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The Physiological Characterization of Connexin41.8 and Connexin39.4, Which Are Involved in the Striped Pattern Formation of Zebrafish*

  • 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
    From the Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,
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  • Risa Sawada
    Affiliations
    From the Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,
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  • Toshihiro Aramaki
    Affiliations
    From the Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,
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  • I. Martha Skerrett
    Affiliations
    the Biology Department, Buffalo State College, Buffalo, New York, 14222, and
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  • Shigeru Kondo
    Affiliations
    From the 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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  • Author Footnotes
    * This work was supported by Japanese Society for the Promotion of Science, KAKENHI Grants 26291049, 26650078 (to M. W.), by Ministry of Education, Culture, Sports, Science, and Technology in Japan, KAKENHI Grants 25111714 (to M. W.) and 22127003 (to S. K.), and by Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (to S. K.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:November 23, 2015DOI:https://doi.org/10.1074/jbc.M115.673129
      The zebrafish has a striped skin pattern on its body, and Connexin41.8 (Cx41.8) and Cx39.4 are involved in striped pattern formation. Mutations in these connexins change the striped pattern to a spot or labyrinth pattern. In this study, we characterized Cx41.8 and Cx39.4 after expression in Xenopus oocytes. In addition, we analyzed Cx41.8 mutants Cx41.8I203F and Cx41.8M7, which caused spot or labyrinth skin patterns, respectively, in transgenic zebrafish. In the electrophysiological analysis, the gap junctions formed by Cx41.8 and Cx39.4 showed distinct sensitivity to transjunctional voltage. Analysis of non-junctional (hemichannel) currents revealed a large voltage-dependent current in Cx39.4-expressing oocytes that was absent in cells expressing Cx41.8. Junctional currents induced by both Cx41.8 and Cx39.4 were reduced by co-expression of Cx41.8I203F and abolished by co-expression of Cx41.8M7. In the transgenic experiment, Cx41.8I203F partially rescued the Cx41.8 null mutant phenotype, whereas Cx41.8M7 failed to rescue the null mutant, and it elicited a more severe phenotype than the Cx41.8 null mutant, as evidenced by a smaller spot pattern. Our results provide evidence that gap junctions formed by Cx41.8 play an important role in stripe/spot patterning and suggest that mutations in Cx41.8 can effect patterning by way of reduced function (I203F) and dominant negative effects (M7). Our results suggest that functional differences in Cx41.8 and Cx39.4 relate to spot or labyrinth mutant phenotypes and also provide evidence that these two connexins interact in vivo and in vitro.

