Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Primary Structure and Differential Gene Expression of Three Membrane Forms of Guanylyl Cyclase Found in the Eye of the TeleostOryzias latipes

1997; Elsevier BV; Volume: 272; Issue: 37 Linguagem: Inglês

10.1074/jbc.272.37.23407

ISSN

1083-351X

Autores

Masato Seimiya, Takehiro Kusakabe, Norio Suzuki,

Tópico(s)

Neurobiology and Insect Physiology Research

Resumo

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. 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. Cyclic GMP (cGMP) is a ubiquitous second messenger in intracellular signaling cascades and responsible for a wide variety of physiological responses. Guanylyl cyclases (GCs) 1The abbreviations used are: GC, guanylyl cyclase; cGMP, guanosine 3′,5′-cyclic monophosphate; OlGC, O. latipes guanylyl cyclase; OlGC, gene encoding O. latipes guanylyl cyclase; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotides; UTR, untranslated region; bp, base pair(s); kb, kilobase(s). 1The abbreviations used are: GC, guanylyl cyclase; cGMP, guanosine 3′,5′-cyclic monophosphate; OlGC, O. latipes guanylyl cyclase; OlGC, gene encoding O. latipes guanylyl cyclase; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotides; UTR, untranslated region; bp, base pair(s); kb, kilobase(s).compose a small family of proteins that catalyze the conversion of GTP to cGMP (1Drewett J.G. Garbers D.L. Endocr. Rev. 1994; 15: 135-162Crossref PubMed Scopus (321) Google Scholar, 2Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar). The cyclases are grouped into two major forms, those found on the plasma membrane (membrane GCs) and those in the cytoplasm (soluble GCs). The soluble GC is a heme-containing heterodimeric protein that is activated by binding of nitric oxide (3Kamisaki Y. Saheki S. Nakane M. Palmieri J.A. Kuno T. Chang B.Y. Waldman S.A. Murad F. J. Biol. Chem. 1986; 261: 7236-7241Abstract Full Text PDF PubMed Google Scholar, 4Humbert P. Niroomand F. Fischer G. Mayer B. Koesling D. Hinsch K.-D. Gausepohl H. Frank R. Schultz G. Böhme E. Eur. J. Biochem. 1990; 190: 273-278Crossref PubMed Scopus (143) Google Scholar). Analysis of cDNA clones for soluble GC subunits have shown that each subunit contains a cyclase catalytic domain which is also conserved in the membrane GCs (5Koesling D. Hertz J. Gausepohl H. Niroomand F. Hinsch K.-D. Mülsch A. Böhme E. Schultz G. Frank R. FEBS Lett. 1988; 239: 29-34Crossref PubMed Scopus (116) Google Scholar, 6Koesling D. Harteneck C. Humbert P. Bosserhoff A. Frank R. Shultz G. Böhme E. FEBS Lett. 1990; 266: 128-132Crossref PubMed Scopus (101) Google Scholar, 7Nakane M. Saheki S. Kuno T. Ishii K. Murad F. Biochem. Biophys. Res. Commun. 1988; 157: 1139-1147Crossref PubMed Scopus (123) Google Scholar, 8Nakane M. Arai K. Saheki S. Kuno T. Buechler W. Murad F. J. Biol. Chem. 1990; 265: 16841-16845Abstract Full Text PDF PubMed Google Scholar).The membrane GCs consist of a single polypeptide with about 150–200 kDa. In mammals, cDNA clones for six membrane GCs (GC-A, GC-B, GC-C, GC-D, GC-E, and GC-F) have been isolated and characterized (9Chinkers M. Garbers D.L. Chang M.-S. Lowe D.G. Chin H. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (881) Google Scholar, 10Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 11Schulz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 12Singh S. Lowe D.G. Thorpe D.S. Rodriguez H. Kuang W.-J. Dangott L.J. Chinkers M. Goeddel D.V. Garbers D.L. Nature. 1988; 334: 708-712Crossref PubMed Scopus (199) Google Scholar, 13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar, 14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). The predicted proteins contain an extracellular region, a kinase-like domain, and a cyclase catalytic domain. GC-A and GC-B are activated by binding of natriuretic peptides to their extracellular domain (2Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar, 9Chinkers M. Garbers D.L. Chang M.-S. Lowe D.G. Chin H. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (881) Google Scholar, 10Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 15Chang M.-S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Garbers D.G. Nature. 1989; 341: 68-72Crossref PubMed Scopus (498) Google Scholar, 16Lowe D.G. Chang M.-S. Hellmiss R. Chen E. Singh S. Garbers D.L. Goeddel D.V. EMBO J. 1989; 8: 1377-1384Crossref PubMed Scopus (313) Google Scholar), and GC-C activity is stimulated by extracellular binding of heat-stable enterotoxin and guanylin (17Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (502) Google Scholar, 18de Sauvage F.J. Keshav S. Kuang W.-J. Gillett N. Henzel W. