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Originally published In Press as doi:10.1074/jbc.M401355200 on June 22, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36993-37003, August 27, 2004
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Connexin 48.5 Is Required for Normal Cardiovascular Function and Lens Development in Zebrafish Embryos*

Shaohong Cheng{ddagger}, Teresa Shakespeare§, Rickie Mui§, Thomas W. White§, and Gunnar Valdimarsson{ddagger}||

From the {ddagger}Department of Zoology and Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and §Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794

Received for publication, February 6, 2004 , and in revised form, June 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gap junctions are clusters of intercellular channels that allow the propagation of small metabolites, signaling molecules, and electrical impulses between adjacent cells. The protein units that make up the gap junction channels in vertebrates are called connexins and are coded for by a multigene family with 20 or more members in mammals (1). Gap junctions play vital roles in the functions of virtually all organs, including the lens and the heart. The evidence to date suggests that distinct sets of connexins may play a role in the development and function of organs like the lens and the heart. Three connexins are expressed in the mammalian lens. Connexin 43 is expressed in the undifferentiated lens epithelium, whereas Cx461 and Cx50 are typically considered lens-specific and are predominantly expressed in the lens fiber cells (for review, see Ref. 2). Both Cx46 and Cx50 knock-out mice developed cataracts (3-5), and the Cx50 knock-out mice also developed small lenses and small eyes.

Four connexins are abundantly expressed in the mammalian heart: Cx43, Cx40, Cx45, and Cx37. Connexin 43 is expressed in the atrial and ventricular working myocardium, Cx40 is found in the atrial working myocardium and the conducting bundle branches and Purkinje fibers, whereas Cx45 is restricted to the atrioventricular conduction system, and Cx37 is restricted to endothelial and endocardial cells (for reviews, see Refs. 6-8). Mouse knock-out experiments have demonstrated that Cx43, Cx40, and Cx45 are all essential for cardiac development and function. For instance, connexin 43 knock-out mice died neonatally from pulmonary outflow tract obstruction (9). Connexin 40-deficient mice on the other hand exhibited slowed cardiac conduction and a partial atrioventricular block (10, 11). Last, Cx45 knock-out mice died by embryonic day 10 displaying the effects of a conduction block, endocardial cushion defects, and abnormalities of vascular development (12, 13). The Cx37 knock-out mice had no apparent cardiovascular abnormalities (14). Although the expression and function of these four heart connexins have been extensively studied, further evidence suggests other connexins may play a role in heart development and function. For example, despite the fact that Cx45 is the earliest connexin known to be expressed in the heart, cardiac function is initiated normally in Cx45 knock-out mice (13). Also, in addition to Cx43, Cx40, Cx45, and Cx37, expression of two other connexins has been reported in the heart. Cx50 was detected in the atrioventricular valves of the rat heart by immunohistochemistry (15), whereas Cx46 mRNA was detected in the rat heart by northern blotting (16), and Cx46 protein was detected in the rabbit sinoatrial node by immunohistochemistry (17). A low amount of Cx46 protein was also found between occasional atrial and ventricular myocytes in the human heart (18). However, no heart defects have been reported in the Cx50 and Cx46 knock-out mice (3-5), and the function of these two connexins in the heart has remained a mystery.

We are using the zebrafish model system to investigate the role of the connexins in vertebrate embryonic development. The zebrafish embryo has unique properties that make it particularly attractive for the study of the cardiovascular and visual systems. The zebrafish cardiovascular system becomes functional by 24 h post-fertilization, but it is not required for the embryo to develop and survive for the first few days (19-21), which allows abnormalities of cardiac morphology and function to be investigated in live embryos. The zebrafish has a prototypical vertebrate heart composed of four continuous structures (sinus venosus, atrium, ventricle, and bulbus arteriosus) and shares many developmental and physiological similarities to the mammalian heart (22-24). The zebrafish also has unique advantages for visual system study, as the eye is relatively large and develops quickly. The zebrafish optic primordium first appears by 12 h post-fertilization, and functional vision has developed by 3 days post-fertilization (dpf) (25, 26).

In a recent study (27) we showed by RT-PCR that Cx48.5, a zebrafish connexin with extensive sequence similarity to mammalian Cx46, is expressed in the adult lens and presented a detailed whole mount in situ hybridization analysis of Cx43, Cx44.1, and Cx48.5 expression in the embryonic lens. In the present study we describe the isolation and further characterization of Cx48.5. We show that in addition to its expression in the embryonic and adult lens it is expressed in the heart (both adult and embryonic), adult testis, and embryonic otic vesicles. In Xenopus oocytes, Cx48.5 forms both gap-junctional intercellular channels and hemichannels with functional properties similar to Cx46 and its orthologues. Gene expression knockdown with Cx48.5 antisense morpholinos resulted in cataract formation as well as the development of both microphakia and microphthalmia and severe cardiovascular abnormalities.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Zebrafish Care—Adult zebrafish maintenance, breeding, and embryo collection were according to The Zebrafish Book (28). The embryos were raised in egg water at 28.5 °C and staged using time elapsed since fertilization. Embryos used for in situ hybridization were treated with 0.003% 1-phenyl-2-thiourea (Sigma, Mississauga, ON, Canada) in egg water beginning at 1 dpf to prevent pigment formation. The protocol for zebrafish use was approved by the University of Manitoba Institutional Animal Care Committee.

Screening the Zebrafish PAC Library—A rat Cx46 cDNA probe (16) was used to screen a zebrafish PAC library (RZDP Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin) at low stringency according to instructions supplied with the library. Two positive PAC clones were digested with PstI and EcoRI and subcloned into the pBluescript II SK +/- vector (Stratagene, La Jolla, CA). Subclones were re-screened with the rat Cx46 probe under the same conditions, and one positive subclone from each of the two original PAC clones was sequenced twice.