      Introduction

      Connexin proteins are the major constituents of gap junctions and are four-pass transmembrane proteins. Six connexin proteins make up a hexamer called a connexon, which acts as a hemichannel at the cell membrane. Each gap junction is a large molecular unit formed by the docking of connexons, allowing ∼1,000-Da small molecules to be transferred between neighboring cells (
      • Kumar N.M.
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      The gap junction communication channel.
      ,
      • Alexander D.B.
      • Goldberg G.S.
      Transfer of biologically important molecules between cells through gap junction channels.
      ). Approximately 20 connexins exist in mammalian genomes, and connexins are categorized into five subgroups (α, β, γ, δ, and ζ), depending on their molecular weight and amino acid sequences (
      • Söhl G.
      • Willecke K.
      Gap junctions and the connexin protein family.
      ,
      • Abascal F.
      • Zardoya R.
      Evolutionary analyses of gap junction protein families.
      ). In zebrafish, ∼36 connexins are predicted to exist (
      • 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.
      ). This higher gene number might be due to genome duplication events during fish evolution. As in mammals, connexins are expressed in tissue-specific but overlapping patterns. A combination of connexins in the same or adjacent cells can lead to several types of gap junctions. Uniform connexins create homomeric homotypic gap junctions, whereas two or more types of connexins form gap junctions that are heteromeric (different connexins within a connexon), heterotypic (different connexins in two opposing connexons), or a combination of both. The composition of heteromeric and heterotypic gap junctions is expected to create gap junctions with a wide range of properties in vivo, although relatively little is known about their importance or complexity due to technical difficulties associated with their analysis (
      • Ebihara L.
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      • Oberti C.
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      Co-expression of lens fiber connexins modifies hemi-gap-junctional channel behavior.
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      Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.
      ,
      • White T.W.
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      • Goodenough D.A.
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      Functional analysis of selective interactions among rodent connexins.
      ,
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      • Klein R.A.
      • Hülser D.F.
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      Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.
      ,
      • Werner R.
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      Formation of hybrid cell-cell channels.
      ,
      • Cao F.
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      A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes.
      • Verselis V.K.
      • Ginter C.S.
      • Bargiello T.A.
      Opposite voltage gating polarities of two closely related connexins.
      ).
      The zebrafish is well known as a model organism for developmental and genetic studies. Because the zebrafish has a striped pattern on its skin (Fig. 1A), this fish is also well known as a useful animal for pattern formation studies (
      • Budi E.H.
      • Patterson L.B.
      • Parichy D.M.
      Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation.
      • Kelsh R.N.
      • Harris M.L.
      • Colanesi S.
      • Erickson C.A.
      Stripes and belly-spots: a review of pigment cell morphogenesis in vertebrates.
      ,
      • McMenamin S.K.
      • Bain E.J.
      • McCann A.E.
      • Patterson L.B.
      • Eom D.S.
      • Waller Z.P.
      • Hamill J.C.
      • Kuhlman J.A.
      • Eisen J.S.
      • Parichy D.M.
      Thyroid hormone-dependent adult pigment cell lineage and pattern in zebrafish.
      ,
      • Singh A.P.
      • Nüsslein-Volhard C.
      Zebrafish stripes as a model for vertebrate colour pattern formation.
      ,
      • Kondo S.
      The reaction-diffusion system: a mechanism for autonomous pattern formation in the animal skin.
      ,
      • Yamaguchi M.
      • Yoshimoto E.
      • Kondo S.
      Pattern regulation in the stripe of zebrafish suggests an underlying dynamic and autonomous mechanism.
      ,
      • Nakamasu A.
      • Takahashi G.
      • Kanbe A.
      • Kondo S.
      Interactions between zebrafish pigment cells responsible for the generation of Turing patterns.
      ,
      • Kondo S.
      • Miura T.
      Reaction-diffusion model as a framework for understanding biological pattern formation.
