AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 286: H1623-H1632, 2004. First published January 2, 2004; doi:10.1152/ajpheart.00800.2003
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Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin

Tara L. Christie,1 Rickie Mui,2 Thomas W. White,2 and Gunnar Valdimarsson1,3

1Department of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; 2Department of Physiology and Biophysics, State University of New York at Stony Brook, New York 11794; and 3Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada

Submitted 21 August 2003 ; accepted in final form 22 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

gap junction; Danio rerio; cardiovascular

GAP JUNCTIONS are collections of intercellular channels that allow the direct exchange of molecules up to about 1,200 Da in size, such as ions, second messengers, and small metabolites (10, 63). Connexins (Cx) are the structural units of gap junctions. Six connexin units join together to form a hemichannel termed a connexon, and two connexons from different cells dock to form an aqueous channel. A gap junction is formed by the aggregation of hundreds of these dodecameric channels into plaques that are 1–2 µm in diameter (66).

The connexins are encoded by a multigene family with representatives found in all vertebrate groups studied. The main connexins found in the mammalian heart are Cx37, Cx40, Cx43, and Cx45, with Cx43 being the most abundant connexin in the working myocardium (15, 25). Each connexin has a specific domain of expression within the heart. For example, Cx40 is abundant in the atrial myocardium of almost all species studied and is also found in the His bundle and proximal bundle branches (32, 33). Not surprisingly, the heart and vascular systems express similar sets of connexins. Generally, blood vessel endothelial cells express Cx40, Cx43, and Cx37, whereas the smooth muscle cells primarily express Cx43 and sometimes Cx40, Cx45, and/or Cx37 (12, 49).

Because gap junctions allow the direct and rapid exchange of small molecules between cells, they provide a simple, yet critical, method for coordinating a wide range of cellular behaviors in multicellular organisms (64). In the heart, a high degree of cell-cell communication is important for the propagation of the rhythmic action potentials generated by the sinoatrial node and also for the coordination of the contractions of the atria and ventricles. Action potentials are prevented from immediately passing from the atria to the ventricles by an insulating connective tissue layer, but they are thought to quickly pass through the His-Purkinje fibers in part due to the high density of gap junctions present in these fibers. Gap junctions also have an important role in the vascular system as they mediate the direct electrical and metabolic intercellular communication among endothelial cells, smooth muscle cells, endothelial cells and smooth muscle cells, and endothelial cells and lymphocytes and monocytes (25, 33, 49). Studies of cardiovascular connexin knockout mice have provided much insight into the roles of connexins in both cardiovascular development and function (5, 26, 36–39, 41, 46, 50–52), yet many questions remain unanswered.

Zebrafish (Danio rerio) are an excellent model system of vertebrate development due to their relatively short generation time, large clutch size, large optically transparent embryos, and easy access to all developmental stages (18). One benefit specific to cardiovascular studies is that early development can continue for a period of time in the absence of functional circulation (42). Even though the cardiovascular system of the zebrafish is functional by 24 h postfertilization (hpf), it is not essential in the early stages because the embryo is small enough to obtain the oxygen it requires through simple diffusion (60).

Although the adult zebrafish heart bears significant dissimilarities anatomically to the adult human heart, embryonic fish hearts are similar to the embryonic hearts of other vertebrates, including mammals (42). The complex process of the development of the vertebrate heart is also remarkably similar in its basic features among all vertebrates. Similarly, the formation of the vasculature in the zebrafish follows a plan similar to that of other vertebrates with only a few unique features attributed to the necessity of gills (31). Despite these differences, many processes and molecular pathways involved in both early heart and vascular development appear to be conserved among vertebrates (31, 42).

Previous studies have shown the zebrafish genome contains orthologues to known vertebrate connexins (11, 17, 21). The zebrafish orthologues of mammalian Cx43 (G. Valdimarsson, unpublished observations) and Cx45 (21), two of the four major connexins expressed in the vertebrate heart, have been cloned and characterized in the zebrafish. In the present study, the zebrafish orthologue of mammalian Cx40 (zfCx45.6) was cloned from a genomic library. The biophysical properties of the new connexin were determined in the Xenopus oocyte expression system, and the mRNA expression profile was determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fish care. Zebrafish were purchased from a local pet store and maintained at 28.5°C by following standard protocols (62). The fish were cared for following the Canadian Council on Animal Care guidelines.

Cloning. Primers for PCR were based on conserved regions of connexin nucleotide sequences corresponding to nucleotides coding for regions in the NH2-terminus (5'-AGTGATCGGAAAGGTTTGGTTAACCGTCCTG-3') and the fourth transmembrane region (5'-CTTTAACTTTTTCCATCCCAGGTGGTAGATCTC-3') (Life Technologies; Burlington, Ontario, Canada). Hotstart PCR was performed with Taq DNA Polymerase (Invitrogen; Carlsbad, CA), zebrafish genomic DNA, and the above primers. A 634-bp PCR product was cloned, sequenced, random primed with [32P]dCTP, and used to screen a zebrafish P1 artificial chromosome (PAC) library spotted on filters (German Human Genome Project, RZPD) (1, 2). The filters were hybridized overnight at 65°C, following the protocol set out by RZPD. Plasmid DNA from two independent clones was isolated from overnight cultures using the Qiagen Large Construct kit (Qiagen; Mississauga, Ontario, Canada) and initially sequenced with primers based on the PCR clone sequence.

Radiation hybrid mapping. ZfCx45.6 was mapped by PCR analysis of the LN54 RH panel (generously supplied by Marc Ekker, Loeb Institute). PCR was performed with 100 ng of DNA from each of the 93 cell lines and controls and primers that amplified a 536-bp nucleotide sequence of zfCx45.6 with the forward primer (5'-TGGATCTGCCTGAGAACAACC-3') in the region encoding for the COOH-terminus (C-tail) and the reverse primer (5'-GCTCTGATACAGATTCTTCCC-3') in the 3'-UTR. The PCR products were run on an agarose gel to determine which hybrids were positive for zfCx45.6. Each cell line was scored, results were entered into the LN54 panel web page (http://mgchdl.nichd.nih.gov:8000/zfrh/beta.cgi), and the linkage group was statistically determined.