RT-PCR Analysis—Total RNA was isolated from adult tissues and whole embryos using TRIzol reagent (Invitrogen, Burlington, ON, Canada). Total RNA (2 µg of each sample) was reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen), and a portion of the reverse transcription reaction was used for PCR with Taq polymerase (Invitrogen). For RT-PCR of 4-dpf embryonic hearts and lenses, tissues were dissected with extra fine needles and transferred to 200 µl PCR tubes with micropipettes. The tissues were washed once with phosphate-buffered saline and then immediately processed for RT-PCR with the Cell-to-cDNA II kit (Ambion, Austin, TX). Two embryonic lenses or hearts were used for each sample, and the tissues were lysed in 6 µl of Cell Lysis II buffer. The lysate was split into two parts; 4 µl were used for the reverse transcription, and the remaining 2 µl were used as a negative control (no reverse transcriptase added). The total volume of each reverse transcription reaction was 8 µl; 4 µl of the reaction was used for Cx48.5 PCR, and the other 4 µl was used for PCR with elongation factor 1{alpha} (eF1{alpha}) control primers. The primers for Cx48.5 were 5'-GCAGACTGTACTTTCTCTCTAG-3' (forward) and 5'-TCTTTCTCCTCCTGGAGC-3' (reverse). The primers for eF1{alpha} were 5'-CAAGGGCTCCTTCAAGTACGCCTG-3' (forward) and 5'-GGCAGAATGGCATCAAGGGCA-3' (reverse). The eF1{alpha} primers anneal to exon sequences flanking an intron in the zfeF1{alpha} gene. The expected size of the elongation factor amplicon is 569 bp from cDNA and 733 bp from genomic DNA.

Whole Mount in Situ Hybridization (WMISH) and Whole Mount Immunohistochemistry—WMISH was carried out as previously described (27). The Cx48.5 probe was 775 bp long extending from 612 nucleotides downstream of the start codon to 81 nucleotides downstream of the stop codon, and the cardiac myosin light chain 2 (cmlc2) probe (a generous gift from Dr. Didier Stainier) was about a 1-kb-long fragment from the 3' end of the cmlc2 cDNA (29). The alkaline phosphatase color reaction was allowed to proceed for 10 and 2 h for the Cx48.5 and the cmlc2 probes, respectively. WMISH embryos were imaged on a Leica MZ8 stereomicroscope. Whole mount immunohistochemistry with the myosin heavy chain (MF20) monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA) was carried out according to the whole mount staining protocol in The Zebrafish Book (28). The secondary antibody used was a goat anti-mouse IgG labeled with Alexa Fluor 546 (Molecular Probes, Eugene, OR). The fluorescence images were collected on the Zeiss Axioskop FS microscope equipped with a rhodamine filter set. All images were captured with a SONY DXC-950 CCD color video camera and Northern Eclipse software (Empix, Mississauga, ON).

Functional Analysis in Xenopus Oocytes—The Cx48.5-coding sequence was subcloned into pCS2+ (30), linearized with NotI, gel-purified, and used as the template (1 µg of DNA) to produce capped cRNAs using the mMessage mMachine kit (Ambion). 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 Cx48.5 cRNA (40 ng/cell) using a Nanoject II Auto/Oocyte injector (Drummond, Broomall, PA). After 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 supplemented to a final Ca2+ concentration of 2.9 mM (31), and manually paired with the vegetal poles apposed. Electrophysiological recordings were made 24-48 h after pairing.

The functional properties of cell-to-cell channels were assessed by dual voltage clamp (32). Current and voltage electrodes (1.2-mm diameter, omega dot; Glass Company of America, Millville, NJ) were pulled to a resistance of 1-2 megaohms 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 ±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. Currents were measured at the end of the voltage pulse, at which time they approached steady state (Ijss), and the macroscopic conductance (Gjss) was calculated by dividing Ijss by Vj. Gjss was then normalized to the values determined at ±20 mV and plotted against Vj. Data describing the relationship of Gjss as a function of Vj were analyzed using Origin 6.0 (Microcal Software, Northampton, MA) and fit to a Boltzmann relation of the form,

(Eq. 1)

where Gjss is the steady-state junctional conductance, Gjmax (normalized to unity) is the maximum conductance, Gjmin is the residual conductance at large values of Vj, and V0 is the transjunctional voltage at which Gjss = (Gjmax - Gjmin)/2. The constant A (=nq/kT) represents the voltage sensitivity as the equivalent number (n) of electron charges (q) moving through the membrane; k is the Boltzmann constant, and T is absolute temperature.

To characterize non-junctional hemichannels, single oocytes were assessed with a two-electrode voltage-clamp procedure (33). Cells were initially clamped at -40 mV. Depolarizing voltage steps (-30 to +60 mV at 10-mV intervals) were imposed for a duration of 4.5 s, and whole-cell currents were recorded. Mean current values were measured at the end of the pulse and plotted against the membrane potential.

Microinjection of Morpholino Oligos—Cx48.5 antisense and control morpholinos were obtained from Gene Tools, LLC (Philomath, OR). Two non-overlapping antisense morpholinos were used. Both were targeted against non-coding sequences in the 5'-untranslated region. The targeted sequences were 5'-GCTTCACGATTTCTAGCCTAGAGAG-3' and 5'-AACAGAGAGGATTCATTGAGAACTA-3'. The standard control morpholino sequence was 5'-CCTCTTACCTCAGTTACAATTTATA-3'. Morpholino stock solutions were prepared according to Nasevicius and Ekker (34), and the working solution was 1 ng/nl. 1-cell-stage zebrafish embryos were orientated in a holding chamber prepared according to The Zebrafish Book (28) and microinjected with a Narishige Pressure Microinjection System (Carsen Medical and Scientific Co. Ltd.) on a Nikon Eclipse TE300 inverted microscope. Injections with a series of volumes ranging from 0.5 to 5 nl were carried out with individual morpholinos and a combination of both morpholinos. For the phenotypic analysis presented in this manuscript embryos were injected with a combination of 1 ng of each antisense morpholino or 2 ng of the control morpholino.

Morphological Analyses—For histological analysis the embryos were embedded in JB-4 (Polysciences, Warrington, PA), sectioned at 3 µm, stained with toluidine blue (1% in aqueous 1% borax), and imaged with a Zeiss Axioskop FS microscope. Observations of live embryos were performed with differential interference contrast optics on a Zeiss Axioskop FS microscope, and dark field observations of dissected lenses were performed with a Leica MZ8 stereomicroscope. All images were acquired with a SONY DXC-950 CCD color video camera and Northern Eclipse Software.

