Takehiro Kusakabe

Primary structure and differential gene expression of three membrane forms of guanylyl cyclase found in the eye of the teleost Oryzias latipes.

Three cDNAs (OlGC3,OlGC4, and OlGC5) encoding membrane guanylyl cyclases were isolated from a medaka (Oryzias latipes) eye cDNA library. An open reading frame for OlGC3 predicted a protein of 1057 amino acids, and those for OlGC4 andOlGC5, 1134 and 1151, respectively. These proteins consist of an apparent signal peptide (21 residues for OlGC3, 50 residues for OlGC4, and 48 residues for OlGC5) and a single transmembrane domain that divides the protein into an amino-terminal extracellular domain and a carboxyl-terminal intracellular domain that further divides into a kinase-like domain and a cyclase catalytic domain. Phylogenetic analysis with amino acid sequences of OlGC3, OlGC4, and OlGC5, as well as those of other membrane guanylyl cyclases, indicated that OlGC3, OlGC4, and OlGC5 are members of the sensory organ-specific guanylyl cyclase family. Reverse transcription-polymerase chain reaction and Northern blot analyses demonstrated that OlGC3,OlGC4, and OlGC5 transcripts are present in the eye, which contains more cGMP than the other organs. In addition to being expressed in the eye, OlGC3 transcripts are also present in the brain, heart, liver, pancreas, and ovary, whileOlGC4 is present in the liver and OlGC5 in the heart. Reverse transcription-polymerase chain reaction analysis with RNA from unfertilized eggs and embryos showed that OlGC3and OlGC5 are expressed both maternally and zygotically, while OlGC4 is expressed only zygotically, and that the zygotic expression of these three genes is differentially activated. These results suggest a structural and functional diversity of sensory organ-specific guanylyl cyclases in vertebrates.

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

Abstract. The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates.n

Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: Implications for conserved mechanisms of chordate development

Abstractu2002The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfMA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3’ untranslated region of BfCA1 contains a sequence that is similar to the ”zipcode” sequence of chicken β-cytoplasmic actin gene, which is thought to direct intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage. In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes.

Tandem Organization of Medaka Fish Soluble Guanylyl Cyclase α1 and β1 Subunit Genes IMPLICATIONS FOR COORDINATED TRANSCRIPTION OF TWO SUBUNIT GENES

We determined the complete nucleotide sequences of the α1 subunit gene (OlGCS-α 1) and the β1 subunit gene (OlGCS-β 1) of medaka fish soluble guanylyl cyclase. In the genome, OlGCS-α 1 andOlGCS-β 1 are organized in tandem. The two genes are only 986 base pairs apart and span approximately 34 kilobase pairs in the order of OlGCS-α 1 andOlGCS-β 1. The nucleotide sequence of a large part of the 5′-upstream region of OlGCS-α 1 is complimentarily conserved in that ofOlGCS-β 1. To analyze the promoter activity of each gene, a fusion gene construct in which the 5′-upstream region was fused with the green fluorescent protein gene was injected into medaka fish 2-cell embryos. When the fusion gene containing theOlGCS-α 1 upstream region was injected, green fluorescent protein fluorescence was detected in the embryonic brain. The 5′-upstream region of OlGCS-β 1alone was insufficient for the reporter gene expression in the embryos. When the OlGCS-α 1 upstream region was located upstream of the OlGCS-β 1-green fluorescence protein fusion gene, the reporter gene was expressed in the brain and trunk region of the embryos. These results suggest that the 5′-upstream region of OlGCS-α 1 can affect the expression of OlGCS-β 1. It is therefore possible that the expression ofOlGCS-α 1 and OlGCS-β 1 is coordinated.

The Guanylyl Cyclase Family in Medaka Fish Oryzias latipes

Abstract Guanylyl cyclase (GC) converts GTP into cGMP, an intracellular second messenger involved in a wide variety of cellular, developmental, and neuronal processes. Medaka fish, a small teleost, Oryzias latipes has been used to study organization and transcriptional regulation of the guanylyl cyclase gene family. Medaka fish expresses virtually all types of GCs found in mammals. Eight membrane GCs (OlGC1-7 and OlGC-R2) have been identified in medaka fish. OlGC1, OlGC2, and OlGC7 belong to the natriuretic peptide receptor subfamily. OlGC6 is a homologue of the mammalian GC-C, an enterotoxin/guanylin receptor, expressed predominantly in the intestine. OlGC3, OlGC4, OlGC5, and OlGC-R2 are members of the sensory organ-specific GC subfamily where they are differentially expressed in rods and cones of the retina and in the pineal organ. Complete genomic DNA sequences have been determined for the OlGC1 and OlGC6 genes. Their exon-intron organization is highly conserved between fish and mammals. The medaka fish genome also contains genes encoding α and β subunits of the cytoplasmic form of GC (soluble GC), which is activated by nitric oxide. The two subunit genes are closely linked in tandem in the order of α and β. Function of cis-regulatory regions of medaka fish GC genes have been investigated in transgenic medaka fish embryos and in mammalian cell lines. The upstream region of the α subunit gene of soluble GC appears to regulate expression of both α and β subunit genes, suggesting a mechanism of coordinated transcription of the two subunit genes. The upstream regions sufficient for the tissue-specific expression of sensory organ GCs also have been determined by transgenic analysis. Readiness for genetics and genetic manipulations in medaka fish would make this small fish a useful experimental system for studying the regulation of gene expression and roles of the guanylyl cyclase family in vertebrates.