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9089-9093Crossref PubMed Scopus (131) Google Scholar, 19Schulz S. Chrisman T.D. Garbers D.L. J. Biol. Chem. 1992; 267: 16019-16021Abstract Full Text PDF PubMed Google Scholar).Sensory neural tissues such as olfactory neurons and retina express specific isoforms of membrane GC. GC-D is specifically expressed in a subpopulation of olfactory sensory neurons (13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar). Two retina-specific GCs (RetGC-1 and RetGC-2) have been isolated and characterized from human retina (20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). These GCs are expressed mostly in the photoreceptor cells, and their expression patterns are indistinguishable. Two membrane GCs, GC-E and GC-F, have also been isolated from a rat eye cDNA library (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). GC-E and GC-F are thought to be orthologs of human RetGC-1 and RetGC-2. Rat GC-E and GC-F show different expression patterns. GC-E is expressed in the eye and pineal gland, whereas the expression of GC-F is confined to the eye (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). Contrary to GC-A, GC-B, and GC-C, no extracellular ligand has been reported for olfactory tissue- and eye-specific membrane GCs. Instead, the existence of two activators, GCAP-1 and GCAP-2, for retina-specific membrane GCs has been reported in bovine rod outer segment membranes (21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar, 22Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Both activators are a Ca2+-binding protein. GCAP-2 activates RetGC-1 and RetGC-2, whereas GCAP-1 activates RetGC-1 only. RetGC-1 is activated through binding of GCAP-2 to the intracellular domain, and the removal of the extracellular and transmembrane domains from RetGC-1 does not significantly alter the activation by GCAP-2 (24Laura R.P. Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 11646-11651Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).In Drosophila, some membrane GC transcripts are present in the unfertilized egg, along with embryos in the early developmental stages (25Gigliotti S. Cavaliere V. Manzi A. Tino A. Graziani F. Malva C. Dev. Biol. 1993; 159: 450-461Crossref PubMed Scopus (20) Google Scholar, 26McNeil L. Chinkers M. Forte M. J. Biol. Chem. 1995; 270: 7189-7196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This suggests that the cGMP signaling pathway mediated by a membrane GC is involved in the early development ofDrosophila. Despite rapidly accumulating information concerning the molecular nature of membrane GCs, there are few reports related to the expression of membrane GCs during vertebrate development or to the molecular phylogenetic relationship among membrane GCs. In this study, to understand the developmental role and diversity of membrane GCs in vertebrates, we begin the characterization of membrane GCs in medaka fish (Oryzias latipes), a species allowing for both classical and molecular genetic analyses.In this first report, we discuss the isolation and characterization of cDNA clones encoding three different membrane GCs expressed in the medaka eye, and the phylogenetic relationship of these membrane GCs. We also report that transcripts of two of the three medaka membrane GCs are present as maternal messages, and that each of these membrane GCs shows distinct expression patterns during embryogenesis and adulthood.DISCUSSIONIn this paper we report the characterization and expression patterns of three genes, OlGC3, OlGC4, andOlGC5, each encoding a different GC in the medaka fish.OlGC3, OlGC4, and OlGC5 are predominantly expressed in the eye, which contains more cGMP than any other organ, suggesting the important role(s) of these GCs in the phototransduction pathway. Hayashi and Yamazaki (49Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (78) Google Scholar) reported multiple forms of photoreceptor GCs in toad, frog, and bovine rods. In mammals, two forms of membrane GC have been identified in the eye (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar, 20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). Our study demonstrated that at least three related membrane GCs encoded by different genes are present in the fish eye. The presence of multiple forms of membrane GC in the eye seems to be common among a wide variety of vertebrates. Since the amino acid sequences of OlGC3, OlGC4, and OlGC5 are related but discrete, the enzyme activity of these GCs may be regulated via different pathways. Alternatively, these membrane GCs might show different cellular and/or subcellular distribution patterns in the eye.The size of the major transcripts for OlGC3,OlGC4, and OlGC5 is 3.5, 8.8, and 6.2 kb, respectively. Although the transcripts of OlGC4 andOlGC5 