Growth and Heart Rate Measurements—For the measurements of the lens, eye, body length, and otic vesicles, embryos were anesthetized according to The Zebrafish Book (28) and then placed in a viewing chamber constructed of a microscope slide and a supported cover glass. The space between the slide and the cover glass was about 1 mm, so that the zebrafish embryos were almost parallel to the slide. Images of lenses, eyes, and otic vesicles were collected as described above and measured with the Northern Eclipse Software. Body lengths were measured by putting the slides against a ruler on a Leica MZ8 stereomicroscope. For the measurements of heart rates, the embryos were placed in 24-well plates and allowed to equilibrate to room temperature (24 °C). The plates were then placed on a Leica MZ8 stereomicroscope, and the heart rates were counted. The embryos destined for heart rate measurements were not anesthetized because this seemed to raise variation in the heart rate. Statistical comparisons were made between control and antisense-injected embryos using an univariate analysis of variance followed by the Tamhane's T2 test. The default {alpha} level was 0.05 unless otherwise specified.

Online Supplemental Material—Supplemental video material is available online. The videos show the heart beat in a 1.5-dpf Cx48.5 morphant embryo (Movie 1) and a 1.5-dpf control embryo (Movie 2). The trunk circulation in a 1.5-dpf Cx48.5 morphant embryo (Movie 3), and a control embryo (Movie 4) is also shown. The movies were collected at 33 frames/s and played at the original speed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of the Zebrafish Cx48.5 Gene—We are using comparative gene function analysis in our continued effort to understand the role of specific connexins in the development and function of the vertebrate ocular lens. We previously reported the cloning and characterization of Cx44.1, a zebrafish connexin with extensive sequence and biophysical similarities to rodent and human Cx50 (35), and here we report the isolation and functional characterization of Cx48.5, a zebrafish connexin with extensive sequence and biophysical similarities to rodent and human Cx46. The isolated genomic sequence contains a coding region of 1305 nucleotides within one single exon, coding for 434 amino acids. The protein has a predicted molecular mass of 48,492 Da and is, therefore, named Cx48.5. Sequence comparisons showed that Cx48.5 has the highest degree of amino acid identity to the Cx46 orthologous group of connexins (61% amino acid identity to mouse Cx46). The amino acid sequence of Cx48.5 is consistent with the typical structure of a connexin (36, 37), with four transmembrane segments, a cytoplasmic loop between transmembrane segments 2 and 3, cytoplasmic amino and carboxyl termini, and two extracellular loops with three conserved cysteines in each. Alignment of the amino acid sequences of Cx48.5, mouse Cx46, and chicken Cx56 (Fig. 1) demonstrated conservation in the location of the six extracellular loop cysteines and the two amino acids that have been shown to be mutated in human Cx46-linked congenital cataracts (Asn-63 and Pro-187) (38, 39). Analysis of the amino acid composition of Cx48.5 revealed that this connexin has a low isoelectric point (pI 5.64) and contains a high proportion of glutamic acid (9.4%). Most members of the Cx46 orthologous group (mouse, sheep, cow, chicken) have a significantly higher pI (7.04-9.85) and much lower proportion of glutamic acid (5.2-6.7%). In contrast to this, most members of the Cx50 orthologous group (human, mouse, sheep, chicken, zebrafish) have low pI (5.01-5.44) and a much higher content of glutamic acid (10.0-13.2%). With respect to pI and glutamic acid content, Cx48.5 therefore resembles the Cx50 orthologous group more closely than the Cx46 orthologous group despite the fact that the overall amino acid comparisons indicated that Cx48.5 is more similar to the Cx46 group than the Cx50 group.



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  		FIG. 1.
Sequence alignment of zebrafish Cx48.5 (zfCx48.5), mouse Cx46 (msCx46), and chicken Cx56 (chCx56). Solid lines over the sequences delineate the four predicted transmembrane regions of connexins. The x symbols indicate the six conserved cysteines within the two predicted extracellular loops. The two bold black dots above the sequences show the conserved Asn and Pro residues that are mutated in humans with congenital cataracts (Asn-63 and Pro-187). Amino acid identity between all three sequences is indicated with an asterisk (*), strong similarity with a colon (:), and weak similarity with dot (.).

 
Cx48.5 mRNA Expression in Embryos and Adult Tissues—RT-PCR was performed with RNA samples isolated from adult tissues, whole embryos, and embryonic hearts and lenses. Cx48.5 transcripts were not detected in 8-h post-fertilization whole embryo RNA but were readily detected at 1 dpf and at later stages (Fig. 2A). In adult tissues Cx48.5 transcripts were detected in the lens and heart, and weak RT-PCR product was also amplified from testis mRNA. Cx48.5 mRNA was not found in the adult liver, ovary, brain, or retina. We also detected Cx48.5 transcripts in RNA isolated from 4 dpf embryonic hearts and lenses (Fig. 2B), but we were not successful in obtaining enough heart material to carry out the analysis at earlier stages due to the fragility of the tissue. Zebrafish eF1{alpha} primers were used to show that comparable amounts of cDNA were included in each reaction and to show the absence of genomic DNA contamination from all the samples. The eF1{alpha} primers span an intron and produce a PCR product of smaller size from cDNA than genomic DNA (569 versus 733 bp) (Fig. 2, A and B).



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  		FIG. 2.
Cx48.5 expression in embryos and adult tissues. RT-PCR was performed with RNA samples isolated from embryos and adult tissues. Cx48.5-specific products were amplified from RNA isolated from 1 to 5 dpf whole embryos (A) and the adult lens, heart, and testis and the embryonic (4-dpf) lens and heart (B). The eF1{alpha} primers amplified a single 569-bp fragment in all the samples, demonstrating the presence of cDNA and the absence of genomic DNA from all samples. Whole mount in situ hybridization detected Cx48.5 transcripts in the otic vesicles (OV) in 1-dpf embryos (C and D) but not in 1.5-dpf embryos. Cx48.5 transcripts were not detected in the lens until 1.5 dpf (E and F). In the 1.5-dpf lens, Cx48.5 is transcribed in the differentiating primary lens fibers (PF) but not in the lateral epithelium (LE).