Functional analysis. The zfCx45.6 coding sequence was PCR amplified using Pwo polymerase (Roche; Laval, Quebec, Canada) and primers flanking the start and stop codons (5'-GTTGTTCCTCAGCAGTGTAG-3 and 5'-GCTTCCAGCTTCTTTTCC-3'). The amplified fragment was cloned into pDrive (Qiagen) and then subcloned into pCS2+ (59). Subclones were sequenced, linearized with NotI, gel purified, and used as a template (1 µg DNA) to produce capped cRNAs using the mMessage mMachine kit (Ambion; Austin, TX). Stage V-VI oocytes were isolated from Xenopus laevis (Nasco; Fort Atkinson, WI), defolliculated by collagenase digestion, and cultured in Modified Barth's medium. Cells were injected with a total volume of 40 nl of either an antisense oligonucleotide (3 ng/cell) to suppress endogenous Xenopus Cx38 or a mixture of antisense plus zfCx45.6 cRNA (40 ng/cell), using a Nanoject II Auto/Oocyte injector (Drummond; Broomall, PA). After an overnight incubation, oocytes were immersed for a few minutes in hypertonic solution to strip the vitelline envelope, transferred to Petri dishes containing Modified Barth's medium, and manually paired with the vegetal poles apposed.

The functional properties of cell-to-cell channels were assessed by dual voltage clamp (53). Current and voltage electrodes (1.2 mm diameter, omega dot; Glass of America, Millville, NJ) were pulled to a resistance of 1–2 M{Omega} with a horizontal puller (Narishige, Tokyo, Japan) and filled with 3 M KCl, 10 mM EGTA, and 10 mM HEPES; pH 7.4. Voltage clamping of oocyte pairs was performed using two GeneClamp 500 amplifiers (Axon Instruments; Foster City, CA) controlled by a PC-compatible computer through a Digidata 1320A interface (Axon Instruments). pCLAMP 8.0 software (Axon Instruments) was used to program stimulus and data collection paradigms. Current outputs were filtered at 50 Hz, and the sampling interval was 10 ms. For simple measurements of junctional conductance, both cells of a pair were initially clamped at –40 mV to ensure zero transjunctional potential, and alternating pulses of ±10–20 mV were imposed to one cell. Current delivered to the cell clamped at –40 mV during the voltage pulse was equal in magnitude to the junctional current and was divided by the voltage to yield the conductance. To determine voltage-gating properties, transjunctional potentials (Vj) of opposite polarity were generated by hyperpolarizing or depolarizing one cell in 20-mV steps (over a range of ±120 mV), while clamping the second cell at –40 mV.

Northern and reverse transcriptase PCR analyses. Total RNA was isolated from embryos staged 0 days postfertilization (dpf) (0 dpf is 0–24 hpf) to 5 dpf using TRIzol Reagent (Invitrogen). PolyA+ mRNA was isolated from total RNA using the Oligotex mRNA Mini kit (Qiagen). Two micrograms of polyA+ mRNA from each sample were run on a 1% agarose denaturing formaldehyde gel and blotted by capillary action onto a nylon Magnacharge membrane (Osmonics; Minnetonka, MN) (47).

A 600-bp zfCx45.6 antisense RNA probe (encompassing nucleotides coding for the 4th transmembrane region and a portion of the C-tail) was created by in vitro transcription with T7 RNA polymerase and [32P]UTP. As a loading control, the blot was probed in the same fashion with a [32P]UTP-labeled antisense RNA probe created from a zebrafish elongation factor 1 {alpha}-clone (zfeF1{alpha}) (a generous gift from Dr. Ross Johnson, Minneapolis, MN).

For RT-PCR, total RNA was isolated from various embryonic stages and adult tissues as above. Residual DNA was removed with RQ1 RNase-free DNase (Promega; Madison, WI). First-strand cDNA was created using Superscript II Reverse Transcriptase (Invitrogen), and PCR was performed with primers amplifying about a 775-bp region coding for a portion of the COOH-tail (5'-GCTCTGTATCCATGTTCAATGC-3') and the 3'-UTR (5'-GCTCTGATACAGATTCTTCCC-3'). A control PCR was set up in a similar fashion using primers that anneal to exon sequences flanking an intron in the zfeF1{alpha} gene. The expected size of the elongation factor amplicon was 569 bp from cDNA and 733 bp from genomic DNA. To increase sensitivity and confirm the identity of the RT-PCR product, the experimental samples were blotted and probed with a [32P]dCTP-labeled DNA probe as in the PAC clone isolation.

Whole mount in situ hybridization. A zfCx45.6 probe that contained the entire coding sequence and ~150 bp each of upstream and downstream sequence was labeled by an in vitro transcription with DIG-11 UTP. Whole mount in situ hybridization (ISH) was performed on various stages of embryos between 0 and 5 dpf and adult hearts following the protocol set out by Thisse et al. (56) with slight modifications. Embryos were dehydrated and mounted in glycerol for whole mount observations on a Zeiss Axioscop FS microscope. Embryos and adult hearts to be sectioned were embedded in JB-4 plastic (Polysciences; Warrington, PA) before 5-µm sections were obtained with a microtome. To verify the localization of zfCx45.6 expression in the embryonic heart, ISH was performed with a cardiac myosin light chain 2 (cmlc2) probe. This marker is known to be expressed at high levels in both the atrial and ventricular myocardium in embryonic zebrafish (67). A 1-kb fragment from the 3' end of the cmlc2 cDNA (generously provided by Dr. D. Stainier) was labeled with DIG-11 UTP as above. Whole mount ISH was performed and sections were obtained as previously described.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning. A genomic clone coding for the zebrafish orthologue to mammalian Cx40 was isolated by taking advantage of the high degree of sequence similarity between the connexins. A 634-bp clone, obtained by hotstart PCR with primers directed against conserved regions of the connexins, was used to probe a zebrafish PAC library at high stringency. Two positive PAC clones from the screen were randomly chosen, and 12-kb HindIII restriction fragments containing the full coding region of the connexin were subcloned. A total of 1,560 bp of sequence was obtained from the HindIII subclones, including a predicted open reading frame of 1,200 bp within one exon. The coding region of the cloned connexin translated into 400 amino acids with an estimated molecular mass of 45.6 kDa. In accordance with the standard connexin naming system (8), the cloned connexin was named zebrafish connexin45.6 (zfCx45.6). Hydrophobicity analysis of the zfCx45.6 amino acid sequence predicted typical connexin membrane topography with four transmembrane regions and internal NH2 and COOH termini.