are larger than those for most known membrane GCs (about 4.0 kb or smaller), the transcript lengths of 8.5 and 11 kb have been reported for the rat eye-specific GCs, GC-E and GC-F, respectively (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). The increase in size seems to be attributable to large 3′- and 5′-UTR sequences (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). Relatively large transcript size may thus be a common feature among vertebrate eye membrane GCs.In addition to the major band of 3.5 kb, a weaker signal of 4.3 kb was detected for OlGC3. Similarly, two weaker bands of 7.2 and 5.3 kb were detected for OlGC4. The OlGC3transcripts of different size may be attributable to transcripts with differently sized 3′-UTR, because we isolated a cDNA clone with a sequence identical to that of OlGC3 except for the size of 3′-UTR. It is uncertain whether the OlGC4 transcripts of different size are due to differently sized UTR. The size of cDNA sequences for OlGC3, OlGC4, and OlGC5determined in the present study is 4110, 4651, and 5839 bp, respectively. These cDNA sequences probably correspond to transcripts of 4.3, 5.3, and 6.2 kb detected in the Northern blot analysis, respectively. The OlGC3 cDNA clone with smaller 3′-UTR may correspond to the 3.5-kb transcripts ofOlGC3.The phylogenetic analysis of intracellular catalytic domains revealed that there are three major groups of membrane GCs in vertebrates: (i) natriuretic peptide receptors, (ii) enterotoxin receptors, and (iii) sensory organ-specific GCs. OlGC3, OlGC4, and OlGC5 are grouped with the sensory organ-specific GCs. These three groups probably represent three distinct subfamilies of membrane GCs that are defined by families of ligands or activators as well as by localization. The phylogenetic tree of extracellular domains also suggests a close relationship among sensory organ-specific GCs, including OlGC3, OlGC4, and OlGC5. Within the sensory organ-specific GC subfamily, relationships among members were different between the phylogenetic trees of intracellular catalytic and extracellular domains. This may reflect different functional constraints on the structure of extracellular and intracellular domains.The relative positions of some amino acids, including six cysteine residues, are highly conserved within the extracellular domains of the sensory organ-specific GCs. Although no extracellular ligands have been reported for the sensory organ-specific GCs, the conservation of these residues suggests a functional importance of the extracellular domains. These conserved cysteine residues may be involved in intramolecular disulfide bond formation and/or oligomerization of the proteins.The RT-PCR analysis showed that OlGC3, OlGC4, andOlGC5 genes are also expressed in a number of organs other than the eye and that the expression patterns in adults are different among the three genes. In contrast, previous studies on mammalian eye- and olfactory neuron-specific GCs revealed that their expression is confined to sensory organs and related tissue (pineal gland) (13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar, 14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar,20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). The difference between the reported expression patterns of medaka GCs and mammalian sensory organ GCs might be attributable to differences between fish and mammals or simply to differences in detection methods. Alternatively, since the sequences of OlGC3, OlGC4, and OlGC5 are relatively diverged from those of any known mammalian sensory organ GCs, there may be unidentified members of the mammalian sensory organ GC subfamily that exhibit sequences and expression patterns similar to those of OlGC3, OlGC4, or OlGC5. The presence of transcripts of OlGC3, OlGC4, and OlGC5in various adult organs suggests that members of sensory organ GC subfamily play previously unrecognized roles in these organs. Since the Northern blot analysis failed to detect OlGC3 andOlGC4 mRNA in the liver and only faint signal forOlGC3 was detected in the brain and ovary, however, these organs may express much smaller amounts of the OlGC3and OlGC4 transcripts than does the eye.There have been few reports on the expression patterns of sensory organ GC genes during vertebrate development. Our results demonstrated that the transcripts of OlGC3 and OlGC5 are present in unfertilized eggs as maternal messages, suggesting that cGMP signaling pathways mediated by these membrane GCs are involved in the early development of fish. Since the OlGC3 transcripts are evident in unfertilized eggs as well as in the adult ovary, OlGC3 may play an important role during oogenesis. In Drosophila, membrane