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

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.

Tandem alternative splicing of zebrafish connexin45.6.

Early studies suggested that most connexin genes share a relatively simple structure with a single intron of variable length interrupting the 5' untranslated region (UTR). Here we report that zebrafish cx45.6 shows six isoforms of alternative 5'UTRs which are generated from multiple promoter usage and alternative pre-mRNA splicing. Interestingly, cx45.6 undergoes tandem alternative splicing, which produces transcripts only differing by 3 nucleotides. This is the first study that has demonstrated tandem alternative pre-mRNA splicing in the connexin gene family. Expression patterns of cx45.6 alternative transcripts were demonstrated by real-time RT-PCR during zebrafish embryonic development and in adult tissues. The complexity of 5'UTR diversity suggests complicated regulatory mechanisms for cx45.6 gene expression at both transcriptional and post-transcriptional levels, and we propose that tandem alternative splicing in cx45.6 5'UTRs could play a role in translational control. These results lay groundwork for further investigations on the regulation and function of cx45.6 gene expression.

Posttranslational modifications in lens fiber connexins identified by off-line-HPLC MALDI-quadrupole time-of-flight mass spectrometry.

PURPOSE: Gap junction intercellular communication is necessary for the development and maintenance of the lens. Lens fiber connexins are known to be posttranslationally modified, but little detail is available regarding the nature of some of these modifications and the specific amino acids affected. The purpose of this study was to identify posttranslational modification in the bovine lens fiber connexins, Cx44 and Cx49. METHODS: Crude preparations of bovine lens membranes were isolated by centrifugation. The membrane preparations were digested with trypsin or chymotrypsin, and the entire mixture of peptides produced was separated by reverse-phase high performance liquid chromatography and then analyzed by mass spectrometry and tandem mass spectrometry. RESULTS: Coverage of significant portions of the cytoplasmic domains of both Cx44 and Cx49 were successfully obtained. Several of the Ser and Thr residues in the C-tail of Cx44 were phosphorylated, whereas in Cx49 only Ser phosphorylation was detected; however, in this connexin, the phosphorylated residues were located in both the C-tail and the central cytoplasmic loop. The data also show that the N-terminal Met residue in each connexin is removed and that the newly exposed N termini become acetylated. In addition, cleavage sites were identified in both proteins. CONCLUSIONS: The study documented the nature and locations of several previously unknown posttranslational modifications in lens fiber connexins. This detailed knowledge of the specific posttranslationally modified sites will allow further work to elucidate the mechanisms that different signaling pathways use to regulate connexins in lens fiber cells.

Developmental regulation and expression of the zebrafish connexin43 gene.

We cloned and sequenced the zebrafish (Danio rerio) connexin43 (Cx43alpha1) gene. The predicted protein sequence shows a high degree of sequence conservation. Transcript analyses revealed multiple transcription start sites and a potential alternative transcript encoding a N-terminally truncated Cx43alpha1 protein. Maternal Cx43alpha1 transcripts were detected, with zygotic expression initiated before gastrulation. In situ hybridization revealed many Cx43alpha1 expression domains, including the notochord and brain, heart and vasculature, many resembling patterns seen in mammalian embryos. Of interest, a reporter construct under control of the mouse Cx43alpha1 promoter was observed to drive green fluorescent protein expression in zebrafish embryos in domains mimicking the native Cx43alpha1 expression pattern in fish and mice. Sequence comparison between the mouse and zebrafish Cx43alpha1 promoter sequences showed the conservation of several transcription factor motifs, which otherwise shared little overall sequence homology. The conservation of protein sequence and developmental gene regulation would suggest that Cx43alpha1 gap junctions are likely to have conserved roles in vertebrate embryonic development.

Biophysical characterization of zebrafish connexin35 hemichannels.

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (I(hc)) that were activated and increased by stepwise depolarization of membrane potential (V(m)) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (gamma(hc): 250-320 pS). The gamma(hc) of Cx35 hemichannels exhibited a pronounced V(m) dependence; i.e., gamma(hc) increased/decreased with relative hyperpolarization/depolarization (gamma(hc) was 72 pS at V(m) = -100 mV and 35 pS at V(m) = 100 mV). Extrapolation to V(m) = 0 mV predicted a gamma(hc) of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of approximately 24 pS. Channel gating was also V(m) dependent: open time declined with negative V(m) and increased with positive V(m). The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.

Connexin 48.5 is required for normal cardiovascular function and lens development in zebrafish embryos.

Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin.

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.

Expression of connexin48.5, connexin44.1, and connexin43 during zebrafish (Danio rerio) lens development.

Connexins (Cx), the protein units of gap junctions, play important roles in lens development and homeostasis. Here, we report the mRNA expression patterns of zebrafish Cx48.5, Cx44.1, Cx43 during lens development. The expression of all three connexins in the adult lens was first confirmed by reverse transcriptase-polymerase chain reaction. By whole-mount in situ hybridization, we detected Cx48.5 expression throughout the lens, except the lateral lens epithelium, at 36 hours postfertilization (hpf). The pattern remained the same at 2 days postfertilization (dpf). By 3 and 4 dpf, Cx48.5 expression was restricted to the differentiating lens fibers in the equatorial and medial regions. Cx44.1 was expressed in a similar manner as Cx48.5 from 36 hpf to 4 dpf. However, Cx44.1 expression was also detected in the lens at 24 hpf. Cx43 expression was detected throughout the lens at 24 and 36 hpf but became restricted to the lateral epithelium at later stages.

Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.