The zfCx45.6 sequence has been deposited in GenBank with accession number AF531762 [GenBank] . A Basic Local Alignment Search Tool (BLAST) search of the nonredundant division of GenBank, using the 1,200-bp coding region of the new connexin as the query sequence, showed that the cloned connexin had the highest degree of sequence similarity to the mammalian Cx40 orthologues. The high degree of similarity between zfCx45.6 and the Cx40 orthologues is highlighted by an alignment of their amino acid sequences (Fig. 1). In this alignment, zfCx45.6 is shown to possess the three cysteines present in the extracellular loops of all connexins in the characteristically conserved sequence of the following: CX6CX3C in the first extracellular loop, and CX4CX5C in the second extracellular loop. The conserved cysteines in the extracellular loops are involved in the formation of intramolecular disulfide bonds that are essential for normal folding and/or channel function (66). The highest sequence divergence between zfCx45.6 and the Cx40 orthologues occurs in the COOH tail. ZfCx45.6 also possesses extra sequence in this region, thus resulting in a higher predicted molecular weight than the mammalian Cx40 orthologues. ZfCx45.6 possesses the highest amino acid similarity to chick Cx42 with 53% identity and 63% similarity. This is followed by human Cx40 (52%/63%), dog Cx40 (51%/62%), rat Cx40 (50%/62%), and mouse Cx40 (49%/62%). This similarity is graphically depicted in Fig. 2, which represents the structural relationships between connexins expressed in the cardiovascular system. ZfCx45.6 clearly groups with the mammalian Cx40 orthologues.



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  		Fig. 1. Amino acid alignment of zfCx45.6 and the connexin (Cx) 40 orthologues. The high degree of amino acid similarity between zfCx45.6 and the Cx40 orthologues is demonstrated by an alignment of the amino acid sequences of this group, whereby asterisks denote identical amino acids and colons denote similar amino acids. Spaces represented by dashed lines have been added to optimize alignment. Shaded regions highlight the four predicted transmembrane regions, and the solid circles highlight the three conserved cysteines in the extracellular loops. The highest divergence in similarity between the members of the group occurs in the cytoplasmic tail, where zfCx45.6 possesses extra sequence.

 


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  		Fig. 2. Phylogenetic tree of Cx expressed in the cardiovascular system. Phylogenetic analysis was performed with CLUSTALX (1.4) (57), and resulting alignment was displayed with TreeView (1.5) (45). The phylogram graphically represents the structural relationships between connexins expressed in the cardiovascular system, whereby the linear distance between sequences is inversely proportional to the level of amino acid similarity. The distance indicated by the scale bar represents 0.1 amino acid substitutions per site. ZfCx45.6 clearly groups with the connexin40 orthologues.

 

Genomic mapping. The genomic location of zfCx45.6 was determined by screening the LN54 radiation hybrid panel by PCR with zfCx45.6-specific primers. The map position was calculated with the RH Mapper software program. The best overall marker linked to zfCx45.6 was found on linkage group (LG) 9 with a logarithm of the odds (LOD) score of 6.9. ZfCx45.6 was mapped zero centirays from fb36h02, a previously mapped EST marker on LG9. The sequences of zfCx45.6 and fb36h02 did not show any significant similarity. The best marker in the second-best LG linked zfCx45.6 to LG15 with a LOD score of 4.6. Further evidence confirming the assignment of zfCx45.6 to LG9 was obtained in the following way. The 1534 nucleotide sequence of zfCx45.6 was used as the query sequence in a BLAST search of finished and unfinished clone sequences produced by the Zebrafish Sequencing Group at the Sanger Institute in Cambridge (http://www.sanger.ac.uk/Projects/D_rerio/blast_server.shtml). This identified one unfinished clone (zK19H21) with very high nucleotide identity to zfCx45.6 (97–99% identity over much of the 1534 nucleotide zfCx45.6 sequence). We then searched for information on this clone from the Zebrafish Genome Fingerprinting Project web page (http://www.sanger.ac.uk/Projects/D_rerio/WebFPC/zebra/small.shtml). This indicated that zK19H21 had been mapped in contig no. 13950 and that the five markers that have been mapped to this contig (etID8281.9, etID17327.9, etID22872.9, etID20388.9, and etID41468.9) have also mapped to LG9.

Functional analysis. To determine whether zfCx45.6 could form functional channels, we used the paired Xenopus oocyte expression system (14). Oocytes had resting potentials ranging between –40 and –50 mV and were clamped at –40 mV for measurements of junctional conductance. As shown in Fig. 3A, zfCx45.6 induced the development of robust levels of electrical coupling 24–48 h after RNA injection. The mean conductance (Gj) of paired zfCx45.6-injected cells was 33.6 µS(n = 30). In contrast, the mean background conductance value, measured between water-injected oocytes, was more than 200-fold lower at 0.12 µS (n = 24). Thus the expression of zfCx45.6 resulted in the formation of functional gap junction channels.



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  		Fig. 3. Analysis of Cx45.6 channel properties. A: junctional conductance (Gj) developed between pairs of Xenopus oocytes as measured by dual voltage clamp. Oocytes were coinjected with the Cx45.6 cRNA and an oligonucleotide antisense to mRNA for Xenopus Cx38 to eliminate the possible contribution of endogenous coupling to the recorded conductances. Antisense-treated water-injected cells were used as negative controls. Cells were then stripped of the vitelline envelope in hypertonic medium and paired with the vegetal poles facing each other 24–48 h before electrophysiological measurements. Bars show means ± SD of the number of pairs indicated. B: voltage gating behavior of gap junction channels formed by Cx45.6. Time-dependent decay of junctional currents (Ij) induced by transjunctional voltage (Vj) steps of 3-s duration applied in 20-mV increments. At Vj steps greater than ±20 mV, Ij decayed symmetrically over the time course of the voltage step. The voltage gating of intercellular channels composed of zebrafish Cx45.6 displayed a high degree of conservation to orthologs such as chicken Cx42 and rat Cx40

 

To further characterize the physiological behavior of channels composed of zfCx45.6, we analyzed their voltage dependence. A representative family of junctional currents (Ij) evoked by voltage steps of opposite polarities and increasing amplitude (Fig. 3B) shows that Ij decreased in a time- and voltage-dependent manner for transjunctional voltages >20 mV. The rate of channel closure, calculated for Vj values of ±80 mV, yielded a time constant ({tau}) on the order of 0.1 s, a value closer to the more rapidly gating mouse channels such as Cx37 or Cx40 than the slower channels like Cx26 and Cx32 (4, 27). Thus the voltage dependence of zfCx45.6 channels was quite similar to that of rodent Cx40 (9, 27).