GCs are expressed during oogenesis and early development (25Gigliotti S. Cavaliere V. Manzi A. Tino A. Graziani F. Malva C. Dev. Biol. 1993; 159: 450-461Crossref PubMed Scopus (20) Google Scholar, 26McNeil L. Chinkers M. Forte M. J. Biol. Chem. 1995; 270: 7189-7196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). cGMP signaling pathways mediated by membrane GCs regulating oogenesis and early development may have been conserved between vertebrates and invertebrates. The RT-PCR analysis revealed that the three medaka GC genes are differentially activated during embryogenesis. The differing expression patterns of medaka GC genes suggest that these GCs have distinct roles during development. Two activators for eye-specific membrane GCs have been reported in bovine rod outer segment (21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar, 22Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Similar activators may regulate the activity of the medaka GCs during early development and in adult organs other than the eye. Cyclic GMP (cGMP) is a ubiquitous second messenger in intracellular signaling cascades and responsible for a wide variety of physiological responses. Guanylyl cyclases (GCs) 1The abbreviations used are: GC, guanylyl cyclase; cGMP, guanosine 3′,5′-cyclic monophosphate; OlGC, O. latipes guanylyl cyclase; OlGC, gene encoding O. latipes guanylyl cyclase; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotides; UTR, untranslated region; bp, base pair(s); kb, kilobase(s). 1The abbreviations used are: GC, guanylyl cyclase; cGMP, guanosine 3′,5′-cyclic monophosphate; OlGC, O. latipes guanylyl cyclase; OlGC, gene encoding O. latipes guanylyl cyclase; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotides; UTR, untranslated region; bp, base pair(s); kb, kilobase(s).compose a small family of proteins that catalyze the conversion of GTP to cGMP (1Drewett J.G. Garbers D.L. Endocr. Rev. 1994; 15: 135-162Crossref PubMed Scopus (321) Google Scholar, 2Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar). The cyclases are grouped into two major forms, those found on the plasma membrane (membrane GCs) and those in the cytoplasm (soluble GCs). The soluble GC is a heme-containing heterodimeric protein that is activated by binding of nitric oxide (3Kamisaki Y. Saheki S. Nakane M. Palmieri J.A. Kuno T. Chang B.Y. Waldman S.A. Murad F. J. Biol. Chem. 1986; 261: 7236-7241Abstract Full Text PDF PubMed Google Scholar, 4Humbert P. Niroomand F. Fischer G. Mayer B. Koesling D. Hinsch K.-D. Gausepohl H. Frank R. Schultz G. Böhme E. Eur. J. Biochem. 1990; 190: 273-278Crossref PubMed Scopus (143) Google Scholar). Analysis of cDNA clones for soluble GC subunits have shown that each subunit contains a cyclase catalytic domain which is also conserved in the membrane GCs (5Koesling D. Hertz J. Gausepohl H. Niroomand F. Hinsch K.-D. Mülsch A. Böhme E. Schultz G. Frank R. FEBS Lett. 1988; 239: 29-34Crossref PubMed Scopus (116) Google Scholar, 6Koesling D. Harteneck C. Humbert P. Bosserhoff A. Frank R. Shultz G. Böhme E. FEBS Lett. 1990; 266: 128-132Crossref PubMed Scopus (101) Google Scholar, 7Nakane M. Saheki S. Kuno T. Ishii K. Murad F. Biochem. Biophys. Res. Commun. 1988; 157: 1139-1147Crossref PubMed Scopus (123) Google Scholar, 8Nakane M. Arai K. Saheki S. Kuno T. Buechler W. Murad F. J. Biol. Chem. 1990; 265: 16841-16845Abstract Full Text PDF PubMed Google Scholar). The membrane GCs consist of a single polypeptide with about 150–200 kDa. In mammals, cDNA clones for six membrane GCs (GC-A, GC-B, GC-C, GC-D, GC-E, and GC-F) have been isolated and characterized (9Chinkers M. Garbers D.L. Chang M.-S. Lowe D.G. Chin H. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (881) Google Scholar, 10Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 11Schulz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 12Singh S. Lowe D.G. Thorpe D.S. Rodriguez H. Kuang W.-J. Dangott L.J. Chinkers M. Goeddel D.V. Garbers D.L. Nature. 1988; 334: 708-712Crossref PubMed Scopus (199) Google Scholar, 13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar, 14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). The predicted proteins contain an extracellular region, a kinase-like domain, and a cyclase catalytic domain. GC-A and GC-B are activated by binding of natriuretic peptides to their extracellular domain (2Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar, 9Chinkers M. Garbers D.L. Chang M.-S. Lowe D.G. Chin H. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (881) Google Scholar, 10Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 15Chang M.-S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Garbers D.G. Nature. 