Expression and function of cGMP-dependent protein kinase type I during medaka fish embryogenesis.
We isolated and characterized cDNA clones (PKG Ialpha and PKG Ibeta) for medaka fish cGMP-dependent protein kinase (PKG) Ialpha and Ibeta, 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 Ialpha gene were present in the spinal cord and gill arch, whereas those of the PKG Ibeta gene were only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG Ialpha-specific morpholino antisense oligonucleotides (Ialpha-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 Ibeta-specific morpholino antisense oligonucleotides (Ibeta-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than Ialpha-MO. The PKG I-overexpressing embryos exhibited smaller eyes and enlargement of the forebrain, a phenotype 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-3beta, 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.
Yamamoto
Takehiro
T
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. take-y@sc.itc.keio.ac.jp
Suzuki
Norio
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000096:biosynthesis* / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D005880:Gills / Q000196:embryology / Q000201:enzymology; D053823:Hedgehog Proteins; D051542:Hepatocyte Nuclear Factor 3-beta; D006822:Hybrid Cells / Q000378:metabolism; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009990:Oryzias; D010641:Phenotype; D010766:Phosphorylation; D016548:Prosencephalon / Q000196:embryology; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013116:Spinal Cord / Q000196:embryology / Q000201:enzymology; D013997:Time Factors; D014018:Tissue Distribution; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D016335:Zinc Fingers
D003470:Culture Media; D004268:DNA-Binding Proteins; C496503:Foxa2 protein, mouse; C496504:Foxa2 protein, rat; D053823:Hedgehog Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569720:Prkg1 protein, mouse