 
WMISH was performed with embryos from 1-4 dpf. A strong signal was detected in the otic vesicles in 1 dpf embryos (Fig. 2, C and D). The otic vesicle expression appeared to be transient since it was not detected at any of the later stages (1.5-4 dpf). In contrast to the signal in the otic vesicles, expression of Cx48.5 in the lens was not detected at 1 dpf (Fig. 2C). Instead, strong expression of Cx48.5 in the lens was observed at 1.5 dpf (Fig. 2, E and F) and later stages. We did not detect Cx48.5 expression in the heart or any other organs by WMISH, probably because of insufficient sensitivity of the method.

Cx48.5 Forms Voltage-sensitive Intercellular Channels—To determine whether Cx48.5 could form junctional channels, we used the paired Xenopus oocyte expression system (40). 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, Cx48.5 induced the development of high levels of electrical coupling 24-48 h after RNA injection. The mean conductance (Gj) of paired Cx48.5-injected cells was 53.7 ± 22.2 microsiemens (mean ± S.D., n = 41). In contrast, the mean background conductance value, measured between oligo-injected control oocytes, was more than 200-fold lower at 0.15 ± 0.18 microsiemens (n = 36). Thus, the expression of Cx48.5 resulted in the formation of functional gap junction channels.



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  		FIG. 3.
Cx48.5 forms functional channels that are gated by transjunctional voltage. A, junctional conductance (Gj) developed between pairs of Xenopus oocytes as measured by dual voltage clamp. Oocytes were co-injected with the Cx48.5 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 the mean ± S.D. of the number of pairs indicated, and symbols show the clustering of all data points. B, voltage gating behavior of gap junction channels formed by Cx48.5. A time-dependent decay of junctional currents (Ij) was induced by transjunctional voltage (Vj) steps. At Vj steps >±40 mV, Ij decayed symmetrically over the time course of the voltage step. The voltage gating of intercellular channels composed of zebrafish Cx48.5 displayed a high degree of conservation to GJA3 orthologs such as chicken Cx56 and rat Cx46.

 
To further characterize the physiological behavior of channels composed of Cx48.5, 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 >40 mV. The rate of channel closure, calculated for Vjs of ± 80 mV, yielded a time constant ({tau}) on the order of 0.3 s, a value intermediate between the slower channels like Cx26 and Cx32 and the more rapidly gating channels such as Cx37 or Cx40 (41, 42). Thus, the voltage dependence of Cx48.5 channels was quite similar to that of rodent Cx46 (31, 33, 43).

The voltage dependence of Cx48.5 channels was further analyzed by plotting junctional conductance (Gj) as a function of transjunctional potential (Vj) (Fig. 4). Gj values for steady-state junctional conductance (Gjss) were normalized to the maximal conductance measured at the lowest Vj (20 mV). No fast gating effects (<5ms) of voltage on these channels were observed (data not shown). In contrast, Gjss was dependent on voltage, and this plot was fitted to a Boltzmann relation (44) whose parameters are given in Table I. For comparison, the Boltzmann parameters for Cx46 and Cx50 orthologs from chicken, rodent, and zebrafish are also shown in Table I. This analysis clearly showed that zebrafish Cx48.5 channels have weak voltage sensitivity. Similar weak voltage sensitivity has been demonstrated for rat Cx46 and chick Cx56 in both the paired Xenopus oocyte system and in transfected mammalian cells (43, 45, 46).



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  		FIG. 4.
Quantitation of Cx48.5 voltage gating. Shown is the relationship of Vj to steady-state junctional conductance (Gjss) normalized to the values obtained at ±20 mV for Cx48.5. The solid line represents the best fit to the Boltzmann equation, whose parameters are given in Table I. The voltage gating of intercellular channels composed of zebrafish Cx48.5 displayed a higher degree of conservation to the Cx46 orthologs than the Cx50 orthologs.

 


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  		TABLE I
Boltzmann parameters of zebrafish, chicken, and rodent lens connexins

Junctional conductance (Gj) developed between pairs of Xenopus oo-cytes was measured by dual voltage clamp in response to increasing transjunctional potentials (Vj) of opposite polarity and normalized to the conductance measured at a Vj of ±20 mV (Gjmax, set as unity), as described under "Experimental Procedures." Data were fit to a Boltzmann equation of the form given under "Experimental Procedures." Gjmin is the minimum conductance value as estimated from the Boltzmann fit, and V0 is the voltage at which a half-maximal decrease of Gj is measured. The cooperativity constant (A), reflecting the voltage sensitivity of the channel, reflects the equivalent number of electron charges moving through the transjunctional field.

 
Cx48.5 Forms Non-junctional Hemichannels in Solitary Oocytes—Cx46 was the first cloned connexin shown to be capable of forming voltage-activated hemichannels in single oocytes (16, 33, 47). Therefore, we tested the ability of Cx48.5 to induce nonjunctional membrane conductance in single oocytes. Whole-cell currents were recorded in response to depolarizing voltage steps that were sequentially imposed from a holding potential of -40 mV. The responses of oocytes expressing Cx48.5 revealed slowly activating outward currents at membrane potentials of >-10 mV that were not seen in water-injected controls (Fig. 5A). Plotting the current-voltage relationship (Fig. 5B) demonstrated that the mean currents recorded at the end of the +60-mV voltage step from Cx48.5-injected cells (2.28 ± 1.17 µA; n = 9) and control oocytes (0.25 ± 0.08 µA; n = 8) were significantly different (p < 0.001; Student's unpaired t test). These data demonstrate that zebrafish Cx48.5 also functions as a membrane channel in single, unpaired oocytes. The current-voltage relationship of zebrafish Cx48.5 hemichannels was nearly identical to that of rat Cx46 (33), activating at membrane potentials -10 mV in the presence of normal modified Barth's medium ([Ca2+] = 0.9 mM). In contrast, Cx50 hemichannels can only be elicited when external Ca2+ has been significantly lowered (48, 49).



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  		FIG. 5.
Cx48.5 forms voltage-gated hemichannels. A, single Xenopus oocytes injected with Cx48.5 cRNA or water were studied by voltage clamp. Cells were initially clamped at -40 mV. Depolarizing voltage steps of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mV were imposed, and the whole-cell currents were recorded. Cx48.5 induced rapidly activating outward currents that exhibited a slower partial inactivation at higher membrane potentials (≥10 mV). In contrast, oligo-injected control cells showed negligible membrane currents. B, current-voltage relationships in Cx48.5 and oligo-injected oocytes. Whole-cell membrane currents (Im) were measured at the end of a voltage step. At membrane potentials (Vm) >-10 mV, Cx48.5 injected cells displayed whole-cell currents not seen in oligo-injected controls. Results are shown as the mean ± S.D. of the indicated number of cells.