1989; 341: 68-72Crossref PubMed Scopus (498) Google Scholar, 16Lowe D.G. Chang M.-S. Hellmiss R. Chen E. Singh S. Garbers D.L. Goeddel D.V. EMBO J. 1989; 8: 1377-1384Crossref PubMed Scopus (313) Google Scholar), and GC-C activity is stimulated by extracellular binding of heat-stable enterotoxin and guanylin (17Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (502) Google Scholar, 18de Sauvage F.J. Keshav S. Kuang W.-J. Gillett N. Henzel W. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9089-9093Crossref PubMed Scopus (131) Google Scholar, 19Schulz S. Chrisman T.D. Garbers D.L. J. Biol. Chem. 1992; 267: 16019-16021Abstract Full Text PDF PubMed Google Scholar). Sensory neural tissues such as olfactory neurons and retina express specific isoforms of membrane GC. GC-D is specifically expressed in a subpopulation of olfactory sensory neurons (13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar). Two retina-specific GCs (RetGC-1 and RetGC-2) have been isolated and characterized from human retina (20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). These GCs are expressed mostly in the photoreceptor cells, and their expression patterns are indistinguishable. Two membrane GCs, GC-E and GC-F, have also been isolated from a rat eye cDNA library (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). GC-E and GC-F are thought to be orthologs of human RetGC-1 and RetGC-2. Rat GC-E and GC-F show different expression patterns. GC-E is expressed in the eye and pineal gland, whereas the expression of GC-F is confined to the eye (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). Contrary to GC-A, GC-B, and GC-C, no extracellular ligand has been reported for olfactory tissue- and eye-specific membrane GCs. Instead, the existence of two activators, GCAP-1 and GCAP-2, for retina-specific membrane GCs has been reported in bovine rod outer segment membranes (21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar, 22Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Both activators are a Ca2+-binding protein. GCAP-2 activates RetGC-1 and RetGC-2, whereas GCAP-1 activates RetGC-1 only. RetGC-1 is activated through binding of GCAP-2 to the intracellular domain, and the removal of the extracellular and transmembrane domains from RetGC-1 does not significantly alter the activation by GCAP-2 (24Laura R.P. Dizhoor A.M. Hurley J.B. J. Biol. Chem. 1996; 271: 11646-11651Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In Drosophila, some membrane GC transcripts are present in the unfertilized egg, along with embryos in the early developmental stages (25Gigliotti S. Cavaliere V. Manzi A. Tino A. Graziani F. Malva C. Dev. Biol. 1993; 159: 450-461Crossref PubMed Scopus (20) Google Scholar, 26McNeil L. Chinkers M. Forte M. J. Biol. Chem. 1995; 270: 7189-7196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This suggests that the cGMP signaling pathway mediated by a membrane GC is involved in the early development ofDrosophila. Despite rapidly accumulating information concerning the molecular nature of membrane GCs, there are few reports related to the expression of membrane GCs during vertebrate development or to the molecular phylogenetic relationship among membrane GCs. In this study, to understand the developmental role and diversity of membrane GCs in vertebrates, we begin the characterization of membrane GCs in medaka fish (Oryzias latipes), a species allowing for both classical and molecular genetic analyses. In this first report, we discuss the isolation and characterization of cDNA clones encoding three different membrane GCs expressed in the medaka eye, and the phylogenetic relationship of these membrane GCs. We also report that transcripts of two of the three medaka membrane GCs are present as maternal messages, and that each of these membrane GCs shows distinct expression patterns during embryogenesis and adulthood. DISCUSSIONIn this paper we report the characterization and expression patterns of three genes, OlGC3, OlGC4, andOlGC5, each encoding a different GC in the medaka fish.OlGC3, OlGC4, and OlGC5 are predominantly expressed in the eye, which contains more cGMP than any other organ, suggesting the important role(s) of these GCs in the phototransduction pathway. Hayashi and Yamazaki (49Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (78) Google Scholar) reported multiple forms of photoreceptor GCs in toad, frog, and bovine rods. In mammals, two forms of membrane GC have been identified in the eye (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar, 20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). Our study demonstrated that at least three related membrane GCs encoded by different genes are present in the fish eye. The presence of multiple