Zebrafish cx30.3: identification and characterization of a gap junction gene highly expressed in the skin

Abstract

We have identified and characterized a zebrafish connexin, Cx30.3. Sequence similarity analyses suggested that Cx30.3 was orthologous to both mammalian Cx26 and Cx30, known to play important roles in the skin and inner ear of mammals. Analysis of mRNA expression showed that Cx30.3 was present in early embryos, and was highly abundant in skin, but also detected in other tissues including fins, inner ear, heart, and the retina. Injection of Cx30.3 cRNA into Xenopus oocytes elicited robust intercellular coupling with voltage gating sensitivity similar to mammalian Cx26 and Cx30. The similarities in functional properties and expression patterns suggest that Cx30.3, like mammalian Cx26 and Cx30, may play a significant role in skin development, hearing, and balance in zebrafish. Thus, zebrafish could potentially serve as an excellent model to study disorders of the skin and deafness that result from human connexin mutations.

Fig. 1. Amino acid sequence alignment of Cx30.3

Zebrafish Cx30.3, and two closely related sequences, human Cx26 (hCx26) and Cx30 (hCx30) were aligned by the CLUSTAL W method. Solid lines under the sequences indicate the four predicted transmembrane domains (M1, M2, M3, and M4). The asterisks indicate the six conserved cysteine residues within the two predicted extracellular loops of Cx30.3. Yellow shading highlights residues identical to Cx30.3.

Fig. 2. Phylogenetic analysis and genomic organization of zebrafish and mammalian connexins

(A). A neighbour-joining tree shows that the zebrafish Cx30.3 amino acid sequence is most closely related to human Cx26 and Cx30. Bootstrap values were based on 1000 neighboring replicates and indicated as node labelling. (B). Similar to a cluster of three connexins (Cx30, Cx26, Cx46) on human chromosome 13, cx48.5 (the zebrafish ortholog of Cx46) is closely linked to cx30.3 on chromosome 9. Connexin genes are represented by arrowheads; gene distance is not drawn in scale. (C). When sequence comparison was limited to Cx26 and Cx30 orthologs, Cx30.3 was most closely related to chicken Cx30 and frog Cx26 sequences, and equally related to mammalian orthologs from bovine, human, mouse and rat. (D). Pair wise sequence comparison of Cx30.3 to both Cx26 and Cx30 orthologs was equally high, and ranged from 55 to 62% identity.

Fig. 3

Intron/Exon Structure of Zebrafish cx30.3 (A). Sequence of the 5′UTR (underlined) and part of the coding region. The translational start site (ATG) identified in the present study is highlighted in a dark box (GenBank accession number: GQ469999). The position of the adenine nucleotide is designated as +1, with its upstream nucleotide as −1 and downstream as +2. The transcriptional start site is indicated by asterisk (*) above the sequence at −1760, and a “TATA” like element was found upstream in the core promoter (indicated in a box). The 5′UTR of cx30.3 is interrupted by a single intron of about 1.6 kb in size (italic letters). The previously reported translational start site (ATG) is highlighted in a gray box (GenBank accession number: AY135446), with its adenine nucleotide located at −90, which is within the intron region identified in the present study. (B). Sketch of intron/exon genomic structure. Filled boxes represent both exons of zebrafish cx30.3 transcript, while solid lines represent genomic sequences, or the intron (not drawn on scale). The blank box represents the sequence between the previously reported translational start site and the second exon identified in the present study.

Fig. 4. Temporal and spatial analyses of cx30.3 mRNA expression

(A) Expression of cx30.3 was detected at the 1 day post fertilization (dpf) stage, and thereafter maintained throughout embryonic development. The zebrafish ef1α gene was used as positive control to demonstrate the integrity and genomic-DNA-contamination-free status of the cDNA samples. (B) cx30.3 mRNA is predominantly expressed in the adult skin, fin, inner ear, ovary, liver, heart, retina, while a relatively low level of cx30.3 expression was also detected in the gill, swim bladder, skeletal muscle, spleen and gut. The relative level of mRNA expression was calculated by comparing ΔCt between mean Cts of zebrafish cx30.3 and ef1α.

Fig. 5. In situ hybridization of Cx30.3 mRNA in embryos

Embryos were stained by whole-mount in situ hybridization with a cx30.3 anti-sense riboprobe. Signal was detected (and highlighted with arrows) in 1 dpf stage embryos, in the otic vesicle (A) and dorsal fins (B). At 2 dpf (C, G, H) or 3 dpf (D, E, F), cx30.3 mRNA signal was readily detected in the skin (C), the inner ear (D), the lateral line (E, G), and the fins (C). Sense-probe controls are shown in (F) and (H).

Fig. 6. Sections of cx30.3 in situ hybridization stained embryos

Embryos were stained by whole-mount in situ hybridization with cx30.3 anti-senses riboprobe, embedded in plastic and 5 μm sections were cut with a microtome. Only sections from embryos at 2 dpf are shown, the staining pattern in later stage embryos is similar. (A) Transverse cross-section of embryonic eyes, showing a lack of staining of the probe. (B) The developing inner ear with probe labeling the otic vesicle (arrow). (C) A transverse section of the embryonic fish tail and fin, with probe accumulating around the peripheral skin and on the fin (far right).

Fig. 7. Cx30.3 forms functional channels that are gated by transjunctional voltage

(A). Junctional conductance (G_j) developed between pairs of Xenopus oocytes as measured by dual voltage clamp. Bars show the mean ± SE of 13–19 pairs. (B). Voltage gating behavior of gap junction channels formed by Cx30.3. A time dependent decay of junctional currents (I_j) was induced by transjunctional voltage (V_j) steps. At V_j steps > ± 60 mV, I_j decayed symmetrically over the time course of the voltage step. (C). The relationship of V_j to steady-state junctional conductance (G_jss) normalized to the values obtained at ± 20 mV for Cx30.3. Solid lines represent the best fits to Boltzmann equations, whose parameters are: V₀=91, G_min=0.51, A=0.09 for +V_js and V₀=−93, G_min=0.43, A=0.06 for −V_js. The voltage gating of intercellular channels composed of zebrafish Cx30.3 displayed a high degree of conservation to human and rat Cx26.