Expression and Function of cGMP-dependent Protein Kinase Type I during Medaka Fish Embryogenesis

Abstract

We isolated and characterized cDNA clones (PKG I␣ and PKG I␤) for medaka fish cGMP-dependent protein kinase (PKG) I␣ and I␤, and demonstrated that both are expressed in the embryos after late gastrula stage. Whole-mount in situ hybridization using each isoformspecific probe revealed that the transcripts of the PKG I␣ gene were present in the spinal cord and gill arch, whereas those of the PKG I␤ gene were only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG I␣-specific morpholino antisense oligonucleotides (I␣-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 I␤-specific morpholino antisense oligonucleotides (I␤-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than I␣-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 PKGdeficient embryos, a sonic hedgehog (shh)-target gene, HNF-3␤, 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.As the intracellular second messengers, the cyclic nucleotides alter various cellular functions predominantly by activation of specific protein kinases. It is known that natriuretic peptides and/or nitric oxide (NO) 1 are involved in many biological events, such as the regulation of vascular smooth muscle tone, reabsorption of salt and fluid into the kidney, platelet aggregation, and synaptic transmission (1). Natriuretic peptides and/or NO activate guanylyl cyclases (GCs) that catalyze the conversion of GTP to cGMP, followed by activation of downstream effectors like cGMP-dependent protein kinase (PKG), phosphodiesterase, and cyclic nucleotide-gated cation channels (1

Visual pattern memory requires foraging function in the central complex of Drosophila

“…On the other hand, for may also affect developmental processes that facilitate visual pattern memory to a certain extent. This is not surprising, though, as PKG has been shown to be involved in the development of the nervous system in Drosophila and other species (Renger et al 1999;Yamamoto and Suzuki 2005;Zhang et al 2005).…”

Role of the ANKMY2-FKBP38 Axis in Regulation of the Sonic Hedgehog (Shh) Signaling Pathway

“…In this regard, it is of note that cGMP activates Shh signaling in mammals (38). Given that protein kinase G (PKG) activated by cGMP phosphorylates Gli3, resulting in activation of Shh signaling (39), it is possible that the FKBP38-ANKMY2 axis controls Shh signaling via the cGMP-PKG pathway.…”

Inhibition of cGMP-Dependent Protein Kinase Reduces the Response to Sonic Hedgehog in Neuralized Embryoid Bodies

“…We and others have demonstrated that an increase of intracellular cGMP concentration enhances the Shh response (13,14). At least two distinct classes of guanylyl cyclases (GCs), which catalyze the conversion of GTP into cGMP, can be activated in response to extracellular cues.…”

“…Six of these sites are present in the sequence of Ci, and several are conserved in Gli1 and Gli2. In NO-sensitive neuroblastoma cells, Gli3 is phosphorylated after treatment of 8-bromo-cGMP (14). Because Gli3 is also phosphorylated by PKA, PKG could enhance Shh signaling either by phosphorylating Gli3 at distinct sites or by interfering with phosphorylation of Gli3 by PKA.…”

“…However, early neural patterning has not been investigated in these animals, and subtler or transient defects may not have been detected, but it remains a possibility that cGMP signaling may not in fact be important for dorsal-ventral patterning in vivo. In support of a role for PKG-I in development, knockdown of PKG-I activity in medaka fish showed a decrease of Shh responsiveness (14). In addition, part of the effects of cGMP may be mediated through PKG-II or PKG-I␤ rather than PKG-I␣, or may be mediated through something other than PKG, such as a cyclic nucleotide-gated channel (38,39) or a phospho-diesterase (PDE) (40).…”