      ,
      • Yamanaka H.
      • Kondo S.
      In vitro analysis suggests that difference in cell movement during direct interaction can generate various pigment patterns in vivo.
      • Watanabe M.
      • Kondo S.
      Is pigment patterning in fish skin determined by the Turing mechanism?.
      ). Zebrafish stripes consist primarily of two types of pigment cells: black pigment cells called melanophores and yellow pigment cells called xanthophores. We proposed that zebrafish stripes are generated by the interaction between melanophores and xanthophores, which satisfies the conditions for the formation of a Turing pattern (
      • Yamaguchi M.
      • Yoshimoto E.
      • Kondo S.
      Pattern regulation in the stripe of zebrafish suggests an underlying dynamic and autonomous mechanism.
      • Nakamasu A.
      • Takahashi G.
      • Kanbe A.
      • Kondo S.
      Interactions between zebrafish pigment cells responsible for the generation of Turing patterns.
      ,
      • Kondo S.
      • Miura T.
      Reaction-diffusion model as a framework for understanding biological pattern formation.
      ,
      • Yamanaka H.
      • Kondo S.
      In vitro analysis suggests that difference in cell movement during direct interaction can generate various pigment patterns in vivo.
      ,
      • Watanabe M.
      • Kondo S.
      Is pigment patterning in fish skin determined by the Turing mechanism?.
      ,
      • Meinhardt H.
      • Gierer A.
      Pattern formation by local self-activation and lateral inhibition.
      • Turing A.
      The chemical basis of morphogenesis.
      ). Several molecules involved in cell-cell interactions for skin pattern formation were identified (
      • Fadeev A.
      • Krauss J.
      • Frohnhofer H.G.
      • Irion U.
      • Nusslein-Volhard C.
      Tight junction protein 1a regulates pigment cell organisation during zebrafish colour patterning.
      • Inoue S.
      • Kondo S.
      • Parichy D.M.
      • Watanabe M.
      Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes.
      ,
      • Lang M.R.
      • Patterson L.B.
      • Gordon T.N.
      • Johnson S.L.
      • Parichy D.M.
      Basonuclin-2 requirements for zebrafish adult pigment pattern development and female fertility.
      • Parichy D.M.
      • Rawls J.F.
      • Pratt S.J.
      • Whitfield T.T.
      • Johnson S.L.
      Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.
      ). The membrane resting potential formed by Kir7.1, the inward rectifier potassium channel 7.1, is important for establishing clear boundaries between melanophores and xanthophores, and this channel functions as a short range factor in the Turing model (
      • Watanabe M.
      • Kondo S.
      Is pigment patterning in fish skin determined by the Turing mechanism?.
      ,
      • Iwashita M.
      • Watanabe M.
      • Ishii M.
      • Chen T.
      • Johnson S.L.
      • Kurachi Y.
      • Okada N.
      • Kondo S.
      Pigment pattern in jaguar/obelix zebrafish is caused by a Kir7.1 mutation: implications for the regulation of melanosome movement.
      ,
      • Inaba M.
      • Yamanaka H.
      • Kondo S.
      Pigment pattern formation by contact-dependent depolarization.
      ). Notch-Delta signaling from xanthophores (Delta) to melanophores (Notch) is required for melanophore survival and acts as a long range factor in this model (
      • Watanabe M.
      • Kondo S.
      Is pigment patterning in fish skin determined by the Turing mechanism?.
      ,
      • Hamada H.
      • Watanabe M.
      • Lau H.E.
      • Nishida T.
      • Hasegawa T.
      • Parichy D.M.
      • Kondo S.
      Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning.
      ). The zebrafish has a third type of pigment cell, the iridophore, which has reflecting internal structures and is involved in skin pattern formation by providing the initial conditions of the pattern formation system (
      • Singh A.P.
      • Schach U.
      • Nüsslein-Volhard C.
      Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish.
      ).
      Figure thumbnail gr1
      FIGURE 1.Skin patterns of zebrafish. A, wild type; B, leopard mutant (cx41.8−/−, leot1 allele, null mutant); C, leopard mutant (cx41.8tq270/tq270, leotq270 allele, Cx41.8I203F); D, luchs mutant (cx39.4−/−; knock-out of cx39.4 by TALEN); E, luchs/leopard double homozygous mutant (cx39.4−/−;cx41.8−/−); F, connexin topology sketch highlighting Cx41.8 mutants used in this study. G, summary of the transgenic experiments; H–J, transgenic zebrafish: Tg(mitfa-cx41.8I203F)cx41.8−/− (H), Tg(mitfa-cx41.8M7)cx41.8−/− (I), and Tg(mitfa-cx41.8M7)cx41.8+/+ (J). H′–J′, magnified images of H–J. Scale bar, 5 mm.
      Gap junctions also play an important role in pattern formation (
