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Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish*

  • Akihiro Misu
    Affiliations
    Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Hiroaki Yamanaka
    Affiliations
    Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Toshihiro Aramaki
    Affiliations
    Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Shigeru Kondo
    Affiliations
    Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

    CREST, Japan Science and Technology Agency, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan
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  • I. Martha Skerrett
    Affiliations
    Biology Department, Buffalo State College, Buffalo, New York 14222
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  • M. Kathryn Iovine
    Affiliations
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015
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  • Masakatsu Watanabe
    Correspondence
    To whom correspondence should be addressed: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-7997; Fax: 81-6-6879-7977.
    Affiliations
    Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
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  • Author Footnotes
    * This work was supported by Ministry of Education, Culture, Sports, Science, and Technology in Japan KAKENHI Grant 22127003 (to S. K.); by the Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (to S. K.); and by Japanese Society for the Promotion of Science KAKENHI Grant 26291049 (to M. W.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:April 25, 2016DOI:https://doi.org/10.1074/jbc.M116.720110
      Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stptl28d). Vertebrae of stptl28d/+ fish look normal in larvae (∼30 days) but are distinctly shorter (59–81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43stp or Cx43sof was reduced compared with Cx43-WT. On the other hand, only Cx43stp induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

      Introduction

      Vertebrate bones develop during embryonic stages and grow continuously as body size increases. Because bone shape and size determine body shape, the mechanisms that generate and maintain bone shape are of great interest to biologists. Recent studies have increased our knowledge of bone formation mechanisms. Interactions among three types of cells—osteoblasts, osteocytes, and osteoclasts—play major roles in bone formation and bone remodeling (
      • Attanasio C.
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      • Zhu Y.
      • Blow M.J.
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      • Liberton D.K.
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      • Holt A.
      • Hosseini R.
      • Phouanenavong S.
      • Akiyama J.A.
      • Shoukry M.
      • Afzal V.
      • Rubin E.M.
      • et al.
      Fine tuning of craniofacial morphology by distant-acting enhancers.
      ,
      • Gluhak-Heinrich J.
      • Ye L.
      • Bonewald L.F.
      • Feng J.Q.
      • MacDougall M.
      • Harris S.E.
      • Pavlin D.
      Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo.
      ,
      • Schoenebeck J.J.
      • Hutchinson S.A.
      • Byers A.
      • Beale H.C.
      • Carrington B.
      • Faden D.L.
      • Rimbault M.
      • Decker B.
      • Kidd J.M.
      • Sood R.
      • Boyko A.R.
      • Fondon 3rd, J.W.
      • Wayne R.K.
      • Bustamante C.D.
      • Ciruna B.
      • et al.
      Variation of BMP3 contributes to dog breed skull diversity.
      ,
      • Bonewald L.F.
      The amazing osteocyte.
      ,
      • Caetano-Lopes J.
      • Canhão H.
      • Fonseca J.E.
      Osteoblasts and bone formation.
      ,
      • Duplomb L.
      • Dagouassat M.
      • Jourdon P.
      • Heymann D.
      Concise review: embryonic stem cells: a new tool to study osteoblast and osteoclast differentiation.
      ,
      • Harada S.
      • Rodan G.A.
      Control of osteoblast function and regulation of bone mass.
      ,
      • Komori T.
      Regulation of osteoblast differentiation by transcription factors.
      ).
      Zebrafish (Danio rerio) is a model organism for developmental and genetic studies, and several mutants related to bone formation have been identified and analyzed (
