Localization of the nitric oxide/cGMP signaling pathway-related genes and influences of morpholino knock-down of soluble guanylyl cyclase on medaka fish embryogenesis

Abstract

To better understand the nitric oxide (NO) / cyclic GMP (cGMP) signaling pathway during embryogenesis, we examined the spatial and temporal expression pattern of the genes for neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase (soluble GC) subunit (OIGCS-α1, OIGCS-α2, and OIGCS-β1), and cGMP-dependent protein kinase (cGK) I and II (cGK I and cGK II) in the medaka fish embryos. OIGCS-β1 and nNOS were expressed maternally and OIGCS-α1, OIGCS-α2, cGK I, and cGK II were expressed zygotically. The zygotic expression of OIGCS-α1 and cGK I was detected at stage 19, while that of OIGCS-α2 was detected at stage 16. Whole-mount in situ hybridization showed that the expression of nNOS or cGK I was localized in tail bud, otic vesicles, thyroid, and brain ventricle, or in thymus, gill arch, and olfactory pits, respectively, and that of OIGCS-α1, OIGCS-α2, or OIGCS-β1 was dim and dispersed throughout the embryos. To clarify the "role of the NO/cGMP signaling pathway in embryogenesis, we examined the influences of morpholino antisense oligonucleotide of the soluble GC subunit gene (α1-MO, α2-MO or β1-MO) on development of medaka fish embryos. Embryos injected with α1-MO or α2-MO mainly exhibited abnormalities in the central nervous system, including defects in the formation of forebrain, eye, and otic vesicles, α2-MO injection caused cell death at the tail bud of the embryos at stage 22, and β1-MO injection inhibited the development of the embryos at late blastula. These results suggest that the NO/cGMP signaling pathway plays critical roles in early embryogenesis.