      • Watanabe M.
      • Kondo S.
      Is pigment patterning in fish skin determined by the Turing mechanism?.
      ,
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ). The gene encoding Cx41.8, an orthologue to the mammalian CX40, was previously identified as a responsible gene for the leopard mutant, which shows a spot pattern phenotype (Fig. 1B) (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). Several alleles in the leopard mutant are known (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ,
      • Maderspacher F.
      • Nüsslein-Volhard C.
      Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions.
      ). For example, cx41.8t1/t1 is a null mutant allele (Fig. 1B), and cx41.8tq270/tq270 (Fig. 1C) is a dominant mutant allele with an I203F amino acid substitution within the fourth transmembrane domain of Cx41.8 (Fig. 1F). This mutant displays a spot pattern phenotype in both heterozygotic and homozygotic situations. Because the connexin mutant cx41.8tq270/tq270 shows a more severe phenotype than that of the Cx41.8 null mutant, it has been predicted that Cx41.8 makes a heteromeric/heterotypic gap junction with unidentified connexin(s) (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). Recently, we successfully observed changes in Turing-related patterning on fish skin by altering the gene encoding Cx41.8 (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ). Cx41.8M7, which has a 6-amino acid deletion from the N terminus of Cx41.8, caused a labyrinth skin pattern when it was introduced into wild-type zebrafish. In summary, Cx41.8 was observed to play a role in tuning the interactions between pigment cells to make Turing patterns in vivo, similar to changing the parameters on the Turing model in silico (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ).
      Most recently, Cx39.4, a novel connexin specific to zebrafish, was identified as a response gene in the zebrafish skin pattern mutant luchs (Fig. 1D) (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ). Cx39.4 is an α-type connexin, as is Cx41.8 (Fig. 2A), and is distributed in the teleost lineage (Fig. 2C). Cx39.4 has an N-terminal sequence 2 amino acids longer, which is predicted to produce unique gap junction characteristics (Fig. 2B). Furthermore, the Cx39.4 null mutant shows a labyrinth pattern (Fig. 1D) (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ), similar to that observed in transgenic zebrafish with the Cx41.8M7 mutation (Fig. 1, J and J′) (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ). The similar phenotype could suggest that Cx39.4 and Cx41.8 interact in vivo (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ).
      Figure thumbnail gr2
      FIGURE 2.Gap junctions in zebrafish and characterization of Cx39.4. A, phylogenic relationship of α-type connexins in rats and zebrafish. B, alignment of N-terminal sequences of α-type connexins in zebrafish; C, alignment of the N-terminal sequences of GJA14 in several teleosts compared with zebrafish Cx41.8: Cs, Cynoglossus semilaevis (XP_008329622.1); El, Esox lucius (XP_010896375); Nc, Notothenia coriiceps (XP_010777882.1); On, Oreochromis niloticus (XP_005453942.1); Ol, Oryzias latipes (XP_004084568.1); Pf, Poecilia Formosa (XP_007559865.1); Sp, Stegastes partitus (XP_008293308.1); Tr, Takifugu rubripes (XP_011607358.1). B and C, blue letters indicate basic residues, and red letters indicate acidic residues. D, exon-intron structure of cx39.4. E, pTol2-BAC construct for analyzing cx39.4 promoter activity. F, the mCherry signals were detected in melanophores (purple arrow) and xanthophores (yellow arrow), driven by the cx39.4-promoter. G, the mRNA expression of connexin genes in isolated melanophores (M) and xanthophores (X). bact, β-actin, a positive control for RT-PCR. dct is a melanophore marker, and aox3 is a xanthophore marker.
      In this study, we performed both transgenic experiments and electrophysiological experiments to assess function and interactions between Cx41.8 and Cx39.4. The oocyte expression system is commonly used to study properties and interactions of mammalian gap junction proteins (
      • Swenson K.I.
      • Jordan J.R.
      • Beyer E.C.
      • Paul D.L.
      Formation of gap junctions by expression of connexins in Xenopus oocyte pairs.
      ), and zebrafish connexins have previously been expressed and analyzed using dual cell voltage clamp methods after expression in oocytes (
      • Dermietzel R.
      • Kremer M.
      • Paputsoglu G.
      • Stang A.
      • Skerrett I.M.
      • Gomes D.
      • Srinivas M.
      • Janssen-Bienhold U.
      • Weiler R.
      • Nicholson B.J.
      • Bruzzone R.
      • Spray D.C.
      Molecular and functional diversity of neural connexins in the retina.
      ).

