Multiple connexins contribute to intercellular communication in the Xenopus embryo

Yosef Landesman1,*, Friso R. Postma1, Daniel A. Goodenough2 and David L. Paul1,{ddagger}

1 Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
2 Department of Cell Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
* Present address: GPC-Biotech Inc., 610 Lincoln Street, Waltham, MA 02451, USA

{ddagger} Author for correspondence (e-mail: dpaul{at}hms.harvard.edu)

Accepted 19 September 2002


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To explore the role of gap junctional intercellular communication (GJIC) during Xenopus embryogenesis, we utilized the host-transfer and antisense techniques to specifically deplete Cx38, the only known maternally expressed connexin. Cx38-depleted embryos developed normally but displayed robust GJIC between blastomeres at 32-128 cell stages, suggesting the existence of other maternal connexins. Analysis of embryonic cDNA revealed maternal expression of two novel connexins, Cx31 and Cx43.4, and a third, Cx43, that had been previously identified as a product of zygotic transcription. Thus, the early Xenopus embryo contains at least four maternal connexins. Unlike Cx38, expression of Cx31, Cx43 and Cx43.4 continue zygotically. Of these, Cx43.4 is the most abundant, accumulating significantly in neural structures including the brain, the eyes and the spinal cord.

Key words: Gap junctions, Dye transfer, Xenopus connexins, Cx30, Cx31, Cx38, Cx41, Cx43, Cx43.4


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gap junctions are collections of intercellular channels that provide a direct connection between the cytoplasm of adjacent cells, allowing exchange of small molecules that influence physiological responses. The intercellular channels are composed of connexins, a highly related family of proteins with >20 members in humans (White and Bruzzone, 2000Go). Individual cells typically express multiple connexins, and the patterns of connexin expression change dramatically during development or in response to stimuli in fully differentiated tissues (Kumar and Gilula, 1996Go; White and Bruzzone, 2000Go; White and Paul, 1999Go).

Four Xenopus connexins have been identified to date. Cx43 and Cx30 transcripts appear at gastrula and tailbud stages, respectively (Gimlich et al., 1988Go; Gimlich et al., 1990Go). Cx41 is not detected until adulthood, where it is restricted to ovarian follicular cells (Bruzzone et al., 1995Go). Cx38 is present in oocytes and eggs and persists in early embryos until neurulation (Ebihara et al., 1989Go; Gimlich et al., 1990Go). Since zygotic transcription in Xenopus does not initiate until midblastula stage (Newport and Kirschner, 1982Go), the Cx38 transcript is clearly a product of the maternal genome. A number of studies suggest that gap junctional intercellular communication (GJIC) may affect embryonic development in Xenopus (Guger and Gumbiner, 1995Go; Guthrie et al., 1988Go; Guthrie, 1984Go; Levin and Mercola, 1998Go; Nagajski et al., 1989Go; Olson et al., 1991Go; Olson and Moon, 1992Go; Warner et al., 1984Go). In addition, the number of communicating cells in the Xenopus embryo increases rapidly during early cleavage stages (Landesman et al., 2000Go). The contribution of Cx38 to this intercellular communication has not been directly explored.

To explore the role of GJIC in Xenopus development, we utilized host-transfer and antisense techniques to ablate connexin expression. Cx38 was targeted because it is the only known maternal connexin and because it has been shown that antisense ablation of Cx38 is sufficient to prevent induction of GJIC when Xenopus oocytes are paired and voltage-clamped in vitro (Barrio et al., 1991Go). Thus, cells in a Cx38-depleted embryo should be incapable of forming gap junctions until the start of zygotic transcription. Surprisingly, although depletion of maternal Cx38 abrogated GJIC in oocyte pair experiments, it did not eliminate GJIC between blastomeres of the embryos developed from those oocytes. Moreover, Cx38-depleted embryos developed normally. These results suggest the presence of maternal connexins in addition to Cx38, and these connexins may be dormant in oocytes and gain activity in the early embryo. Therefore, we screened a fertilized egg cDNA library and discovered two more maternal connexins, Cx31 and Cx43.4. In addition, RT-PCR revealed the previously overlooked maternal expression of Cx43. Expression analysis indicated that Cx38 and Cx43.4 were the most abundant maternal connexins. However, unlike Cx38, whose levels decline at the start of zygotic transcription, Cx43.4 levels increased significantly. In situ hybridization showed a gradual accumulation of Cx43.4 in the neural folds and later in the neural tube and the brain of the embryo, consistent with an involvement of Cx43.4 in neural development.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
cDNA library
A fertilized egg cDNA library in a gt22A- vector was made by Michael W. King at the Biochemistry and Molecular Biology, IU School of Medicine, Terre Haute, IN.