Ascidian Actin Genes: Developmental Regulation of Gene Expression and Molecular Evolution

Abstract Actin is a ubiquitous protein in eukaryotic cells and plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. Multiple genes encoding muscle or nonmuscle actins have been isolated from several species of ascidians and their expression patterns have been investigated. Sequence and expression analyses of muscle actin genes have shown that ascidians have at least two distinct isoforms of muscle actin, the larval muscle and body-wall isoforms. In the ascidian Halocynthia roretzi, two clusters of actin genes are expressed in the larval muscle cells. The HrMA2/4 cluster contains at least five actin genes and the HrMA1 cluster contains a pair of actin genes whose expression is regulated by a single bidirectional promoter. cis-Regulatory elements essential for muscle-specific expression of a larval muscle actin gene HrMA4a have been identified. The adult body-wall muscle actin is clearly distinguished from the larval muscle actin by diagnostic amino acids. The adult muscle actin genes may be useful tools to investigate the mechanisms of muscle development in ascidian adults. The evolution of chordate actin genes has been inferred by comparing the organization and sequences of actin genes and performing molecular phylogenetic analysis. The results suggest a close relationship between ascidian and vertebrate actins. The chordate ancestor seems to have evolved the “chordate-type” cytoplasmic and muscle actins before its divergence into vertebrates and urochordates. The phylogenetic analysis also suggests that the vertebrate muscle actin isoforms evolved after the separation of the vertebrates and urochordates. Muscle actin genes have been used to investigate the mechanism of muscle cell regression during the evolution of anural development. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression. Actin genes have provided a unique system to study developmental and evolutionary mechanisms in chordates.

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203-215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon-intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages, and gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.

Localization of mRNAs encoding α and β subunits of soluble guanylyl cyclase in the brain of rainbow trout: comparison with the distribution of neuronal nitric oxide synthase

Abstract Detailed distribution of mRNAs encoding α and β subunits of soluble guanylyl cyclase (sGC) was examined in the brain of rainbow trout by in situ hybridization. In addition, distribution of nitric oxide synthase (NOS) was mapped in adjacent parallel sections by neuronal NOS (nNOS) immunocytochemistry and NADPH-diaphorase (NADPHd) histochemistry. Following application of digoxigenin-labeled riboprobes for sGC α and β subunit mRNAs, we found comparatively intense hybridization signals in the telencephalon, preoptic area, thalamus, hypothalamus, pretectum and tegmentum. Both nNOS immunocytochemistry and NADPHd histochemistry showed extensive distribution of nitroxergic neurons in various brain areas, although various degrees of dissociation of nNOS immunoreactivity (ir) and NADPHd staining were detected. In comparison with sGC subunit mRNAs, nNOS signals were more widely distributed in many neurons, including parvocellular neurons in the preoptic area, nucleus anterior tuberis in the hypothalamus, periventricular neurons in the optic tectum, most of the rhombencephalic neurons and pituitary cells. However, wide overlaps of sGC mRNA-containing neurons and nNOS-positive neurons were observed in the olfactory bulb, telencephalon, preoptic area, thalamus, hypothalamus, pretectum, optic tectum, tegmentum and cerebellum. The widespread overlapping in sGC subunit mRNAs and nNOS distribution suggests a role for sGC in various neuronal functions, such as processing of olfactory and visual signals and neuroendocrine function, possibly via NO/cGMP signaling in the brain of rainbow trout.

Photoreceptors and olfactory cells express the same retinal guanylyl cyclase isoform in medaka: visualization by promoter transgenics1

We examined the spatial expression patterns of two orphan receptor guanylyl cyclase genes OlGC4 and OlGC5 during embryogenesis of medaka and characterized the 5′ flanking region required for tissue‐specific expression of OlGC4 by introducing promoter–GFP fusion constructs into medaka embryos. Expression of OlGC5 is confined to retinal photoreceptor cells, while OlGC4 is expressed in the retina, pineal organ, and olfactory pits. The OlGC4 upstream region between −2374 and +343 is sufficient to drive the sensory organ‐specific gene expression. Mutations in the consensus binding sequences for OTX/CRX transcription factors did not impair the reporter gene expression. Our results suggest that the same isoform of guanylyl cyclase is utilized in both photoreceptors and olfactory cells, and that transcription factors other than OTX/CRX primarily activate the OlGC4 expression.

A cis-regulatory element essential for photoreceptor cell-specific expression of a medaka retinal guanylyl cyclase gene.

Abstract. We examined the spatial expression pattern of an orphan receptor guanylyl cyclase gene, OlGC3, during embryogenesis of medaka, and we analyzed the cis-regulatory region required for its photoreceptor cell-specific expression by introducing promoter-green fluorescent protein gene (GFP) fusion constructs into medaka embryos. Zygotic expression of OlGC3 first appears at stage 36 and is confined to retinal photoreceptor cells. The 5′ flanking sequence up to –388 is sufficient to drive the photoreceptor cell-specific gene expression. When a mutation was introduced into the orthodenticle-related homeobox (OTX)-binding consensus sequence at –36/–31, reporter gene expression was completely abolished in retinal photoreceptor cells. The mutation seemed not to impair promoter elements for general transcription factors, because ectopic GFP expression in yolk epithelium was not diminished by the same mutation. These results suggest that the proximal cis-regulatory element containing a consensus binding sequence for OTX transcription factors is essential for OlGC3 expression in photoreceptor cells.