      Results

      As noted above, Irion et al. (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ) recently identified cx39.4 as a gene that is responsible for a zebrafish skin pattern mutant called luchs, which shows an irregular, labyrinth-like skin pattern. They suggested that cx39.4 is a zebrafish-specific gene, and Cx39.4 might partner with Cx41.8 to make a heteromeric gap junction, although no analysis was performed or reported. Here, to examine the relationship between Cx41.8 and Cx39.4 in skin pattern formation, we performed electrophysiological experiments involving Cx41.8 and Cx39.4.

      Characterization of cx39.4 Gene

      Fig. 2B shows the amino acid alignment of the N-terminal sequences of α-type connexins in zebrafish and indicates that Cx39.4 has an N terminus that is 2 amino acids longer than other connexins. Because Cx39.4 belongs to the GJA subfamily of gap junctions but does not have a mammalian orthologue, we used the designation “GJA14” for Cx39.4 (Fig. 2A). Database analysis revealed that Cx39.4 is not zebrafish-specific but a teleost-specific connexin (Fig. 2C) and that the EXXXE motif, a predicted polyamine binding site, is well conserved among Cx39.4 proteins in teleosts (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ,
      • Watanabe M.
      • Watanabe D.
      • Kondo S.
      Polyamine sensitivity of gap junctions is required for skin pattern formation in zebrafish.
      ). Next, we examined the exon-intron structure of cx39.4 using 5′-RACE methods and found 102 bp of exon 1 and 7.7 kb of intron 1 located upstream of exon 2, as shown in Fig. 2D. Exon 1 includes the 5′-UTR sequence but does not include the ORF sequence, which is encoded only by exon 2. This gene structure is very common for connexin genes (
      • Söhl G.
      • Willecke K.
      Gap junctions and the connexin protein family.
      ). Next, we examined the gene expression of cx39.4, not by in situ hybridization or immunostaining, but by transgenic and RT-PCR experiments, because it is very difficult to detect small amounts of mRNA or protein in pigment cells (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ). We generated a reporter construct using a BAC clone to detect the promoter activity of cx39.4. The ORF sequence for cx39.4 was replaced with the mCherry gene, and the pTol2 cassette was inserted into the BAC plasmid, as shown in Fig. 2E. The BAC plasmid was co-injected with Tol2 mRNA into fertilized eggs at the one-cell stage, and mCherry signals were detected in pigment cells, melanophores, and xanthophores in adult fish skin at the F0 generation (Fig. 2F). Next, we confirmed the gene expression of cx39.4 by RT-PCR experiments. In this study, we examined the gene expression of α-type connexins and found that cx41.8 and cx39.4 were expressed in both melanophores and xanthophores, and the other α-type connexins tested were not detected (Fig. 2G). To rule out the possibility of cross-contamination of melanophores and xanthophores, the expression levels of dct and aox3 were also examined. Connexins belonging to GJA4 (Fig. 2A) were not analyzed in this study because their exon-intron structures were not elucidated.