Northern and RT-PCR analyses. ZfCx45.6 was detected through Northern analysis as a 2.4-kb transcript in 1 to 5 dpf embryos (Fig. 4A). The Northern blot results were confirmed by RT-PCR (not shown). Because it appeared from this initial RT-PCR analysis that zfCx45.6 could be detected at some point before 24 hpf, RT-PCR was performed with cDNA samples generated from embryos at different developmental stages throughout the first day of development (0 dpf). For each stage, RT-PCR was performed at least twice using RNA isolated from two independently collected batches of embryos. ZfCx45.6 expression was consistently detected in embryos before 2 hpf and in 12.5–13 hpf and older embryos (Fig. 4B). Only very weak zfCx45.6 expression was detected between 3.5 and 12.5 hpf. PCR with zfeF1{alpha} primers was performed as a positive control and resulted in amplification from all the stages, although the signal was significantly weaker at 3.5–4 hpf, presumably reflecting the loss of maternal transcripts at the midblastula transition (34, 35).



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  		Fig. 4. Northern blot and RT-PCR analyses of zfCx45.6 expression. A: to determine the expression patterns of zfCx45.6, Northern blot analysis was performed with a blot composed of polyA+ mRNA isolated from various embryonic stages. ZfCx45.6 was detected as a 2.4-kb transcript in embryos 1 to 5 days postcoitum (dpf). The loading control (zfeF1{alpha}) indicated that polyA+ mRNA was present in all of the lanes tested but not necessarily in equal amounts. B: to more accurately determine when zfCx45.6 is expressed, RT-PCR was performed with cDNA isolated from various staged embryos throughout 0 dpf. The PCR products were blotted onto a membrane and probed with a zfCx45.6-specific DNA probe. Overall, zfCx45.6 was consistently detected before 2 h postfertilzation (hpf) and after 12.5 hpf. ZfCx45.6 expression was not strongly detected between 2.5 and 12 hpf. PCR with the zfeF1{alpha} primers produced a 569-bp cDNA-derived amplicon and no 733-bp genomic DNA-derived amplicons, indicating that cDNA was present in all samples tested and that no genomic DNA contamination was present. C: to confirm the adult spatial expression pattern of zfCx45.6, RT-PCR was performed with cDNA generated from various adult tissues. ZfCx45.6 expression was detected in the heart, liver, brain, and ovary. ZfCx45.6 expression was not detected in the lens. The loading control (zfeF1{alpha}) indicated cDNA was present in all samples tested, and there was no genomic contamination.

 

RT-PCR was also performed multiple times using cDNA generated from adult heart, lens, liver, brain, and ovary. ZfCx45.6 expression was always strongly detected in the heart and more weakly in the liver, brain, and ovary but not in the lens (Fig. 4C).

Whole mount in situ hybridization. No signal was detected with the zfCx45.6 sense probe in any of the ISH experiments (Fig. 5A). ZfCx45.6 expression was detected before 24 hpf, but no specific staining patterns were observed at these early stages. At 1 dpf specific patterns of zfCx45.6 expression were beginning to emerge. At this stage zfCx45.6 expression was primarily detected in the dorsal aorta (Fig. 5B), where expression continued to be detected until 5 dpf (Fig. 5C). Ventral views of the 1-dpf embryos also revealed expression in tail somites (Fig. 5D). The somite expression was not detected at later stages. Expression was also detected in the head, but no specific patterns were visible in this region until later stages of development.



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  		Fig. 5. Whole mount in situ hybridization analysis: embryonic expression of zfCx45.6. Whole mount in situ hybridization (ISH) detected zfCx45.6 in all areas of the embryo in regions corresponding to the location of major blood vessels. Staining was not visible in control embryos (A). By 1 dpf a distinct band of expression was located in the tail just below the notochord, corresponding to the location of the dorsal aorta (short arrow in B). This expression continued to be detected through to 5 dpf (short arrow in C). In a ventral view of the 1 dpf embryos only, zfCx45.6 was also detected in the somites (D). By 1 dpf expression was detected in the heads of embryos, but specific patterns of expression were not detected until 2 dpf. Expression of zfCx45.6 was detected in a structure surrounding the ventromedial one-third of the embryonic eye just outside the retina (long arrow in F-H). ZfCx45.6 was also detected as localized areas of expression at the posterior/ventral corner of the eye in embryos 2 dpf and older (small arrowhead in G). The most distinct expression patterns of zfCx45.6 were visible in the branchial arch region. Expression was detected by 2 dpf (large arrowhead in E) and continued to be detected to 5 dpf (large arrowheads in F, H–J). ZfCx45.6 expression was also apparent in the limb bud of embryos 1 dpf and older (small arrow in H and I). In all panels anterior is to the left. Scale bar represents 50 µm in D, 100 µm in E–H, and J, 200 µm in C and I, and 400 µm in A and B. A. 1 dpf (~30 hpf), whole control embryo; B: 1 dpf (~24 hpf), whole embryo; C: 5 dpf, lateral view tail; D: 1 dpf, ventral view tail; E: 2 dpf, head ventral view; F and G: 3 dpf, head ventral view; H: 4 dpf, head, oblique view; I: 5 dpf, head ventral view; J: 5 dpf, head lateral view.

 

By 2 dpf and through to 5 dpf, distinct patterns of expression emerged in areas that correlate with the location of major embryonic blood vessels. More specifically, zfCx45.6 expression was visible around the eyes, in the branchial arch region, in the limb buds, in the tail, and over the yolk sac. The identity of these zfCx45.6-positive vessels was provisionally determined with the aid of confocal microangiograph figures of developing zebrafish vasculature (31) and "The 3-D Interactive Atlas of Zebrafish Vascular Anatomy" web page (http://dir.nichd.nih.gov/lmg/uvo/atlas.html).

Expression of zfCx45.6 around the eyes was mainly visible in two areas. In embryos 2 dpf and older, a vessel was visible in proximity to the medioventral portion of each eye, just outside the retina (Fig. 5, E–G and I). This vessel was best observed from a ventral view but was also visible in the lateral view as a small line just below the eye (Fig. 5H). Localized, bilateral "dot-like" zfCx45.6 expression was also visible at the posterior/ventral corner of the eyes of 2 dpf and older embryos. At 2 dpf, only one "dot" was visible, but by 3 and 4 dpf, three "dots" were visible (Fig. 5G). This expression pattern was evident in both the ventral and lateral view. The dynamic nature of the cephalic circulation during these early stages of vascular development (31) makes it difficult to identify with certainty these zfCx45.6-positive vessels. They likely correspond to connections between vessels and expansions of vessels, previously identified in angiograms of the area around the eye (31).