 
Antisense Morpholino Knock-down of Cx48.5 Expression—Microinjection of antisense morpholinos into 1-2-cell stage zebrafish embryos has been widely used for targeted inhibition of zebrafish gene expression (Ref. 34; for review, see Refs. 50-52). Embryos that have had the expression of a specific gene inhibited with morpholinos are termed morphants (51). In this study two non-overlapping antisense morpholinos (MO-1 and MO-2) were targeted against sequences upstream of the Cx48.5-coding sequence. Comparisons of the morpholino sequences to all zebrafish sequences in GenBankTM failed to identify any sequences with significant similarities to either morpholino. In preliminary dose-response experiments the effective dose for producing a specific phenotype was 3-5 ng when either MO-1 or MO-2 was injected individually. However, the same phenotype was produced with only 1 ng of each morpholino when the two morpholinos were co-injected. Synergistic effects between two non-overlapping morpholinos are frequently observed in zebrafish embryos (50). The morphant data presented in the following sections were derived from embryos co-injected with 1 ng of each antisense morpholino. Whether individually injected or co-injected, the morpholinos clearly produced a similar phenotype, whereas embryos injected with the same amount of nonspecific control morpholino never displayed any developmental abnormalities. Hence, we conclude that the phenotype obtained is specific for Cx48.5 targeting. However, the age at which the phenotype became detectable and the severity of the phenotype varied slightly between individual embryos (a maximum of a 6 h delay in the time of onset, except for the onset of cataract formation, which varied by as much as 2 days), presumably due to slight variability in the volume injected and individual differences in the rate of development.

Cataract Formation, Microphakia, and Microphthalmia in Cx48.5 Morphants—The center region of the lens in Cx48.5 morphants became visibly abnormal in live embryos by 3-4 dpf. The lenses in live Cx48.5 morphants appeared uneven and rough when viewed with differential interference contrast optics, in contrast to the very smooth appearance of the lenses in the control embryos (Fig. 6, A and B). Sectioning of the morphant eyes also revealed histological abnormalities. In the control lenses at 3 dpf, the primary lens fibers in the center region were mature and less intensely stained with toluidine blue than the cortical fibers. The less intense staining in the central fibers in the normal control lenses reflects the loss of basophilic materials such as ribosomes and nuclei during the differentiation process. The differentiating secondary fiber cells elongate medially from the equatorial region and form the long, smooth, and tightly packed fibers of the normal lens (Fig. 6D). However, this differentiation process was disrupted in the Cx48.5 morphants. The primary and secondary fibers in the Cx48.5 morphants did not gain the same long, thin, and smooth shape by 3 dpf. Instead, most of the fibers remained nucleated, and the entire core of the lens appeared disorganized (Fig. 6C). Cx48.5 morphant and control lenses were dissected from embryos at a series of stages and observed with a microscope equipped with darkfield optics. Nuclear opacities (cataracts) first appeared in the Cx48.5 morphant lenses by 5.5-7.5 dpf. The cataractous lens shown in Fig. 6E was photographed at 9.5 dpf. The size of the cataracts in the lenses of the Cx48.5 morphants also varied. The retina also appeared to be abnormal in the Cx48.5 morphants. All the retinal cell layers were present in the Cx48.5 morphants, but they appeared to be thinner and less well developed than in the control retinas (Fig. 6C).



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  		FIG. 6.
Cx48.5 morphants developed cataracts and smaller lenses and eyes. Microscopic observations of the Cx48.5 morphants with differential interference contrast (DIC) optics revealed marked irregularities and roughness in the lens core by 3 dpf (A), which was in strong contrast to the smoothness of the control lenses (B). Sections of those lenses revealed that lens fiber differentiation was abnormal and immature in the Cx48.5 morphants (C), whereas the lens fibers in the core region of the control lenses had fully differentiated by this time (D). Note also that the retina in the Cx48.5 morphants (C) is smaller than in the controls. Lenses were dissected from 9.5 dpf embryos and observed with a darkfield microscope. The morphant lenses developed nuclear opacities (cataract, E), whereas the control lens is transparent (F). Lens, eye, and otic vesicle size as well as body length were compared between the Cx48.5 morphants and the controls. The Cx48.5 morphant has significantly smaller lenses and eyes at 3.5 and 6.5 dpf (G and H, p < 0.001). However, no significant difference in whole body length was detected between the Cx48.5 morphants and the controls at 3.5 and 6.5 dpf (I) and neither does the size of the otic vesicles (OV) differ between the morphants and the controls at 2.5 and 3.5 dpf (J). Data are presented as the means ± S.D.; n = 12. Note that the y axes in G-J do not go to 0. Bar, 50 µm.

 
Previous work has demonstrated that Cx50, but not Cx46, is required for the development of normal lens and eye size in mice (3-5, 53). Measurements of lens and eye size revealed that both were significantly smaller in the Cx48.5 morphants than in the controls (p < 0.001). At 3.5 dpf, when the lens abnormality first became noticeable, the Cx48.5 morphant lenses were 6.3% smaller in diameter than the control lenses (Fig. 6G). By using the lens diameter measurements to calculate lens volume (assuming that the lens is a sphere), the lenses in the Cx48.5 morphants were found to be 17.7% smaller in volume than the controls at 3.5 dpf. By 6.5 dpf the morphant lenses were 13.7% smaller in diameter than the controls and 35.7% smaller in volume (Fig. 6G). The size of the eye was also significantly smaller in the Cx48.5 morphants (Fig. 6H). The anterior-posterior diameter of the morphant eyes was 10.5 and 7.2% smaller than the diameter of the control eyes at 3.5 and 6.5 dpf, respectively. No significant difference was observed in the length of the whole embryo between the Cx48.5 morphants and the controls (Fig. 6I), indicating that the smaller lens and eye in Cx48.5 morphants was not due to undergrowth of the embryo as a whole. Because we observed Cx48.5 expression in the otic vesicles only early on, we measured their size at 2.5 and 3.5 dpf. No significant difference was found in the size of otic vesicles between the Cx48.5 morphants and the controls (Fig. 6J).

