during liver formation. Tg(lfabp:GFP) morphants were dissociated and the percentage of GFP positive hepatocytes was analyzed by FACS.

n = 4; ANOVA, p = 0.041, p = 0.002 in comparison to control. Results in (F) show mean ± SEM.