forms of membrane GC in the eye seems to be common among a wide variety of vertebrates. Since the amino acid sequences of OlGC3, OlGC4, and OlGC5 are related but discrete, the enzyme activity of these GCs may be regulated via different pathways. Alternatively, these membrane GCs might show different cellular and/or subcellular distribution patterns in the eye.The size of the major transcripts for OlGC3,OlGC4, and OlGC5 is 3.5, 8.8, and 6.2 kb, respectively. Although the transcripts of OlGC4 andOlGC5 are larger than those for most known membrane GCs (about 4.0 kb or smaller), the transcript lengths of 8.5 and 11 kb have been reported for the rat eye-specific GCs, GC-E and GC-F, respectively (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). The increase in size seems to be attributable to large 3′- and 5′-UTR sequences (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). Relatively large transcript size may thus be a common feature among vertebrate eye membrane GCs.In addition to the major band of 3.5 kb, a weaker signal of 4.3 kb was detected for OlGC3. Similarly, two weaker bands of 7.2 and 5.3 kb were detected for OlGC4. The OlGC3transcripts of different size may be attributable to transcripts with differently sized 3′-UTR, because we isolated a cDNA clone with a sequence identical to that of OlGC3 except for the size of 3′-UTR. It is uncertain whether the OlGC4 transcripts of different size are due to differently sized UTR. The size of cDNA sequences for OlGC3, OlGC4, and OlGC5determined in the present study is 4110, 4651, and 5839 bp, respectively. These cDNA sequences probably correspond to transcripts of 4.3, 5.3, and 6.2 kb detected in the Northern blot analysis, respectively. The OlGC3 cDNA clone with smaller 3′-UTR may correspond to the 3.5-kb transcripts ofOlGC3.The phylogenetic analysis of intracellular catalytic domains revealed that there are three major groups of membrane GCs in vertebrates: (i) natriuretic peptide receptors, (ii) enterotoxin receptors, and (iii) sensory organ-specific GCs. OlGC3, OlGC4, and OlGC5 are grouped with the sensory organ-specific GCs. These three groups probably represent three distinct subfamilies of membrane GCs that are defined by families of ligands or activators as well as by localization. The phylogenetic tree of extracellular domains also suggests a close relationship among sensory organ-specific GCs, including OlGC3, OlGC4, and OlGC5. Within the sensory organ-specific GC subfamily, relationships among members were different between the phylogenetic trees of intracellular catalytic and extracellular domains. This may reflect different functional constraints on the structure of extracellular and intracellular domains.The relative positions of some amino acids, including six cysteine residues, are highly conserved within the extracellular domains of the sensory organ-specific GCs. Although no extracellular ligands have been reported for the sensory organ-specific GCs, the conservation of these residues suggests a functional importance of the extracellular domains. These conserved cysteine residues may be involved in intramolecular disulfide bond formation and/or oligomerization of the proteins.The RT-PCR analysis showed that OlGC3, OlGC4, andOlGC5 genes are also expressed in a number of organs other than the eye and that the expression patterns in adults are different among the three genes. In contrast, previous studies on mammalian eye- and olfactory neuron-specific GCs revealed that their expression is confined to sensory organs and related tissue (pineal gland) (13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar, 14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar,20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). The difference between the reported expression patterns of medaka GCs and mammalian sensory organ GCs might be attributable to differences between fish and mammals or simply to differences in detection methods. Alternatively, since the sequences of OlGC3, OlGC4, and OlGC5 are relatively diverged from those of any known mammalian sensory organ GCs, there may be unidentified members of the mammalian sensory organ GC subfamily that exhibit sequences and expression patterns similar to those of OlGC3, OlGC4, or OlGC5. The presence of transcripts of OlGC3, OlGC4, and OlGC5in various adult organs suggests that members of sensory organ GC subfamily play previously unrecognized roles in these organs. Since the Northern blot analysis failed to detect OlGC3 andOlGC4 mRNA in the liver and only faint signal forOlGC3 was detected in the brain and ovary, however, these organs may express much smaller amounts of the OlGC3and OlGC4 transcripts than does the eye.There have been