Some of the most distinctive expression patterns of zfCx45.6 occurred in the branchial arch region. Expression was visible by 2 dpf as bands through this area (Fig. 5E). As the embryo grew and the branchial area enlarged, the expression pattern of zfCx45.6 changed, corresponding to branchial arch remodeling and development. By 3 dpf, zfCx45.6 expression was clearly visible in the branchial arches (Fig. 5F). The zfCx45.6 expression pattern in this region became more intricate by 4 dpf, when expression could be seen in the hypobranchial artery, the opercular artery, and the third, fourth, fifth, and/or sixth branchial arches (Fig. 5H). The expression of zfCx45.6 continued to be visible in this area through to 5 dpf from both a ventral (Fig. 5I) and lateral view (Fig. 5J).

ZfCx45.6 expression was also distinct in other areas of the embryo such as the limb buds (from 1 to 5 dpf) (Fig. 5, H and I) and the common cardinal vein over the yolk sac in the 1-dpf embryos (figure not shown because the location of the yolk sac interfered with image capture).

In the majority of cases, the embryonic zebrafish heart appeared to express low amounts of zfCx45.6, but visualization was difficult due to the location of the heart with respect to the yolk. Semithin plastic sections were obtained from 3-dpf embryos, but zfCx45.6 staining in the heart (Fig. 6, A and C) was not as distinct as seen in sections from other areas expressing zfCx45.6 such as the limb buds or branchial arch vessels (Fig. 6, A and B). This is in contrast to the very dense staining obtained with the cmlc2 probe (Fig. 6D), a marker of myocardial differentiation known to be expressed at high levels in the embryo (67).



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  		Fig. 6. Expression of ZfCx45.6 in the embryonic branchial arch region, and embryonic and adult heart. To confirm and localize expression in the embryonic and adult zebrafish heart, semithin sections were obtained from 3 dpf embryos and adult hearts probed by whole mount ISH. ZfCx45.6 staining in the embryonic heart (large arrowhead in A and C) was weaker than in areas such as the branchial arches (arrow in A and B) and also much weaker than cmlc2 staining in the embryonic heart (large arrowhead in D). In the adult heart, whole mount ISH detected zfCx45.6 in the myocytes of both the ventricle (arrowheads in E–G), and atrium (arrowhead in H). ZfCx45.6 was more strongly expressed in the compact layer of the ventricle (between large arrows in E). Staining was not visible in hearts hybridized with the sense control probe (I). The scale bar represents 20 µm in I; 50 µm in C, E, F, H; and 100 µm in A, B, D. A: 3 dpf, sagittal section through branchial arches and heart; B and C: 4 dpf, sagittal section through branchial arches and heart; D: 3 dpf, cmlc2 expression, sagittal section through branchial arches and heart; E, F and G: adult ventricle; H: adult atrium; I: control, adult ventricle.

 

To localize the expression of zfCx45.6 in the adult heart, whole mount ISH was performed on adult hearts in a similar manner as above. Because the adult heart tissue is thicker and more compact than that of embryos, hearts were subjected to a more intensive proteinase K treatment than used for the embryos to ensure penetration of the probe. In some experiments the whole heart was cut sagitally after fixation but before whole mount ISH. Moderate expression of zfCx45.6 was detected in the myocytes of both the atria (Fig. 6H) and the compact (Fig. 6 E) and trabeculated layers (Fig. 6, F and G) of the ventricle of the adult heart. ZfCx45.6 was more strongly expressed in the ventricular compact layer than the trabeculated layer, presumably due to the additional expression detected in the endothelial cells lining the coronary vessels of the compact layer. Within the compact layer, stronger staining was visible in the ventral portion of the ventricle, near the bulbous arteriosus, although zfCx45.6 staining was not detected in the bulbous arteriosus itself. Staining was not visible in hearts hybridized with the sense control probe (Fig. 6I).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cloning and genomic mapping of ZfCx45.6. To learn more about the role connexins play in cardiovascular development and function, one of the four main connexins expressed in the mammalian and avian cardiovascular systems was cloned from zebrafish. Genomic clones containing the full coding region of the new connexin zfCx45.6 were isolated from a PAC library. Similarities in sequence, RNA expression patterns, and voltage dependence indicate a new member of the Cx40 orthologous group has been cloned.

Using the LN54 radiation hybrid panel, we mapped zfCx45.6 to LG9 with a LOD score of 6.9, whereas the best marker on the second-best linkage group was found on LG15 (LOD 4.4). For a marker to be conclusively placed on the LN54 radiation hybrid map, it must have a first LOD score >5, and the second LOD score must be at least 3 LOD units away from the first (30). From these criteria, our radiation hybrid mapping data did not allow us to conclusively assign the zfCx45.6 gene to LG9. However, the new clone-based sequencing and mapping data from the zebrafish genome sequencing project strongly support the assignment of zfCx45.6 to LG9. The observation that ZfCx45.6 and fb36h02 had no significant sequence similarity despite mapping to the same position is not unexpected because this panel 1 centiray equals 118 kb (30), and the sequence obtained from zfCx45.6 was only 1.5 kb.

Functional analysis. Consistent with its high sequence identity to the mammalian Cx40 orthologues, the functional behavior of zfCx45.6 in paired Xenopus oocytes was also similar to this subgroup of connexins. Like the rat or mouse Cx40 orthologues (9, 27), zfCx45.6 induced robust levels of intercellular coupling between paired cells that was highly sensitive to transjunctional voltage. A similar functional conservation of gating was seen in members of the Cx45 orthologous subgroup from four species (zebrafish, chicken, mouse, and human), although some species-specific gating differences were also observed (3). Thus zfCx45.6 is a functional orthologue of chick Cx42 and rat Cx40, in addition to sharing high sequence identity.

Expression analysis of ZfCx45.6. In the zebrafish, zygotic transcription does not begin until the midblastula transition, which is initiated at about the 1,000 cell stage or 3 hpf (34, 35). The fact that zfCx45.6 is detected before 2.5 hpf indicates this message is maternally supplied to the embryo. It is well known that in many species maternally inherited molecules stored within the oocyte during oogenesis regulate the earliest stages of embryogenesis and that zygotic expression only becomes important once these maternal molecules have decayed at the so called maternal-zygotic transition (23, 55). Genes may be expressed only maternally, maternally and zygotically, or only zygotically (19). From the present study it appears that zfCx45.6 is expressed both maternally and zygotically and could thus be crucial for the earliest stages of embryogenesis, as well as later in development. To our knowledge this is the first evidence of a Cx40 orthologue being maternally supplied.