Abnormal Cardiac Contractions and Circulation Blockage in Cx48.5 Morphants—In addition to the ocular abnormalities described above, we also observed severe cardiovascular dysfunction in the Cx48.5 morphants. The abnormal cardiovascular function became apparent about 1.5 dpf, much earlier than the lens dysmorphology. Prior to 1.5 dpf, cardiovascular development in the Cx48.5 morphants appeared normal, including the initiation of cardiac contractions around 22-24 h post-fertilization. At 1.5 dpf the normal zebrafish heart is a simple tubular structure, and neither the bulbus arteriosus nor the cardiac valves have developed yet. Blood is pumped unidirectionally through the heart by alternate atrial and ventricular contractions. In each chamber contractions are initiated caudally, and a wave of contractions then travels anteriorly, whereas the posterior portion of the chamber remains contracted. This process culminates in the near obliteration of the chamber lumen, thereby minimizing backflow (this study, and Refs. 23 and 54) (Fig. 7, F-H; Supplemental Movie 2). By contrast, contractions in the morphant atria appeared less coordinated, and as a consequence the full contraction of the entire atrium as seen in the controls was never achieved. Frequently the posterior end of the atrium was already initiating relaxation by the time the contractile wave reached the anterior end, thereby causing the blood to move back and forth within the atrium (Fig. 7, A-C; Supplemental Movie 1). In control embryos the ventricles could be seen to expand in volume and fill very rapidly and forcefully during diastole (Fig. 7, I-J; Supplemental Movie 2). This is likely aided by the rapid fall in intraventricular pressure to near zero that occurs at the end of systole (23). Contractions in the morphant ventricles appeared very uncoordinated. In addition, the ventricle appeared to expand much less than the control ventricles during diastole, and as a result the extent of ventricular filling was much lower than normal (Fig. 7, D-E; Supplemental Movie 1).



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  		FIG. 7.
Cx48.5 morphants exhibited cardiac abnormalities by 1.5 dpf. All panels are ventral views, head up. Panels A-C (Cx48.5 morphant embryo) and F-H (control embryo) show images of atria at different stages of the cardiac cycle. The atria are demarcated with dashed lines. The contractile wave in the Cx48.5 morphant is initiated normally at the posterior end of the atrium (B), but because of slow conduction, as it travels toward the anterior end the posterior portion of the atrium begins to recover and relax (C). Arrowheads show a region of the morphant atrium undergoing contraction, whereas other parts of the atrium are in a relaxed state. In contrast, the posterior portion of the atrium in the control embryos remains contracted as the wave travels in the anterior direction such that the entire atrium is contracted at the end of the cycle (G and H). Panels D-E (Cx48.5 morphant embryo; for complete video, see Supplemental Movie 1) and I-J (control embryo; for complete video, see Supplemental Movie 2) show images of ventricles at two stages of the cardiac cycle. The ventricles are outlined with dash lines. The atria are labeled with a small font letter A. Panel I shows a fully distended ventricle in a control embryo at the end of diastole, and panel J shows the same ventricle at the end of systole. These panels show that the volume of the ventricle increases significantly during diastole. However, in the Cx48.5 morphant the ventricular volume difference between diastole and systole was greatly reduced (D and E). The differences in the shape of the morphant (D and E) and the control ventricles (I and J) are partly due to variation in orientation of the embryos but also due to the lack of movement of blood through the morphant heart. Bar, 100 µm.

 
Accompanying the cardiac abnormality was a block in the systemic circulation (compare Supplemental Movies 1 and 2 and Movies 3 and 4) that was evident between 1.5 and 3.5 dpf. Blood cells did not move through the heart during this period, and starting from 1.5 dpf, blood accumulation was seen in the common cardinal vein distal to the atrium in the Cx48.5 morphants (Fig. 8, A and B). Between 2 and 4 dpf, blood accumulation was seen in the trunk veins of the morphant embryos and in the atria of some of them (Fig. 8C). Arrhythmia, tachycardia, enlarged atria, and pericardial edema developed frequently between 3 and 5 dpf; these are possibly secondary effects of the primary cardiac dysfunction evident at 1.5-2 dpf. However, most of the Cx48.5 morphants recovered from the cardiovascular symptoms by 5-6 dpf. The recovery of the Cx48.5 morphants from cardiac dysfunction is possibly due to withdrawal of the antisense morpholino effects. Antisense morpholinos are typically effective for the knock-down of translation of the targeted mRNA during first a few days of zebrafish development only (50, 52).



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  		FIG. 8.
Cardiac dysfunction and blood circulation blockage in the Cx48.5 morphants. By 1.5 dpf blood had begun to accumulate in the common cardinal vein in the Cx48.5 morphants (A and B, arrowhead). The morphology of the heart appeared normal at this stage (B and E). Blood accumulation was also seen in the caudal vein and other vessels in the trunk (C). Blood circulation was almost completely blocked at this stage (see Supplemental Movie 3). The Cx48.5 morphants had significant slower heart rate than the controls at 1.5 and 2.5 dpf (G, p < 0.001) but had faster heart rates than the controls at 3.5 dpf (G, p < 0.01). Data are presented as the means ± S.D.; n = 15. Panels A-F are lateral views, with the head to the left. DIC, differential interference contrast.

 
Overall, the duration of each cardiac cycle appeared longer in the morphant hearts (compare Supplemental Movies 1 and 2). We quantified this difference by comparing the heart rates in the Cx48.5 morphants and the control embryos. The Cx48.5 morphants had significantly slower heart rates than the control embryos at 1.5 and 2.5 dpf (p < 0.001), but at 3.5 dpf the rates had become faster than the controls (Fig. 8G) (p < 0.01). Heart rate measurements were not carried out with older embryos because the morphant cardiac phenotype was much more varied in later stages (including arrhythmia, tachycardia, pericardial edema, and occasional bradycardia), rendering group comparisons less meaningful. The lengthening of the cardiac cycle in the morphants appeared to arise primarily from the slowing of the caudal to cephalic waves of atrial and ventricular contractions. To verify this we quantified three phases of the atrial contractile cycle, the length of time for contractions to travel from posterior to anterior end, the length of time from the start of relaxation to full relaxation, and the duration of the full relaxation. These data are summarized in Table II. The time it took the contraction to travel from the posterior to the anterior end was significantly longer in the morphant atria compared with the controls (p < 0.001). The duration of atrial relaxation (the stage that the blood flows in the atrium) was significantly shorter in the morphant atria than in the controls (p < 0.01). No significant difference was found in the length of time from the start of relaxation to full relaxation. Overall, the length of the contractile cycle was significantly longer in the morphant atria than in the control atria (p < 0.05).