      Zebrafish Mutants

      Mutations in Cx41.8 are known to alter skin patterns in zebrafish (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ,
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). In this study, we characterize two Cx41.8 mutations: I203F, a mutant involving an amino acid substitution in the fourth transmembrane domain (Fig. 1F), which induces a spot pattern rather than stripes (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ), and Cx41.8M7, an artificially designed mutant with a 6-amino acid deletion from its N terminus (Fig. 1F). The ectopic expression of Cx41.8M7 in wild-type pigment cells caused a labyrinth skin pattern in zebrafish (Fig. 1, J and J′) (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ). In this study, we found that the ectopic expression of Cx41.8M7 on the cx41.8−/− background induced an unusual pattern of very small spots (Fig. 1, I and I′). The same pattern was observed in the luchs/leopard double homozygous mutant (cx39.4−/−;cx41.8−/−) (Fig. 1E). Taken together, the similarity of the phenotypes between mutants of luchs or luchs/leopard and Cx41.8M7 transgenic fish lead to the hypothesis that Cx41.8 and Cx39.4 interact in vivo.
      The two mutations Cx41.8I203F and Cx41.8M7 were introduced into the cx41.8 null background fish, and their effects were assessed in the F1 generation (Fig. 1, G–I). When Cx41.8I203F was introduced into the cx41.8 null mutant, the leopard phenotype was partially rescued (Fig. 1, H and H′). However, when Cx41.8M7 was introduced into the cx41.8 null mutant, the mutant phenotype was not rescued; instead, the transgenic fish showed a more severe phenotype (Fig. 1, I and I′) than the Cx41.8 null mutant (Fig. 1B): a smaller spot pattern. These results show that Cx41.8I203F retained the gap junction function to form stripes, but Cx41.8M7 did not, and that Cx41.8M7 had a negative effect on other connexin(s) in vivo.

      Expression in Oocytes

      To examine the gap junction properties of Cx41.8 and Cx39.4, the connexins were expressed in Xenopus oocytes. In paired oocytes, junctional currents were induced by both Cx41.8 and Cx39.4. The voltage sensitivity of Cx41.8 was similar to that of zebrafish Cx45.6 (
      • Christie T.L.
      • Mui R.
      • White T.W.
      • Valdimarsson G.
      Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin.
      ), a paralogous connexin in zebrafish, and rat CX40 (
      • Bruzzone R.
      • Haefliger J.A.
      • Gimlich R.L.
      • Paul D.L.
      Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.
      ), an orthologous connexin in mammals (Fig. 3, A and A′). Cx41.8 displayed relatively strong sensitivity to the transjunctional voltage and relatively quick time-dependent inactivation in response to application of transjunctional voltage (Fig. 3A). As shown in Fig. 3B, Cx39.4 formed a functional gap junction between Xenopus oocytes but showed weaker sensitivity to transjunctional voltage than Cx41.8 (Fig. 3B′); gap junction channels induced by Cx39.4 required higher transjunctional voltages for inactivation and displayed a slower time-dependent decay of the junctional current compared with Cx41.8. For instance, when currents induced by transjunctional voltage of ±100 mV were fitted to the exponential curve, y = A0 + A1exp(−kx), [k] values were [k]Cx41.8 = 2.9 × 10−2 and [k]Cx39.4 = 7.7 × 10−3 (1/s), which were consistent with this observation.
      Figure thumbnail gr3
      FIGURE 3.Electrophysiological analysis of homomeric-homotypic gap junctions. Junctional current traces of homotypic gap junctions of Cx41.8 (A), Cx39.4 (B), Cx41.8I203F (C), and Cx41.8M7 (D). A′ and B′, plots of normalized steady state junctional conductance versus transjunctional voltages (A′, n = 24; B′, n = 30). For these experiments, 40 ng of mRNA for Cx41.8 or its mutant or 5 ng of mRNA for Cx39.4 was injected into each oocyte.
      We also examined the formation of heterotypic gap junctions by expressing Cx41.8 and Cx39.4 in apposing cells. When an oocyte injected with mRNA for Cx39.4 was paired with an oocyte injected with mRNA for Cx41.8, transjunctional current with asymmetric sensitivity to voltage was induced (Fig. 4, A and A′).
      Figure thumbnail gr4
      FIGURE 4.Cx39.4 and Cx41.8 form heterotypic gap junction channels. Junctional current traces induced by heterotypic gap junctions resulting from the expression of Cx41.8 and Cx39.4 in apposing cells. A′, plot of normalized steady state junctional conductance versus transjunctional voltages (n = 7).
      We further assessed the roles and interactions of Cx41.8 and Cx39.4 by expressing Cx41.8 mutants in oocytes. Neither of the Cx41.8 mutants (neither Cx41.8I203F nor Cx41.8M7) induced junctional currents in oocytes (Fig. 3, C and D). To further elucidate differences between Cx41.8I203F and Cx41.8M7, we co-expressed wild-type and mutant connexins in the same oocyte. When 40 ng of mRNA for Cx41.8I203F was co-injected with the same amount of mRNA for WT Cx41.8, a small current was consistently detected (Fig. 5, B and B′). In contrast, when 40 ng of mRNA for Cx41.8M7 was co-injected with mRNA for WT Cx41.8, no current was detected (Fig. 5D). This result indicates that the ability to rescue the leopard phenotype correlates with the formation of gap junction channels in oocytes. The Cx41.8M7 inhibits the function of Cx41.8 junctions in oocytes (Fig. 1, H and I) and induces a small-spot phenotype in the Cx41.8 null mutant (Fig. 1I). This suggests that Cx41.8M7 forms heteromeric channels with Cx41.8.
      Figure thumbnail gr5
      FIGURE 5.Electrophysiological analysis of heteromeric gap junctions. Junctional current traces of heteromeric gap junctions of Cx41.8 + Cx39.4 (A), Cx41.8 +Cx41.8I203F (B), Cx39.4 + Cx41.8I203F (C), Cx41.8 + Cx41.8M7 (D), and Cx39.4 + Cx41.8M7 (E). A′–C′, plots of normalized steady state junctional conductance versus transjunctional voltages (A′, n = 8; B′, n = 11; C′, n = 15).
      We also used the Xenopus oocyte expression system to examine the possibility that Cx41.8 and Cx39.4 interact, creating heteromeric channels. The junctional currents induced after co-expression of the two connexins displayed intermediate sensitivity to voltage (Fig. 5, A and A′), weaker time- and voltage-dependent inactivation than Cx41.8 (Fig. 3A) but stronger than Cx39.4 (Fig. 3B). This is consistent with the formation of heteromeric channels but not conclusive. The co-expression of mutants such as Cx41.8I203F and Cx41.8M7 with wild type Cx41.8 and Cx39.4 provided stronger evidence for heteromerization (Fig. 5, B–D). Co-expression of the N-terminal mutant abolished currents induced by Cx39.4 (Fig. 5E) and Cx41.8 (Fig. 5D). These results provide strong evidence for interactions between Cx39.4 and Cx41.8 and are consistent with in vivo experiments (Fig. 6).
      Figure thumbnail gr6
      FIGURE 6.Electrophysiological analysis of Cx41.8 mutants. Cx41.8 mutants exhibit negative effects on WT Cx41.8 (A) and Cx39.4 (B) in heteromeric gap junctions. Current values of −100 mV at the steady state were used for the comparison. Error bar, S.E. *, p < 0.01; **, p < 0.05, calculated by Student's t test.

