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

Abstract

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 nonjunctional (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.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 ϳ1,000-Da small molecules to be transferred between neighboring cells (1,2). Approximately 20 connexins exist in mammalian genomes, and connexins are categorized into five subgroups (␣, ␤, ␥, ␦, and ), depending on their molecular weight and amino acid sequences (3, 4). In zebrafish, ϳ36 connexins are predicted to exist (5). 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 (6 -12).The zebrafish i...

Instructive role of melanocytes during pigment pattern formation of the avian skin

“…S10). To determine whether cx40 inhibition changes the pigment pattern, we constructed a dominant negative form of JQ cx40 (DNcx40) by deleting six N-terminal amino acids based on the zebrafish study (25,26). Electroporation of DNcx40 into JQ embryos produced ectopic black pigmentation, consistent with results of the inhibitor experiment ( Fig.…”

“…5 A and B). This type of connection could be mediated with gap junction channels as seen in the pigment cells of zebrafish, where a mutant study showed that connexin is involved in stripe formation (25,26). Therefore, we set up an explant culture system in which pigment stripes form and tested the role of ion channels and gap junctions in pigment patterning.…”

Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish

“…Double mutants for leo and luc loss-of-function alleles display a very severe phenotype, the pattern is completely dissolved with single melanophores scattered on a uniform light sheet of epithelial-like dense iridophores covered by a net of xanthophores. Mutants homozygous for the strongest of the dominant leo alleles, leo tK3 , show the same strong phenotype, arguing that both connexins can form heteromeric and homomeric gap junctions (Irion et al, 2014a; Maderspacher and Nüsslein-Volhard, 2003), which was confirmed by in vitro studies (Watanabe et al, 2015). This suggests that the communication between xanthophores and melanophores via heteromeric gap junctions provides signals to the dense iridophores to induce the transition into the loose shape required for dark stripe formation.…”

Evolution of the potassium channel geneKcnj13underlies colour pattern diversification inDaniofish

“…Several genes are autonomously required in the chromatophores for these heterotypic interactions 11,12,20,41-43 . These genes typically encode integral membrane proteins such as adhesion molecules 42 , channels 20 , or components of cellular junctions, some of which mediate direct cell contacts 43,44 . In Meox1 ( choker ) mutants, lacking the horizontal myoseptum as anatomical landmark, the horizontal orientation is lost, but stripes form of normal width and composition (Fig.…”

“…In D. rerio loss-of-function mutations in the gap junction genes Cx39 . 4 ( luchs ) 44,46 and Cx41 . 8/Gja5b ( leopard ) 41,46,47 as well as mutations in Igsf11 ( seurat ) 42 , which codes for a cell adhesion molecule, lead to melanophore spots (Fig.…”