A maternal supply of connexin transcripts has been documented before, but the functional significance of these transcripts is not clear. For example, in Xenopus, maternal transcripts for four connexins (Cx38, Cx31, Cx43.4, and Cx43) have been detected, and three of these (Cx31, Cx43.4, and Cx43) are transcribed zygotically as well (20, 24, 40). Because zfCx45.6 is detected in the adult ovary, and only for a short time in the early embryo, it is possible that the zfCx45.6 mRNA detected before 2.5 hpf is residual mRNA required during oocyte maturation but without function in the embryo. The zfCx45.6 signal detected in the adult ovary by RT-PCR may be partially attributable to oocyte expression but is probably mostly derived from blood vessel expression. In rodents, ovarian Cx40 expression appears to be entirely endothelial in origin (44, 65). Any possible function of zfCx45.6 before 2.5 hpf could be studied through the use of antisense technologies.

ZfCx45.6 expression was detected by whole mount ISH and RT-PCR in the cardiovascular system of the zebrafish starting at 1 dpf. Whole mount ISH did not detect specific expression patterns in the embryo before 1 dpf; however, RT-PCR, a more sensitive method, detected zygotic zfCx45.6 transcripts by 12.5 hpf in the whole embryo. This is similar to the situation in the mouse, where Cx40 (mCx40) was not detected by whole mount ISH in the heart or cardiovascular system until 9.5 days postcoitum (dpc) even though RT-PCR indicated low levels of expression by 8.5 dpc (16). In the zebrafish, cardiac progenitors begin to arrive at the embryonic axis by 13 hpf (8 somite stage), and by about 16.5 hpf two tubular heart primordia have formed in the lateral plate mesoderm (54). Vascularization begins by 20–22 hpf (24 somite stage), and by 24 hpf (28 somite stage) a simple circulatory loop has been set up (22, 31, 61). The detection of the zfCx45.6 transcript in the whole embryo before these times and the subsequent localization of the transcript in the embryonic and adult cardiovascular system suggest that zfCx45.6 plays a role in zebrafish cardiovascular function and development.

The detection of zfCx45.6 in the heart and major vessels is similar to the embryonic expression patterns of Cx40 in the mouse. In the vasculature of the 9.5-dpc mouse, Cx40 is expressed at high levels in the endothelial cells in a number of the same vessels in which we detected zfCx45.6 expression (16). During the following days of development the Cx40 vascular expression pattern changes corresponding to the arrangement of the vascular tree. In the 9.5-dpc mouse heart, low levels of Cx40 expression is initially detected in myocytes of the undivided common atrium and ventricle. It is interesting to note that the 9.5-dpc mouse heart shares many structural features with the adult zebrafish heart. Levels of expression in the mouse heart increase by 10.5 dpc and remain high in both atria after septation and into adulthood. Later in development, Cx40 expression continues to be detected in the atria, but expression disappears from the ventricular myocytes and becomes restricted to the ventricular conduction system. Cx40 is also expressed in the endothelial cells of the ventricular coronary vessels but not the endocardium (13, 16). We did not observe a regionalized pattern of zfCx45.6 expression in the adult zebrafish heart, which could correspond to a conduction system. This is not surprising because studies have shown that although activation patterns in the adult hearts of zebrafish and higher vertebrates are similar, there is an absence of anatomically distinct conduction system in the zebrafish, with the trabeculae instead serving as the functional equivalent of the His-Purkinje system of higher vertebrates (48).

The observation that zfCx45.6 expression in the embryonic heart appears to be at a much lower level than in the adult heart suggests that cardiac zfCx45.6 expression may be developmentally regulated. ZfCx45.6 may play more of a physiological role in heart function and therefore not be highly expressed until later stages of development when detection via whole mount ISH is less efficient. Real-time quantitative RT-PCR of a developmental series of embryonic heart RNA could be used to confirm this.

ZfCx45.6 may also be regulated at the protein level. Even though mRNA levels of zfCx45.6 are not easily detected in the embryonic heart, high levels of protein could still be produced. Analysis of the distribution of mCx40 suggests this posttranscriptional regulation, because the amount of mRNA detected in the lungs is greater than that detected in the heart, but the reverse is true for the levels of protein (58). Western analysis and/or immunohistochemistry would help to determine whether zfCx45.6 expression is translationally regulated.

It is also possible that in the zebrafish a connexin other than zfCx45.6 is expressed at high levels in the heart and, at least partially, taking over the role played by Cx40 in the mammalian heart. In some cases there appears to be significant interspecies variability in the expression pattern of specific connexin orthologues. For example, in the adult chick, Cx42 is the major connexin expressed in the myocardium of the atria and ventricles, and little, if any, Cx43 is expressed in the myocytes (6, 43). Interestingly enough, studies with antibodies raised against mammalian connexin peptide sequences suggest that Cx32 is the major connexin expressed in the goldfish heart and that Cx40 and Cx43 are not expressed at all (6).

ZfCx45.6 was not detected in the bulbous arteriosus of the adult zebrafish heart. The bulbous arteriosus is one of the four segments in the zebrafish heart located between the ventricle and ventral aorta (29). In the zebrafish, the bulbous arteriosus is noncontractile. It stretches with ventricular systole but regains its original shape during ventricular diastole. The bulbous arteriosus therefore acts as a capacitator to ensure a constant and regulated flow of blood into the gills. This noncontractile nature is due to the fact that whereas the cells of the bulbous arteriosus start out with myocardial characteristics, they transition to smooth muscle by 4 wk (28). This may explain why zfCx45.6 was not detected in the bulbous arteriosus, because zfCx45.6 was detected in the myocardial and endothelial cells of the ventricle and atrium.

The zfCx45.6 detected by RT-PCR in the adult brain, eye minus the lens, liver, and ovary most likely reflects zfCx45.6 expression in the vasculature of these organs, although this would best be confirmed through immunohistochemistry. ZfCx45.6 was not detected in the embryonic or adult zebrafish lens. The fact that zfCx45.6 was not detected in the lens is consistent with other studies (7). It is also consistent with the fact that even though Cx40 expression in different organisms is not always the same, its expression does appear to be restricted to the cardiovascular system. Because the lens is an avascular tissue, one would not expect to find zfCx45.6 expressed there.

Now that three of the four main connexins expressed in the mammalian cardiovascular system have been cloned in the zebrafish, studies can be performed to determine the relative levels of connexin expression in different parts of the system. These studies could possibly help to decipher the roles of connexins in zebrafish cardiovascular development and function and provide insights into how to prevent certain forms of heart disease.


   ACKNOWLEDGMENTS
 
Present address for T. L. Christie: Dept. of Biochemistry and Molecular Biology, University of Calgary, Calgary AB, T2N 4N1, Canada.