      • Haffter P.
      • Granato M.
      • Brand M.
      • Mullins M.C.
      • Hammerschmidt M.
      • Kane D.A.
      • Odenthal J.
      • van Eeden F.J.
      • Jiang Y.J.
      • Heisenberg C.P.
      • Kelsh R.N.
      • Furutani-Seiki M.
      • Vogelsang E.
      • Beuchle D.
      • Schach U.
      • et al.
      The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
      ,
      • Kimmel C.B.
      • Miller C.T.
      • Moens C.B.
      Specification and morphogenesis of the zebrafish larval head skeleton.
      ,
      • Neuhauss S.C.
      • Solnica-Krezel L.
      • Schier A.F.
      • Zwartkruis F.
      • Stemple D.L.
      • Malicki J.
      • Abdelilah S.
      • Stainier D.Y.
      • Driever W.
      Mutations affecting craniofacial development in zebrafish.
      ,
      • Piotrowski T.
      • Schilling T.F.
      • Brand M.
      • Jiang Y.J.
      • Heisenberg C.P.
      • Beuchle D.
      • Grandel H.
      • van Eeden F.J.
      • Furutani-Seiki M.
      • Granato M.
      • Haffter P.
      • Hammerschmidt M.
      • Kane D.A.
      • Kelsh R.N.
      • Mullins M.C.
      • et al.
      Jaw and branchial arch mutants in zebrafish II: anterior arches and cartilage differentiation.
      ,
      • Schilling T.F.
      • Walker C.
      • Kimmel C.B.
      The chinless mutation and neural CREST cell interactions in zebrafish jaw development.
      ). Mutant bone formation phenotypes can be categorized into two types: bone development mutants and bone growth mutants. Genes corresponding to bone development mutants are usually expressed at early developmental stages of bone formation (type 1), and bone malformations are detected when the bones first appear in embryo (
      • Clément A.
      • Wiweger M.
      • von der Hardt S.
      • Rusch M.A.
      • Selleck S.B.
      • Chien C.B.
      • Roehl H.H.
      Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher.
      ,
      • Cooper K.L.
      • Oh S.
      • Sung Y.
      • Dasari R.R.
      • Kirschner M.W.
      • Tabin C.J.
      Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.
      ,
      • Gray R.S.
      • Wilm T.P.
      • Smith J.
      • Bagnat M.
      • Dale R.M.
      • Topczewski J.
      • Johnson S.L.
      • Solnica-Krezel L.
      Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.
      ). On the other hand there are several zebrafish bone growth mutants, in which bone development looks almost normal at early stages, but mutant phenotypes appear at late stages (type 2) (
      • Huycke T.R.
      • Eames B.F.
      • Kimmel C.B.
      Hedgehog-dependent proliferation drives modular growth during morphogenesis of a dermal bone.
      ,
      • Perathoner S.
      • Daane J.M.
      • Henrion U.
      • Seebohm G.
      • Higdon C.W.
      • Johnson S.L.
      • Nüsslein-Volhard C.
      • Harris M.P.
      Bioelectric signaling regulates size in zebrafish fins.
      ,
      • Iovine M.K.
      • Johnson S.L.
      Genetic analysis of isometric growth control mechanisms in the zebrafish caudal Fin.
      ).
      In this study, we focused on the stoepsel (stp) mutant fish (
      • Haffter P.
      • Odenthal J.
      • Mullins M.C.
      • Lin S.
      • Farrell M.J.
      • Vogelsang E.
      • Haas F.
      • Brand M.
      • van Eeden F.J.
      • Furutani-Seiki M.
      • Granato M.
      • Hammerschmidt M.
      • Heisenberg C.P.
      • Jiang Y.J.
      • Kane D.A.
      • et al.
      Mutations affecting pigmentation and shape of the adult zebrafish.
      ) to study bone morphogenesis in adult stages. The stp mutant was originally isolated by a large scale screen of zebrafish mutants induced by ENU
      The abbreviations used are: ENU
      N-ethyl-N-nitrosourea
      AP
      anterior-posterior
      dpf
      days after fertilization
      DV
      dorsoventral
      TALEN
      transcription activator-like effector nucleases.
      (
      • Haffter P.
      • Odenthal J.
      • Mullins M.C.
      • Lin S.
      • Farrell M.J.
      • Vogelsang E.
      • Haas F.
      • Brand M.
      • van Eeden F.J.
      • Furutani-Seiki M.
      • Granato M.
      • Hammerschmidt M.
      • Heisenberg C.P.
      • Jiang Y.J.
      • Kane D.A.
      • et al.
