Expression and function of cGMP-dependent protein kinase type I during medaka fish embryogenesis

We isolated and characterized cDNA clones (PKG Iα and PKG Iβ) for medaka fish cGMP-dependent protein kinase (PKG) Iα and Iβ, and demonstrated that both are expressed in the embryos after late gastrula stage. Whole-mount in situ hybridization using each isoform-specific probe revealed that the transcripts of the PKG Ia gene were present in the spinal cord and gill arch, whereas those of the PXG Iβ gene were only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG Iα-specific morpholino antisense oligonucleotides (Iα-MO) into two-cell stage medaka fish embryos caused severe abnormalities in the developing embryos, such as the development of a hammer-like head, fusion of the developing eyes, and degeneration of cells around the eyes, whereas injection of PKG Iβ-specific morpholino antisense oligonucleotides (Iβ-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than Iα-MO. The PKG I-overexpressing embryos exhibited smaller eyes and enlargement of the forebrain, a pheno-type similar to that observed in the cAMP-dependent protein kinase (PKA)-depressed embryos. In the PKG-deficient embryos, a sonic hedgehog (shh)-target gene, HNF-3β, was expressed weakly, and this phenotype was similar to that observed in the PKA-overexpressing embryos suggesting that the cGMP/PKG signaling pathway is involved in some steps of shh signaling. We also demonstrated that Gli proteins, shh-downstream molecules, are phosphorylated by the NO/cGMP signaling pathway, probably by PKG in NG108-15 neuroblastoma cells. These results imply that PKG and PKA share common substrates and work in an opposite manner during the early embryogenesis of medaka fish.