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  		TABLE II
Contractile properties of the morphant and control atria

Contractions of the morphant and control atria were videotaped. Three phases of the contractile cycle were measured from the videos using the pause-forward VCR function. Data were analyzed using univariate analysis of variance followed by the Tamhane's T2 test and are presented as the means ± S.D.; n = 6.

 
Although the cardiovascular function of the Cx48.5 morphants was severely impaired, no gross morphological abnormalities were seen in the morphant hearts in the early stages (1.5-3 dpf). However, some of the morphant hearts became morphologically atypical later (3-5 dpf), possibly induced by the early stage heart dysfunction. To confirm whether the morphant hearts were morphologically normal and whether the myocardium had differentiated normally in terms of expression of contractile proteins, we examined the expression of two cardiac markers in the Cx48.5 morphants, cmlc2 (29) and MF20 (55, 56). These markers are expressed in both the atrium and the ventricle (29). We carried out a whole mount in situ hybridization with a cmlc2 antisense RNA probe at 1.5 dpf, and the MF20 antibody was used in whole mount immunohistochemistry at 1.75 dpf. The expression of cmlc2 and MF20 appeared to be typical in the Cx48.5 morphants (Fig. 9, A and C). Hence, we conclude that the functional cardiovascular defect displayed by the Cx48.5 morphants at the early stages is not caused by abnormal cardiac morphology nor did it delay activation of the genes coding for the myocardial contractile proteins.



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  		FIG. 9.
The expression of cardiomyocyte markers is normal in the early Cx48.5 morphant hearts. The expression of the cardiomyocyte differentiation markers cmlc2 and MF20 was examined at 1.5 and 1.75 dpf, respectively. Both markers were expressed normally in the Cx48.5 morphants (A and C), indicating that differentiation of the myocardium was not disrupted by the knock-down of Cx48.5 expression. The difference in the appearance of the hearts in C and D is due to a slight difference in the orientation of the embryos. V, ventricle; A, atrium. All panels are ventral views with the head pointing up.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we set out to isolate and characterize the zebrafish orthologue of murine and human Cx46 and in the process identified Cx48.5, a novel member of the zebrafish connexin gene family. The predicted amino acid sequence of Cx48.5 is consistent with all the typical features of connexin family members, such as four {alpha}-helical transmembrane segments and two extracellular loops with three conserved cysteine residues in each. Sequence comparisons indicated that Cx48.5 shared the highest degree of sequence identity with the Cx46 orthologous group of connexins. An unusual feature of Cx48.5 is its low isoelectric point and high content of glutamic acid residues. With regard to these two features Cx48.5 resembles more closely the Cx50 orthologous group of connexins than the Cx46 subgroup. How these differences might influence the function of Cx48.5 is not clear, but the fact that the ocular growth defects we observed in the Cx48.5 knock-down embryos resemble the ocular defects in the Cx50 knock-out mice more than in the Cx46 knock-out mice may be at least partly attributable to these features of the primary sequence.

Previous research on the in vivo function of Cx46 and Cx50 family members has primarily focused on the expression and role of these connexins in the development and maintenance of the ocular lens. In fact, these two connexins have frequently been considered lens-specific. This view has been reinforced by the apparent absence of non-ocular abnormalities in the Cx46 and Cx50 knock-out mice (3, 4). However, several studies have documented the expression of Cx46 and Cx50 in tissues other than the lens, including heart (15-18), kidney (16, 57), alveolar epithelial cells (58), osteoblastic cells (59), retina (60, 61), and degenerating Schwann cells (62). In this study we demonstrate that in addition to the lens, Cx48.5 is expressed in the embryonic and adult heart, in the early otic vesicles, and adult testes. We have recently reported a detailed description of the Cx48.5 WMISH expression pattern in the developing zebrafish lens (27). Although it is somewhat perplexing that we did not see any Cx48.5 staining in the heart by WMISH at developmental times when the antisense morpholinos disrupted cardiovascular function most severely, as we pointed out in the results section, we feel that the absence a cardiac Cx48.5 signal in these embryos is due to insufficient sensitivity of the technique rather than lack of Cx48.5 expression. Consistent with this interpretation is the fact that we did not detect Cx48.5 cardiac staining at 4 dpf either (data not shown), whereas we were able to detect Cx48.5 in 4-dpf hearts by RT-PCR. It seems clear that the level of Cx48.5 mRNA in the embryonic heart is very low, but the morpholino results show that functionally it is still very significant. We did not observe any gross morphological abnormality in the otic vesicles of the Cx48.5 morphants, and the question of whether Cx48.5 has a role in inner ear development and function is yet to be answered. Neither do we know what possible role Cx48.5 might play in the testis because the antisense morpholino effects have worn off by the time the testes develop.

Consistent with its high sequence identity to the mammalian Cx46 orthologues, the functional behavior of zebrafish Cx48.5 in single and paired Xenopus oocytes was also similar to this subgroup of connexins. Like Cx46 and its orthologues in other species (16, 33, 47, 63), Cx48.5 induced robust hemichannel activity in solitary cells. Recently there has been renewed interest in the physiological relevance of hemichannel activity (for review, see Refs. 64 and 65), including it playing roles in apoptosis, cellular signaling, and neural transmission. Although it is unlikely that the lens generates sufficient voltage gradients to activate Cx48.5 hemichannels, it will be of great interest to resolve whether the cardiac phenotype in Cx48.5 morphants is due to the loss of intercellular channel activity, hemichannel activity, or a combination of these two discrete functions. Hemichannel activity can be modulated by other physiological stimuli, most notably external calcium concentration, so further characterization of Cx48.5 hemichannels may provide additional clues to their relevance to heart and lens physiology.