Host transfer and dye transfer
Defolliculated Xenopus oocytes were injected with partially phosphorothioate-modified oligonucleotides then incubated for 60 hours to allow degradation of endogenous Cx38 protein before transplantation into a host female. The procedure was a slight modification of that described by Heasman et al. (Heasman et al., 1991Go). We found that the vital dyes Neutral red and Nile blue increased fluorescent background of sectioned embryos and therefore could not be used for subsequent dye-transfer studies. Instead, nonvital dyed oocytes from pigmented frogs were transplanted into albino female frogs. Dye-transfer experiments were performed as described by Landesman et al. (Landesman et al., 2000Go).

RT-PCR oligonucleotides
All sequences are listed in 5' to 3' orientation:

  1. Cx30 Sense (277): CAT ATC CGT CTG TGG GCC
    Antisense (563): CAC TTA AGA AGT CGG ATG
  2. Cx31 Sense (320): GGG CCC TCC AGC TTA TCT TTG TTA
    Antisense (640): TTT CTC TGT GGG ACG GGC TAT GTA
  3. Cx38 Sense (132): GGA ATT ACT AAA GCT CTT
    Antisense (548): TAT GTG CAC ATC AAG GGC
  4. Cx41 Sense (503): GAT GAC AAA GAG CAA GTT
    Antisense (899): TGG CAG GGA TGT ATT GTG
  5. Cx41 Sense (1936): TGC CCT CCA CCG TCC TCT G
    Antisense (2382): ATG CAA AGT TGG GCC TGG TTA GTA
  6. Cx43 Sense (159): AGT GCC TTA GGA AGA CTT
    Antisense (508): CCA CCT TCG TTC TGA ACC
  7. Cx43.4 Sense (55): GCG TCC GAG CAA GTG AAG
    Antisense (451): CTT CAT AGT CTC TCA TGG
  8. Histone H4 (Niehrs et al., 1994Go) Sense: CGG GAT AAC ATT CAG GGT A
    Antisense: TCC ATG GCC GTA ACT GTC
  9. Vg1 (Weeks and Melton, 1987Go) Sense: CCC TCA ATC CTT TGC GGT G
    Antisense: CAG AAT TGC ATG GTT GGA CCC

RT-PCR conditions
For all primers the initial denaturation was carried at 95°C for 5 minutes. For Cx31, Cx43.4, Cx41 (1936+2382), denaturation was at 94°C, 30 seconds, annealing at 65°C, 30 seconds and extension at 72°C for 30 seconds (35 cycles). For Cx30, Cx41 (503+899): denaturation was at 94°C, 30 seconds, annealing at 60°C, 30 seconds and extension at 72°C for 30 seconds (35 cycles). For Histone H4, denaturation was at 94°C, 30 seconds, annealing at 60°C, 30 seconds and extension at 72°C for 30 seconds (21 cycles). For Vg1, denaturation was at 94°C, 30 seconds, annealing at 60°C, 30 seconds and extension at 72°C for 30 seconds (21 cycles).

Phosphorothioate-modified Cx38 sense and antisense oligonucleotides
Phosphorothioate-modified Cx38 sense and antisense oligonucleotides (Bio-Synthesis, INC.) were HPLC purified or desalted. Asterisks represent phosphorothioate linkages:

Functional expression of connexins
For expression in oocyte pairs, sense cRNA was transcribed from linearized templates in SP64T using an SP6 mMessage mMachine kit (Ambion) according to the manufacturer's directions. 500 pg of cRNA was injected into the vegetal hemisphere of Xenopus oocytes. Oocytes were processed, injected, paired and voltage clamped as described previously (Landesman et al., 1999Go).