few reports on the expression patterns of sensory organ GC genes during vertebrate development. Our results demonstrated that the transcripts of OlGC3 and OlGC5 are present in unfertilized eggs as maternal messages, suggesting that cGMP signaling pathways mediated by these membrane GCs are involved in the early development of fish. Since the OlGC3 transcripts are evident in unfertilized eggs as well as in the adult ovary, OlGC3 may play an important role during oogenesis. In Drosophila, membrane GCs are expressed during oogenesis and early development (25Gigliotti S. Cavaliere V. Manzi A. Tino A. Graziani F. Malva C. Dev. Biol. 1993; 159: 450-461Crossref PubMed Scopus (20) Google Scholar, 26McNeil L. Chinkers M. Forte M. J. Biol. Chem. 1995; 270: 7189-7196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). cGMP signaling pathways mediated by membrane GCs regulating oogenesis and early development may have been conserved between vertebrates and invertebrates. The RT-PCR analysis revealed that the three medaka GC genes are differentially activated during embryogenesis. The differing expression patterns of medaka GC genes suggest that these GCs have distinct roles during development. Two activators for eye-specific membrane GCs have been reported in bovine rod outer segment (21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar, 22Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Similar activators may regulate the activity of the medaka GCs during early development and in adult organs other than the eye. In this paper we report the characterization and expression patterns of three genes, OlGC3, OlGC4, andOlGC5, each encoding a different GC in the medaka fish.OlGC3, OlGC4, and OlGC5 are predominantly expressed in the eye, which contains more cGMP than any other organ, suggesting the important role(s) of these GCs in the phototransduction pathway. Hayashi and Yamazaki (49Hayashi F. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4746-4750Crossref PubMed Scopus (78) Google Scholar) reported multiple forms of photoreceptor GCs in toad, frog, and bovine rods. In mammals, two forms of membrane GC have been identified in the eye (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar, 20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). Our study demonstrated that at least three related membrane GCs encoded by different genes are present in the fish eye. The presence of multiple forms of membrane GC in the eye seems to be common among a wide variety of vertebrates. Since the amino acid sequences of OlGC3, OlGC4, and OlGC5 are related but discrete, the enzyme activity of these GCs may be regulated via different pathways. Alternatively, these membrane GCs might show different cellular and/or subcellular distribution patterns in the eye. The size of the major transcripts for OlGC3,OlGC4, and OlGC5 is 3.5, 8.8, and 6.2 kb, respectively. Although the transcripts of OlGC4 andOlGC5 are larger than those for most known membrane GCs (about 4.0 kb or smaller), the transcript lengths of 8.5 and 11 kb have been reported for the rat eye-specific GCs, GC-E and GC-F, respectively (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). The increase in size seems to be attributable to large 3′- and 5′-UTR sequences (14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar). Relatively large transcript size may thus be a common feature among vertebrate eye membrane GCs. In addition to the major band of 3.5 kb, a weaker signal of 4.3 kb was detected for OlGC3. Similarly, two weaker bands of 7.2 and 5.3 kb were detected for OlGC4. The OlGC3transcripts of different size may be attributable to transcripts with differently sized 3′-UTR, because we isolated a cDNA clone with a sequence identical to that of OlGC3 except for the size of 3′-UTR. It is uncertain whether the OlGC4 transcripts of different size are due to differently sized UTR. The size of cDNA sequences for OlGC3, OlGC4, and OlGC5determined in the present study is 4110, 4651, and 5839 bp, respectively. These cDNA sequences probably correspond to transcripts of 4.3, 5.3, and 6.2 kb detected in the Northern blot analysis, respectively. The OlGC3 cDNA clone with smaller 3′-UTR may correspond to the 3.5-kb transcripts ofOlGC3. The phylogenetic analysis of intracellular catalytic domains revealed that there are three major groups of membrane GCs in vertebrates: (i) natriuretic peptide receptors, (ii) enterotoxin receptors, and (iii) sensory organ-specific GCs. OlGC3, OlGC4, and OlGC5 are grouped with the sensory organ-specific GCs. These three groups probably represent three distinct subfamilies of membrane GCs that are defined by families of ligands or activators as well as by localization. The