      Properties of Non-junctional Currents

      Non-junctional currents carried by connexin channels are often referred to as hemichannel currents. These currents occur across the plasma membrane of single oocytes and were assessed in oocytes injected with RNA encoding either Cx39.4 or Cx41.8 and also oocytes co-injected with RNA encoding both connexins. In addition, both of the Cx41.8 mutants were expressed in single oocytes, and their membrane currents were assessed. Currents induced by Cx41.8, Cx41.8I203F, and Cx41.8M7 were small but differed significantly from each other and from oligonucleotide-injected controls. (Figs. 7 (A–D) and 8A). The relative levels of current induced were well correlated with the junctional current levels induced by Cx41.8, Cx41.8I203F, and Cx41.8M7 (Figs. 6A and 8A and Table 2). In contrast, Cx39.4 RNA induced a large hemichannel current, which increased with membrane depolarization (Fig. 7E). When the Cx41.8 mutants were co-expressed with Cx39.4, membrane currents were reduced significantly compared with those induced by Cx39.4 alone. Specifically, 5 ng of mRNA encoding Cx39.4 was co-injected with 40 ng of RNA for Cx41.8 or the Cx41.8 mutants, and in all cases, current levels were significantly lower than in cells expressing Cx39.4 alone. Fig. 7F shows that the Cx39.4 hemichannel current was reduced by co-expression of Cx41.8, and Fig. 7, G and H, shows that the Cx39.4 hemichannel current was reduced by Cx41.8I203F and Cx41.8M7 (Fig. 8B). These results provide strong evidence that Cx41.8 interacts with Cx39.4 to form heteromeric hemichannels. Furthermore, the results are consistent with the observation that co-expression of Cx41.8M7 significantly reduced gap junction currents carried by Cx39.4 in paired oocytes.
      Figure thumbnail gr7
      FIGURE 7.Non-junctional current recordings. For these experiments, 40 ng of mRNA for the Cx41.8 or Cx41.8 mutant and/or 5 ng of mRNA for Cx39.4 was injected into each oocyte. ■, 0 mm [Ca2+]; □, 2 mm [Ca2+]. A, Cx41.8; ■, n = 15; □, n = 14. B, H2O; ■, n = 14; □, n = 13. C, Cx41.8I203F; ■, n = 20; □, n = 28. D, Cx41.8M7; ■, n = 10; □, n = 10. E, Cx39.4; ■, n = 17; □, n = 12. F, Cx39.4 + Cx41.8; ■, n = 14; □, n = 12. G, Cx39.4 + Cx41.8I203F; ■, n = 18; □, n = 15. H, Cx39.4 + Cx41.8M7; ■, n = 15; □, n = 7. Error bars, S.E.
      Figure thumbnail gr8
      FIGURE 8.Hemichannel activities of Cx41.8 and Cx39.4. A, Cx41.8 and Cx41.8 mutants. B, effect of Cx41.8 and Cx41.8 mutants on the activity of Cx39.4 in heteromeric hemichannel. Current values of 30 mV at the instantaneous state of recording were used for the comparison. Error bars, S.E.; *, p < 0.01; **, p < 0.05, calculated by Student's t test.
      TABLE 2Corresponding table of phenotype and properties of gap junction and hemichannel