      Mutations affecting pigmentation and shape of the adult zebrafish.
      ). This mutant looks normal until ∼35 days after fertilization (dpf); however, the vertebrae of the mutant fish become shorter than those of wild type fish at later growth stages. The stp allele is dominant and homozygous lethal. We re-examined the stp phenotypes in detail using modern techniques, and we performed positional cloning to identify the stp gene. Surprisingly, we isolated a mutation in the connexin43 gene, which is known as the sof (short-of-fin) gene in zebrafish (
      • Iovine M.K.
      • Higgins E.P.
      • Hindes A.
      • Coblitz B.
      • Johnson S.L.
      Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins.
      ). sof is a well studied type 2 zebrafish mutant, in which fin segments are short in the caudal fin (
      • Iovine M.K.
      • Higgins E.P.
      • Hindes A.
      • Coblitz B.
      • Johnson S.L.
      Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins.
      ). Four sof mutant alleles were isolated by mutagenesis screening, and three single mutations that cause amino acid substitutions in Connexin43 protein were identified (
      • Hoptak-Solga A.D.
      • Klein K.A.
      • DeRosa A.M.
      • White T.W.
      • Iovine M.K.
      Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication.
      ). In the sofb123 allele, no amino acid substitution was identified in the connexin43 gene, but cx43 expression was reduced in the mutant fish (
      • Iovine M.K.
      • Higgins E.P.
      • Hindes A.
      • Coblitz B.
      • Johnson S.L.
      Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins.
      ,
      • Sims Jr., K.
      • Eble D.M.
      • Iovine M.K.
      Connexin43 regulates joint location in zebrafish fins.
      ,
      • Hoptak-Solga A.D.
      • Nielsen S.
      • Jain I.
      • Thummel R.
      • Hyde D.R.
      • Iovine M.K.
      Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration.
      ).
      Connexin proteins are four-pass transmembrane proteins that are subunits of gap junctions (
      • Kumar N.M.
      • Gilula N.B.
      The gap junction communication channel.
      ). Approximately 20 connexin genes are known from the mammalian genome, and ∼36 connexin genes are predicted in the zebrafish genome (
      • Abascal F.
      • Zardoya R.
      Evolutionary analyses of gap junction protein families.
      ,
      • Eastman S.D.
      • Chen T.H.
      • Falk M.M.
      • Mendelson T.C.
      • Iovine M.K.
      Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes.
      ). Six connexin proteins make a hexamer called a connexon; docking of two connexons on adjacent cells creates a gap junction. Gap junctions allow movement of small molecules (<1000 Da; e.g. ATP, inositol trisphosphate, ions, etc.) between neighboring cells; a connexon acts as a hemichannel on the cytoplasmic membrane (
      • Kumar N.M.
      • Gilula N.B.
      The gap junction communication channel.
      ). connexin43 is one of the most studied connexin genes because it is linked to several human diseases (
      • Becker D.L.
      • Phillips A.R.
      • Duft B.J.
      • Kim Y.
      • Green C.R.
      Translating connexin biology into therapeutics.
      ,
      • Merrifield P.A.
      • Laird D.W.
      Connexins in skeletal muscle development and disease.
      ,
      • Bai D.
      Structural analysis of key gap junction domains-Lessons from genome data and disease-linked mutants.
      ). Oculodentodigital dysplasia is one Cx43-related human disease, which causes small eyes, underdeveloped teeth, and malformed fingers (
      • Laird D.W.
      Syndromic and non-syndromic disease-linked Cx43 mutations.
      ,
      • Pizzuti A.
      • Flex E.
      • Mingarelli R.
      • Salpietro C.
      • Zelante L.
      • Dallapiccola B.
      A homozygous GJA1 gene mutation causes a Hallermann-Streiff/ODDD spectrum phenotype.
      ). To identify the cause of the substantial difference in phenotype between the sof and stp alleles of cx43, we performed electrophysiological experiments and found that the growth-dependent malformation of stp vertebrae is likely caused by aberrant hemichannel activity of Cx43stp rather than reduced gap junction intercellular conductance observed in Cx43sof.