The Cx48.5 knock-down embryos reproduced the cataract phenotype seen in the Cx46 knock-out mice (3). Somewhat surprisingly, however, the Cx48.5 morphants also developed abnormally small lenses and eyes, features that were observed in the Cx50, but not the Cx46 knock-out mice (3-5). Further insight into the contribution of Cx46 and Cx50 to ocular growth has been gained from recent knock-in and knock-over experiments. Homozygous replacement of the Cx50-coding sequence with the Cx46-coding sequence did not rescue the growth defect regardless of whether the native Cx46 locus was intact or not (53, 66). On the other hand lens growth was normal in mice with one native Cx50 allele intact, whether the second allele was deleted or replaced with Cx46 (53, 66). Thus, it appears that the role of connexins in lens growth in mice is primarily served by Cx50 and that the role of Cx46 in this process is negligible. From the present study it is clear that zebrafish Cx48.5 has a growth regulatory role in the lens. The question of whether Cx44.1 also plays a role in lens growth is presently unanswered and awaits future knock-down experiments. Our data also suggest a role for Cx48.5 in retinal development, since smaller retinas developed in the Cx48.5 morphants. Currently we do not know the underlying cause of the retinal defect, but it may be that the aberrant development of the Cx48.5 knock-down lenses interferes with signaling between the lens and the retina. A number of studies have demonstrated that signals emanating from the lens are required for normal growth of the retina (67-69).

Although Cx46 mRNA or protein has been detected in the adult rat, rabbit, and human heart (16-18), no cardiac defects were reported in the Cx46 knock-out mice (3). Several possible explanations exist for the apparent discrepancy between the mouse and zebrafish data with respect to cardiac function. The most straightforward explanation is that Cx46 is either not expressed or does not carry out an essential physiological function in the fetal, neonatal, or adult mouse heart. We are currently investigating this possibility. Alternatively, Cx46 may have an essential role in the heart at some stage in the life of the mouse, but in its absence this role can be covered by one of the other connexins expressed in the heart. Another possibility is that the Cx46 knock-out mice may have suffered a cardiac abnormality in early fetal life that was both transient and non-lethal and, therefore, went undetected. We cannot exclude the possibility that the role of Cx48.5 in zebrafish cardiovascular function is transient and non-essential as well, because we do not know whether the recovery of function we observed is due to reduction in the knock-down effect caused be cellular dilution of the morpholino or due to a diminished role for Cx48.5 in cardiac function as the heart matures. Finally, although Cx48.5 and Cx46 share many structural and functional features, we are unable to conclusively assign an orthologous relationship to these two genes at this time. In fact, Cx48.5 may be a unique fish gene that arose from the extra genome duplication event that took place in the ray-finned fish lineage after its separation from the rest of the vertebrate lineages. The proper assignment of orthologous relationships between zebrafish and mammalian connexins will have to await the identification of more zebrafish connexin genes, the identification of a number of connexin genes in an appropriate out-group (e.g. sharks or lampreys), and a more thorough establishment of syntenic relationships between zebrafish and mouse and human chromosomes (70).

The abnormalities we observed in the cardiovascular system is most consistent with a defect in some aspect of the generation or propagation of chamber activation. The underlying molecular and cellular mechanisms are not known at this time, but it is interesting to note that several of the mutants identified in the initial large scale screens displayed cardiovascular defects resembling the cardiovascular problems of the Cx48.5 morphants (71, 72). Adult zebrafish hearts do not appear to contain a histologically discrete conduction system such as the His-Purkinje system of mammals. Instead, the ventricular trabeculae appear to be the functional equivalents of the specialized conduction system in mammals (73). Although an extensive network of ventricular trabeculae began to form by 5 dpf, at 2 dpf, when the cardiovascular abnormalities in the Cx48.5 morphants were at their peak, the ventricle was devoid of trabeculae (23). The conduction requirements of the 2-3-dpf zebrafish heart may, therefore, be quite different from 5-dpf hearts in which trabeculae have formed. Perhaps these early requirements are partially fulfilled by Cx48.5, but once trabeculae form, impulse conduction can proceed through gap junctions composed of other connexins. A similar situation may exist in mice. Cx45 is the earliest known connexin to be expressed in the heart tube, yet cardiac contractions are initiated at the correct time in Cx45 knock-out mice (13). It will be interesting to determine whether the cardiac contractions in the early Cx45 knock-out embryos are mediated by Cx46. In this report we have placed less emphasis on the cardiovascular abnormalities observed in later stage Cx48.5 morphants, because it is not possible based on our data to distinguish whether these were primary effects due to Cx48.5 knock-down or secondary effects arising from the earlier defects. In particular, it has been shown that the fluid forces from the blood circulation are necessary for the zebrafish heart to develop properly (54). Hence, the developmental and functional abnormalities observed at later stages were possibly at least partially due to secondary effects and, therefore, less indicative to the function of Cx48.5 in the zebrafish heart.


   FOOTNOTES
 
* This work was supported by research grants (to G. V.) and scholarships (to S. C.) from the Natural Sciences and Engineering Research Council of Canada and the Manitoba Health Research Council and by National Institutes of Health Grants EY13163 and DC06652 (to T. W. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental videos. Back

|| To whom correspondence should be addressed: Dept. of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. Tel.: 204-4746170; Fax: 204-4747588; E-mail: valdimar{at}cc.umanitoba.ca.

1 The abbreviations used are: Cx, connexin; cmlc2, cardiac myosin light chain 2; MF20, myosin heavy chain; dpf, days post-fertilization; eF1{alpha}, elongation factor 1{alpha}; WMISH, whole mount in situ hybridization; RT, reverse transcriptase. Back


   ACKNOWLEDGMENTS
 
We Dr. Erwin Huebner for allowing use of the Narishige Microinjection System, Dr. Ted J. Wiens for lending the Nikon Eclipse TE300 inverted microscope, and Tara Christie for preparing the cmlc2 probe. We also thank Dr. Didier Stainier for generously providing the cmlc2 cDNA. The MF20 monoclonal antibody developed by Dr. D. A. Fischman was obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 under contract NO1-HD-7-3263 from the NICHD, National Institutes of Health.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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