      Discussion

      When we identified cx41.8 as a gene responsible for the leopard mutant, one or more connexins were predicted to be involved in the zebrafish skin pattern formation (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). In addition, the leotq270 and leotw28 alleles of the leopard mutant, which have the amino acid substitutions I203F and I31F, respectively, showed dominant phenotypes (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). In particular, the leotq270 allele showed smaller spots than did the cx41.8 null mutant (
      • Watanabe M.
      • Iwashita M.
      • Ishii M.
      • Kurachi Y.
      • Kawakami A.
      • Kondo S.
      • Okada N.
      Spot pattern of leopard Danio is caused by mutation in the zebrafish connexin41.8 gene.
      ). Hence, previous results suggested that connexin interactions might be important for skin pattern formation. Specifically, it was hypothesized that the Cx41.8I203F mutant forms a heteromeric and/or heterotypic gap junction with another connexin protein(s), reducing gap junction function. We have shown that the Cx41.8M7 mutant creates a very severe phenotype when introduced into the cx41.8 null mutant, which also suggests the involvement of other connexin(s) in pattern formation. Recently, Irion et al. (
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ) successfully showed that Cx39.4 is also involved in skin pattern formation and that Cx39.4 functions in melanophores and xanthophores. The expression of cx39.4 in melanophores was independently reported by the Johnson group (
      • Higdon C.W.
      • Mitra R.D.
      • Johnson S.L.
      Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.
      ). Cx39.4 expression was detected by the transcriptome analysis of melanophores using NGS (next-generation sequencing) (
      • Higdon C.W.
      • Mitra R.D.
      • Johnson S.L.
      Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.
      ). Our RT-PCR results and transgenic experiments also suggested the expression of Cx39.4 in pigment cells.
      In this study, we showed that Cx39.4 has different properties from Cx41.8 in both gap junctions and hemichannels. Furthermore, we showed that currents carried by gap junctions and hemichannels correlate to skin pattern phenotypes, although the question of which function is required or whether both are required for pattern formation remains unresolved. By the analysis of hemichannel currents (Fig. 7), we found that Cx41.8 and the mutants Cx41.8I203F and Cx41.8M7 induced very small currents when expressed alone in single oocytes (Fig. 8A). However, both Cx41.8 and Cx41.8I203F mutant reduced Cx39.4 activity when co-expressed in single oocytes (Fig. 8B).
      Transplantation experiments suggest that Cx41.8 and Cx39.4 form gap junction(s) between melanophores and xanthophores (
      • Maderspacher F.
      • Nüsslein-Volhard C.
      Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions.
      ,
      • Irion U.
      • Frohnhöfer H.G.
      • Krauss J.
      • Çolak Champollion T.
      • Maischein H.M.
      • Geiger-Rudolph S.
      • Weiler C.
      • Nüsslein-Volhard C.
      Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.
      ). The mathematical model also suggests interactions between melanophores and xanthophores. By modeling different interactions between melanophores and xanthophores, various skin patterns were easily generated in silico, as can be achieved by changing the function of gap junctions in vivo (
      • Watanabe M.
      • Kondo S.
      Changing clothes easily: connexin41.8 regulates skin pattern variation.
      ). We conclude that Cx39.4 and Cx41.8 function in a coordinated manner in the interaction between melanophores and xanthophores to create the striped pattern of zebrafish.
      The gating mechanisms of gap junctions are quite diverse and include responses to cytoplasmic calcium, protons, and phosphorylation as well as transjunctional (Vj) and transmembrane voltage (
      • Harris A.L.
      Emerging issues of connexin channels: biophysics fills the gap.
      ). Two gating models prevail in the literature. One is a ball and chain model, in which the N-terminal domains of six connexins form a plug structure; this plug controls the open/closed states of the gap junction, depending on the resting potential of the cells (
      • Oshima A.
      • Tani K.
      • Hiroaki Y.
      • Fujiyoshi Y.
      • Sosinsky G.E.
      Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule.
      ,
      • Maeda S.
      • Nakagawa S.
      • Suga M.
      • Yamashita E.
      • Oshima A.
      • Fujiyoshi Y.
      • Tsukihara T.
      Structure of the connexin 26 gap junction channel at 3.5 Å resolution.
      • Oshima A.
      Structure and closure of connexin gap junction channels.
      ). The other is the subunit rotation model, which controls the open/closed states of the gap junction by changing the three-dimensional conformation of the gap junction to alter the channel pore size, depending on the calcium ion concentration (
      • Oshima A.
      Structure and closure of connexin gap junction channels.
      ) and modification of the C-terminal domain of connexin (
      • Grosely R.
      • Kopanic J.L.
      • Nabors S.
      • Kieken F.
      • Spagnol G.
      • Al-Mugotir M.
      • Zach S.
      • Sorgen P.L.
      Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain.
      ,
      • Kjenseth A.
      • Fykerud T.A.
      • Sirnes S.
      • Bruun J.
      • Yohannes Z.
      • Kolberg M.
      • Omori Y.
      • Rivedal E.
      • Leithe E.
      The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.
      ). We used the N terminus deletion mutant Cx41.8M7 in this study because it was previously predicted that deletion of the N terminus domain would cause a closed-state gap junction (
      • Kyle J.W.
      • Minogue P.J.
      • Thomas B.C.
      • Domowicz D.A.
      • Berthoud V.M.
      • Hanck D.A.
      • Beyer E.C.
      An intact connexin N-terminus is required for function but not gap junction formation.
      ,
      • Oshima A.
      • Tani K.
      • Toloue M.M.
      • Hiroaki Y.
      • Smock A.
      • Inukai S.
      • Cone A.
      • Nicholson B.J.
      • Sosinsky G.E.
      • Fujiyoshi Y.
      Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.
      ). The possibility that the N-terminal deletion might function as a dominant negative connexin form was supported by our electrophysiological analyses showing that Cx41.8M7 inhibits both the gap junction and hemichannel function of Cx41.8 and Cx39.4.
      We previously hypothesized that gap junctions involving Cx41.8 might have rectification properties because Cx41.8 has the EXXXE motif in its N terminus, such as rat CX40 (
      • Musa H.
      • Fenn E.
      • Crye M.
      • Gemel J.
      • Beyer E.C.
      • Veenstra R.D.
      Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions.
      ), which is recognized to be a polyamine binding motif. In the case of zebrafish pigment cells, melanophores express Kir7.1, and this expression is required to make a clear boundary between melanophores and xanthophores in fish skin (
      • Inaba M.
      • Yamanaka H.
      • Kondo S.
      Pigment pattern formation by contact-dependent depolarization.
      ). If Kir7.1 is mutated, melanophores and xanthophores intermingle, and the boundary becomes obscure. Because it is well known that polyamine is required for the rectifier property of Kir channels (
      • Hibino H.
      • Inanobe A.
      • Furutani K.
      • Murakami S.
      • Findlay I.
      • Kurachi Y.
      Inwardly rectifying potassium channels: their structure, function, and physiological roles.
      ), polyamine is hypothesized to localize around the cell membranes of melanophores and possibly control the channel function of Kir7.1 and gap junctions involving Cx41.8. In Cx39.4, redundant EXXXE motifs exist at the N terminus, and this motif is well conserved among Cx39.4 orthologues in teleosts. This observation suggests that Cx39.4 and Cx41.8 might work together to generate a unidirectional signal flow from xanthophores to melanophores.
      This is the first study aimed at correlating gap junction channel function and skin pattern phenotype in zebrafish (Fig. 1). There was a strong correlation between the behavior of connexins expressed in Xenopus oocytes and the phenotypes observed in zebrafish. Both systems provide evidence for interactions between Cx41.8 and Cx39.4. This is also the first study to reveal that Cx39.4 induces calcium-sensitive transmembrane currents that can be regulated (reduced) by co-expression of Cx41.8. This finding may be incorporated into models of pattern formation and suggests that further investigation of connexin function is warranted.

      Author Contributions

      M. W. and S. K. conceived the study. M. W, R. S., and T. A. performed transgenic experiments. M. W. and I. M. S. performed electrophysiological experiments and analyzed data. M. W. wrote the paper.

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