Fig. 6

The Mg²⁺-sensitive Socs3a pathway is involved in the proliferative defect of the swd mutants. Supplementary Mg²⁺ improves the exocrine pancreas phenotype of the swd mutants that have repression of socs3a. (A) swd^p75fm mutants were incubated in E3 medium with or without supplementary 40 mM MgCl₂ for 72 or 96 hpf and analyzed for socs3a mRNA by real-time PCR. (B) swd^p75fm mutants and WT were incubated in E3 medium (containing 1.65 mM Mg²⁺) for 72 or 96 hpf, and the socs3a mRNA levels were determined by real-time PCR. (C) Embryos collected from swd^p75fm/+ intercross were microinjected with socs3a-ATG-MO or control MO, incubated until 4 dpf and the exocrine pancreas (red arrows) was analyzed by whole-mount in situ hybridization using anti-trypsin riboprobes. The image is representative of 20 larvae in each group from three independent experiments. The swd^p75fm mutants injected with either non-targeting control MO or socs3a-5-mispair MO are indistinguishable in the exocrine pancreas by in situ hybridization using anti-trypsin riboprobes. (D) socs3a-ATG-MO- or control-MO-injected larvae were incubated until 72 hpf, injected with BrdU and then analyzed for the proportion of cells in S phase (% BrdU+ nuclei). (E) socs3a-ATG-MO- or control-MO-injected larvae were incubated until 96 hpf and analyzed for cell growth (area, in μm², per cell). *P<0.05 indicates statistically significant difference. (C-E) The swd^p75fm mutants were identified on the basis of their hypopigmented skin, which was not grossly affected by socs3a-ATG-MO.