GRANTS

This research was supported by research grants from the Natural Sciences and Engineering Research Council of Canada and the Manitoba Health Research Council to G. Valdimarsson and National Institutes of Health grants to T. W. White.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Valdimarsson, Dept. of Zoology, Univ. of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (E-mail address: valdimar{at}cc.umanitoba.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Amemiya CT, Zhong TP, Silverman GA, Fishman MC, and Zon LI. Zebrafish YAC, BAC, and PAC genomic libraries. Methods Cell Biol 60: 235–258, 1999.[ISI][Medline]
  2. Amemiya CT and Zon LI. Generation of a zebrafish P1 artificial chromosome library. Genomics 58: 211–213, 1999.[CrossRef][ISI][Medline]
  3. Barrio LC, Capel J, Jarillo JA, Castro C, and Revilla A. Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes. Biophys J 73: 757–769, 1997.[Abstract/Free Full Text]
  4. Barrio LC, Suchyna T, Bargiello T, Xu LX, Roginski RS, Bennett MVL, and Nicholson BJ. Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci USA 88: 8410–8414, 1991.[Abstract/Free Full Text]
  5. Becker DL, Bonness V, Catsicas M, and Mobbs P. Changing patterns of ganglion cell coupling and connexin expression during chick retinal development. J Neurobiol 52: 280–293, 2002.[CrossRef][ISI][Medline]
  6. Becker DL, Cook JE, Davies CS, Evans WH, and Gourdie RG. Expression of major gap junction connexin types in the working myocardium of eight chordates. Cell Biol Int 22: 527–543, 1998.[CrossRef][ISI][Medline]
  7. Beyer EC. Molecular cloning and developmental expression of two chick embryo gap junction proteins. J Biol Chem 265: 14439–14443, 1990.[Abstract/Free Full Text]
  8. Beyer EC, Paul DL, and Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105: 2621–2629, 1987.[Abstract/Free Full Text]
  9. Bruzzone R, Haefliger JA, Gimlich RL, and Paul DL. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell 4: 7–20, 1993.[Abstract]
  10. Bruzzone R, White TW, and Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 238: 1–27, 1996.[ISI][Medline]
  11. Cason N, White TW, Cheng S, Goodenough DA, and Valdimarsson G. Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein. Dev Dyn 221: 238–247, 2001.[CrossRef][ISI][Medline]
  12. Christ GJ, Spray DC, el Sabban M, Moore LK, and Brink PR. Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res 79: 631–646, 1996.[Abstract/Free Full Text]
  13. Coppen SR, Kaba RA, Halliday D, Dupont E, Skepper JN, Elneil S, and Severs NJ. Comparison of connexin expression patterns in the developing mouse heart and human foetal heart. Mol Cell Biochem 242: 121–127, 2003.[CrossRef][ISI][Medline]
  14. Dahl G, Miller T, Paul D, Voellmy R, and Werner R. Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA. Science 236: 1290–1293, 1987.[Abstract/Free Full Text]
  15. Davis LM, Kanter HL, Beyer EC, and Saffitz JE. Distinct gap junction protein phenotypes in cardiac tissues with disparate conduction properties. J Am Coll Cardiol 24: 1124–1132, 1994.[Abstract]
  16. Delorme B, Dahl E, Jarry-Guichard T, Briand JP, Willecke K, Gros D, and Théveniau-Ruissy M. Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system. Circ Res 81: 423–437, 1997.[Abstract/Free Full Text]
  17. Dermietzel R, Kremer M, Paputsoglu G, Stang A, Skerrett IM, Gomes D, Srinivas M, Janssen-Bienhold U, Weiler R, Nicholson BJ, Bruzzone R, and Spray DC. Molecular and functional diversity of neural connexins in the retina. J Neurosci 20: 8331–8343, 2000.[Abstract/Free Full Text]
  18. Driever W and Fishman MC. The zebrafish: heritable disorders in transparent embryos. J Clin Invest 97: 1788–1794, 1996.[ISI][Medline]
  19. Dworkin MB and Dworkin-Rastl E. Functions of maternal mRNA in early development. Mol Reprod Dev 26: 261–297, 1990.[CrossRef][ISI][Medline]
  20. Ebihara L, Beyer EC, Swenson KI, Paul DL, and Goodenough DA. Cloning and expression of a Xenopus embryonic gap junction protein. Science 243: 1194–1195, 1989.[Abstract/Free Full Text]
  21. Essner JJ, Laing JG, Beyer EC, Johnson RG, and Hackett PBJ. Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos. Dev Biol 177: 449–462, 1996.[CrossRef][ISI][Medline]
  22. Fouquet B, Weinstein BM, Serluca FC, and Fishman MC. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol 183: 37–48, 1997.[CrossRef][ISI][Medline]
  23. Gandolfi TA and Gandolfi F. The maternal legacy to the embryo: cytoplasmic components and their effects on early development. Theriogenology 55: 1255–1276, 2001.[CrossRef][ISI][Medline]
  24. Gimlich RL, Kumar NM, and Gilula NB. Differential regulation of the levels of three gap junctional mRNAs in Xenopus embryos. J Cell Biol 110: 597–605, 1990.[Abstract/Free Full Text]
  25. Gros DB and Jongsma HJ. Connexins in mammalian heart function. Bioessays 18: 719–730, 1996.[CrossRef][ISI][Medline]
  26. Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien KR, Stuhlmann H, and Fishman GI. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88: 333–339, 2001.[Abstract/Free Full Text]
  27. Hennemann H, Suchyna T, Lichtenberg-Fraté H, Jungbluth S, Dahl E, Schwarz J, Nicholson BJ, and Willecke K. Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung. J Cell Biol 117: 1299–1310, 1992.[Abstract/Free Full Text]
  28. Hu N, Sedmera D, Yost HJ, and Clark EB. Structure and function of the developing zebrafish heart. Anat Rec 260: 148–157, 2000.[CrossRef][Medline]
  29. Hu N, Yost HJ, and Clark EB. Cardiac morphology and blood pressure in the adult zebrafish. Anat Rec 264: 1–12, 2001.[CrossRef][Medline]
  30. Hukriede N, Fisher D, Epstein J, Joly L, Tellis P, Zhou Y, Barbazuk B, Cox K, Fenton-Noriega L, Hersey C, Miles J, Sheng X, Song A, Waterman R, Johnson SL, Dawid IB, Chevrette M, Zon LI, McPherson J, and Ekker M. The LN54 radiation hybrid map of zebrafish expressed sequences. Genome Res 11: 2127–2132, 2001.[Abstract/Free Full Text]
  31. Isogai S, Horiguchi M, and Weinstein BM. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230: 278–301, 2001.[CrossRef][ISI][Medline]