      Discussion

      In this paper we analyzed a zebrafish mutation that disrupts normal vertebra formation. Homozygous (stptl28d/tl28d) fish die; heterozygous (stptl28d/+) fish grow to adults but show a specific phenotype of shorter vertebrae along only the AP axis. Because this phenotype is not seen in young fish (∼35 dpf) and becomes evident only after ∼60 dpf, we deduced that the mutation caused some defect in the control of bone growth. To identify the gene altered in the stp mutant, we performed positional cloning and TALEN knock-out of the genomic sequence and found that a single base mutation in cx43 causes the phenotype.
      Curiously, a previous paper reported that another mutation in cx43 is responsible for the sof phenotype, in which fins are short but vertebrae are normal. To determine why these alleles of the same gene differed in their effects, we investigated two different functions of connexins: as gap junctions and hemichannels. Both Cx43stp and Cx43sof showed reduced gap junction currents relative to wild type, which confirmed that the sof phenotype was caused by the defect in gap junction activity, as suggested by Hoptak-Solga et al. (
      • Hoptak-Solga A.D.
      • Klein K.A.
      • DeRosa A.M.
      • White T.W.
      • Iovine M.K.
      Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication.
      ). On the other hand, Cx43stp hemichannels showed abnormally high (50-fold higher than wild type) conductance, whereas Cx43sof hemichannel conductance was almost the same as Cx43-WT. These results are consistent with the hypothesis that extremely high hemichannel activity causes the vertebral phenotype of the stp allele.
      Although the mechanism by which gap junctions and hemichannels affect different bones is unknown, we assume that it is related to the difference in the bone forming process between vertebrae and fin bones. Fins are mechanically supported by fin rays arrayed in parallel. Each fin ray is composed of short segments of bone called lepidotrichia. Each segment is almost the same size along the AP axis (for tail fins). When the fin grows, a new fin ray segment is made at the tip of each fin ray. The newly made segments are nearly as long as the old segments along the AP axis and do not grow longer with time or with increasing fin length. On the other hand, vertebrae initially appear at ∼14 days post fertilization and continuously enlarge during the growth of the fish. Therefore, we infer that Cx43 gap junctions are involved in initial bone formation, and Cx43 hemichannels are involved in later bone growth.
      One proposed mechanism of mechanical stress sensing by osteocytes is as follows. When bones are exposed to mechanical stress, they deform slightly, causing movement of the fluid surrounding the osteocytes. This movement of fluid causes shear stress that induces effusion of prostaglandin E2 through hemichannels (
      • Cherian P.P.
      • Siller-Jackson A.J.
      • Gu S.
      • Wang X.
      • Bonewald L.F.
      • Sprague E.
      • Jiang J.X.
      Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin.
      ). Prostaglandin E2 promotes osteoclast differentiation by down-regulating Osteoprotegerin (
      • Suda K.
      • Udagawa N.
      • Sato N.
      • Takami M.
      • Itoh K.
      • Woo J.T.
      • Takahashi N.
      • Nagai K.
      Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation.
      ), a protein that blocks RANKL-RANK interaction and promotes osteoclast differentiation.
      We hypothesize that the stp allele reduces vertebral elongation by mimicking high mechanical stress. We found that cx43 expression in zebrafish vertebrae is highest in the growing edges (Fig. 4, E and E′). Because these parts of the vertebra contact the adjacent vertebra, we expect that mechanical stress is highest there. Therefore, one possible explanation for the effect of the stp mutation on vertebral shape is that it enhances hemichannel activity, allowing more prostaglandin E2 effusion. This would mimic the effects of high mechanical stress and lead to increased osteoclast differentiation and, hence, increased rates of bone breakdown at the edges of the centrum.
      The major finding of our study is that there are two distinct functions of Cx43 in bone formation, one of which affects early development only and one of which affects subsequent growth only. These two functions correlate with differences in the electrophysiological properties of gap junctions and hemichannels among cx43 alleles. We hypothesize that the differences in gap junction and hemichannel function among alleles cause their differing effects on bone development and growth. Future studies are needed to test this hypothesis in vivo.

      Author Contributions

      A. M., H. Y., S. K., and M. W. designed the experimental strategy. A. M. performed positional cloning experiments. A. M. and T. A. performed transgenic experiments. A. M., H. Y., S. K., and M. K. I analyzed bone development. M. W. and I. M. S. performed electrophysiological experiments and analyzed data. A. M. prepared the manuscript, and all authors commented on the manuscript.

      Acknowledgments

      We thank the Shimadzu Corporation and the Osaka Bioscience Institute for lending CT scanners. We also thank H. Shirota and C. Sato at Osaka University for technical assistance in the positional cloning experiment, and Dr. T. W. White at SUNY Stony Brook for technical advice on the oocyte clamp experiments.

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