phylogenetic tree of extracellular domains also suggests a close relationship among sensory organ-specific GCs, including OlGC3, OlGC4, and OlGC5. Within the sensory organ-specific GC subfamily, relationships among members were different between the phylogenetic trees of intracellular catalytic and extracellular domains. This may reflect different functional constraints on the structure of extracellular and intracellular domains. The relative positions of some amino acids, including six cysteine residues, are highly conserved within the extracellular domains of the sensory organ-specific GCs. Although no extracellular ligands have been reported for the sensory organ-specific GCs, the conservation of these residues suggests a functional importance of the extracellular domains. These conserved cysteine residues may be involved in intramolecular disulfide bond formation and/or oligomerization of the proteins. The RT-PCR analysis showed that OlGC3, OlGC4, andOlGC5 genes are also expressed in a number of organs other than the eye and that the expression patterns in adults are different among the three genes. In contrast, previous studies on mammalian eye- and olfactory neuron-specific GCs revealed that their expression is confined to sensory organs and related tissue (pineal gland) (13Fülle H.J. Vassar R. Foster D.C. Yang R.-B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (231) Google Scholar, 14Yang R.-B. Foster D.C. Garbers D.L. Fülle H.-J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 602-606Crossref PubMed Scopus (217) Google Scholar,20Shyjan A.W. de Sauvage F.J. Gillett N.A. Goeddel D.V. Lowe D.G. Neuron. 1992; 9: 727-737Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar). The difference between the reported expression patterns of medaka GCs and mammalian sensory organ GCs might be attributable to differences between fish and mammals or simply to differences in detection methods. Alternatively, since the sequences of OlGC3, OlGC4, and OlGC5 are relatively diverged from those of any known mammalian sensory organ GCs, there may be unidentified members of the mammalian sensory organ GC subfamily that exhibit sequences and expression patterns similar to those of OlGC3, OlGC4, or OlGC5. The presence of transcripts of OlGC3, OlGC4, and OlGC5in various adult organs suggests that members of sensory organ GC subfamily play previously unrecognized roles in these organs. Since the Northern blot analysis failed to detect OlGC3 andOlGC4 mRNA in the liver and only faint signal forOlGC3 was detected in the brain and ovary, however, these organs may express much smaller amounts of the OlGC3and OlGC4 transcripts than does the eye. There have been few reports on the expression patterns of sensory organ GC genes during vertebrate development. Our results demonstrated that the transcripts of OlGC3 and OlGC5 are present in unfertilized eggs as maternal messages, suggesting that cGMP signaling pathways mediated by these membrane GCs are involved in the early development of fish. Since the OlGC3 transcripts are evident in unfertilized eggs as well as in the adult ovary, OlGC3 may play an important role during oogenesis. In Drosophila, membrane GCs are expressed during oogenesis and early development (25Gigliotti S. Cavaliere V. Manzi A. Tino A. Graziani F. Malva C. Dev. Biol. 1993; 159: 450-461Crossref PubMed Scopus (20) Google Scholar, 26McNeil L. Chinkers M. Forte M. J. Biol. Chem. 1995; 270: 7189-7196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). cGMP signaling pathways mediated by membrane GCs regulating oogenesis and early development may have been conserved between vertebrates and invertebrates. The RT-PCR analysis revealed that the three medaka GC genes are differentially activated during embryogenesis. The differing expression patterns of medaka GC genes suggest that these GCs have distinct roles during development. Two activators for eye-specific membrane GCs have been reported in bovine rod outer segment (21Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (233) Google Scholar, 22Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Gorczyca W.A. Polans A.S. Surgucheva I.G. Subbaraya I. Baehr W. Palczewski K. J. Biol. Chem. 1995; 270: 22029-22036Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Similar activators may regulate the activity of the medaka GCs during early development and in adult organs other than the eye. We thank the staff members of the Research Center for Molecular Genetics and the Center for Animal Experiments, Hokkaido University for facilitating the use of equipment for radiolabeling probes and culturing medaka fishes. We thank Dr. H. Hori for his suggestion regarding the 3′-UTR sequence ofOlGC3.

Referência(s)