  32. Jongsma HJ. Diversity of gap junctional proteins: does it play a role in cardiac excitation? J Cardiovasc Electrophysiol 11: 228–230, 2000.[ISI][Medline]
  33. Jongsma HJ and Wilders R. Gap junctions in cardiovascular disease. Circ Res 86: 1193–1197, 2000.[Abstract/Free Full Text]
  34. Kane DA. Cell cycles and development in the embryonic zebrafish. Methods Cell Biol 59: 11–26, 1999.[ISI][Medline]
  35. Kane DA and Kimmel CB. The zebrafish midblastula transition. Development 119: 447–456, 1993.[Abstract]
  36. Kirchhoff S, Kim JS, Hagendorff A, Thonnissen E, Kruger O, Lamers WH, and Willecke K. Abnormal cardiac conduction and morphogenesis in connexin40 and connexin43 double-deficient mice. Circ Res 87: 399–405, 2000.[Abstract/Free Full Text]
  37. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, and Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr Biol 8: 299–302, 1998.[CrossRef][ISI][Medline]
  38. Kruger O, Plum A, Kim JS, Winterhager E, Maxeiner S, Hallas G, Kirchhoff S, Traub O, Lamers WH, and Willecke K. Defective vascular development in connexin 45-deficient mice. Development 127: 4179–4193, 2000.[Abstract]
  39. Kumai M, Nishii K, Nakamura K, Takeda N, Suzuki M, and Shibata Y. Loss of connexin45 causes a cushion defect in early cardiogenesis. Development 127: 3501–3512, 2000.[Abstract]
  40. Landesman Y, Postma FR, Goodenough DA, and Paul DL. Multiple connexins contribute to intercellular communication in the Xenopus embryo. J Cell Sci 116: 29–38, 2003.[Abstract/Free Full Text]
  41. Liao Y, Day KH, Damon DN, and Duling BR. Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice. Proc Natl Acad Sci USA 98: 9989–9994, 2001.[Abstract/Free Full Text]
  42. Lohr JL and Yost HJ. Vertebrate model systems in the study of early heart development: Xenopus and zebrafish. Am J Med Genet 97: 248–257, 2000.[CrossRef][ISI][Medline]
  43. Minkoff R, Rundus VR, Parker SB, Beyer EC, and Hertzberg EL. Connexin expression in the developing avian cardiovascular system. Circ Res 73: 71–78, 1993.[Abstract]
  44. Okuma A, Kuraoka A, Iida H, Inai T, Wasano K, and Shibata Y. Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary. J Reprod Fertil 107: 255–264, 1996.[Abstract]
  45. Page RD. TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357–358, 1996.[Free Full Text]
  46. Reaume AG, de Sousa PA, Kulkarni S, Langille BL, Zhu D, Davies TC, Juneja SC, Kidder GM, and Rossant J. Cardiac malformation in neonatal mice lacking connexin43. Science 267: 1831–1834, 1995.[Abstract/Free Full Text]
  47. Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 1989.
  48. Sedmera D, Reckova M, DeAlmeida A, Sedmerova M, Biermann M, Volejnik J, Sarre A, Raddatz E, McCarthy RA, Gourdie RG, and Thompson RP. Functional and morphological evidence for a ventricular conduction system in the zebrafish and Xenopus heart. Am J Physiol Heart Circ Physiol 284: H1152–H1165, 2003.[Abstract/Free Full Text]
  49. Severs NJ, Rothery S, Dupont E, Coppen SR, Yeh HI, Ko YS, Matsushita T, Kaba R, and Halliday D. Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system. Microsc Res Tech 52: 301–322, 2001.[CrossRef][ISI][Medline]
  50. Simon AM, Goodenough DA, and Paul DL. Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block. Curr Biol 8: 295–298, 1998.[CrossRef][ISI][Medline]
  51. Simon AM and McWhorter AR. Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40. Dev Biol 251: 206–220, 2002.[CrossRef][ISI][Medline]
  52. Simon AM and McWhorter AR. Decreased intercellular dye-transfer and downregulation of non-ablated connexins in aortic endothelium deficient in connexin37 or connexin40. J Cell Sci 116: 2223–2236, 2003.[Abstract/Free Full Text]
  53. Spray DC, Harris AL, and Bennett MVL. Gap junctional conductance is a simple and sensitive function of intracellular pH. Science 211: 712–715, 1981.[Abstract/Free Full Text]
  54. Stainier DY, Lee RK, and Fishman MC. Cardiovascular development in the zebrafish. I Myocardial fate map and heart tube formation. Development 119: 31–40, 1993.[Abstract]
  55. Telford NA, Watson AJ, and Schultz GA. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 26: 90–100, 1990.[CrossRef][ISI][Medline]
  56. Thisse C, Thisse B, Schilling TF, and Postlethwait JH. Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119: 1203–1215, 1993.[Abstract]
  57. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882, 1997.[Abstract/Free Full Text]
  58. Traub O, Eckert R, Lichtenberg-Fraté H, Elfgang C, Bastide B, Scheidtmann KH, Hulser DF, and Willecke K. Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells. Eur J Cell Biol 64: 101–112, 1994.[ISI][Medline]
  59. Turner DL and Weintraub H. Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8: 1434–1447, 1994.[Abstract/Free Full Text]
  60. Warren KS and Fishman MC. Physiological genomics: mutant screens in zebrafish. Am J Physiol Heart Circ Physiol 275: H1–H7, 1998.[Abstract/Free Full Text]
  61. Weinstein BM, Stemple DL, Driever W, and Fishman MC. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nat Med 1: 1143–1147, 1995.[CrossRef][ISI][Medline]
  62. Westerfield M. The Zebrafish Book. Eugene, OR: University of Oregon Press, 1995.
  63. White TW, Bruzzone R, and Paul DL. The connexin family of intercellular channel forming proteins. Kidney Int 48: 1148–1157, 1995.[ISI][Medline]
  64. White TW and Paul DL. Genetic disease and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61: 283–310, 1999.[CrossRef][ISI][Medline]
  65. Wright CS, Becker DL, Lin JS, Warner AE, and Hardy K. Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation. Reproduction 121: 77–88, 2001.[Abstract]
  66. Yeager M, Unger VM, and Falk MM. Synthesis, assembly and structure of gap junction intercellular channels. Curr Opin Struct Biol 8: 517–524, 1998.[CrossRef][ISI][Medline]
  67. Yelon D, Horne SA, and Stainier DY. Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev Biol 214: 23–37, 1999.[CrossRef][ISI][Medline]




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