Cx43.4 channel activity was also examined using dual whole cell patch clamp in transiently transfected N2A cells. To produce a construct for expression of Cx43.4 in N2A cells, a PCR fragment containing the complete coding region was subcloned into the StuI site of pCS2+. N2A neuroblastoma cells were grown in DMEM supplemented with 10% fetal calf serum. On reaching 80% cell density, cells were washed with DMEM/F12 and transfected using lipofectamine (GIBCO) according to the manufacturer's directions. 1 µg of Cx43.4 in pCS2+ was co-transfected with equivalent amounts of vectors expressing either enhanced green fluorescent protein (eGFP) or enhanced yellow fluorescent protein (eYFP) (Clontech) so that transfected cells could be identified for patch clamping. 12 hours after transfection, cells were trypsinized, mixed in a 1:1 ratio, and reseeded in low density. Recordings of fluorescent cell pairs were routinely initiated after 3 hours.

Double patch-clamp recordings were carried out as described by Srinivas et al. (Srinivas et al., 1999Go). Briefly, glass capillaries were pulled to a resistance of ~5 Mohm (Sutter Instr., CA) and filled with (in mM) CsCl 140, Hepes 10, MgCl2 1, EGTA 5, CaCl 0.5, and pH 7.2. The extracellular solution contained (in mM) NaCl 140, Hepes 10, CaCl 2, MgCl 1, CsCl 5, D-Glucose 10, pH 7.4 Patch amplifiers (EPC-7, Heka Electronik, Lambrecht/Pfalz, Germany and Axoclamp 200B; Axon Instr. Union City, CA) were interfaced to a PC running pClamp 8 via the digidata1200 (Axon Instr.). After obtaining gigaohm seals, cell pairs were voltage clamped at 0 mV. Junctional conductance was determined by imposing 10 mv transjunctional potentials. Pairs with junctional conductances exceeding 6 nS were discarded. At the start of each sweep a 10 mV pulse was applied to normalize variance in resistance within one experiment. Off-line data analysis was performed using Clampfit (Axon instruments) and Excel (Microsoft). Curve fitting was performed using Origin 6.1 (Originlab, Northampton MA).

RNA probes, in situ hybridization and histology
Antisense RNA probes for northern blots were synthesized from a full-length template of Cx38 (Ebihara et al., 1989Go), and the C-terminal coding region of Cx43.4 (SacI/EcoRI fragment). The same Cx43.4 template was used to synthesize digoxigenin-labeled probe for in situ hybridization and used as described previously (Harland, 1991Go). Embryos were photographed before or after clearing in benzyl benzoate/benzyl alcohol (1:1). Some embryos were embedded in Paraplast and 12 micron sections were cut.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Parameters for antisense oligonucleotide depletion of Cx38
Consistent with previous reports (Ebihara et al., 1989Go; Gimlich et al., 1990Go), levels of Cx38 mRNA in the oocyte and during early cleavage stages were relatively high and could be detected by a northern blot (Fig. 1). To determine if maternal Cx38 mRNA was asymmetrically distributed, RNA was prepared separately from animal and vegetal halves of 16-cell stage embryos. Cx38 mRNA appeared to be equally distributed along the animal-vegetal axis (Fig. 1). To confirm that animal and vegetal poles were correctly separated, the filter was reprobed for Vg-1, a transcript that has been previously shown to be restricted to the vegetal cortex of the oocyte and egg (Weeks and Melton, 1987Go). The Vg-1 signal reflected the expected distribution (Fig. 1).



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Fig. 1. Cx38 RNA was symmetrically distributed in the 16-cell stage embryo. Embryos were separated into animal and vegetal parts and RNA was analyzed by northern blot. The Vg-1 antisense probe was used as a marker for transcripts from vegetal cells. Cx38 transcripts were equally abundant in animal and vegetal cells. Ribosomal RNA was used as a loading control (data not shown).

 

Four different antisense oligonucleotides were screened for their ability to deplete endogenous Cx38 mRNA and reduce GJIC in oocyte pairs (Fig. 2). Groups of defolliculated oocytes were injected with 2 ng of sense or antisense oligonucleotides. Twelve hours after the injection, total RNA was extracted from samples and analyzed by a northern blot. All the antisense oligonucleotides tested in this study significantly reduced Cx38 transcript levels compared with those in uninjected and sense-injected controls. Antisense treatment generally resulted in a loss of hybridizing bands, although in one case a specific cleavage giving rise to a faster migrating band occurred (oligo-1, Fig. 2). Faint non-specific hybridization of the 18S ribosome subunit is evident as a band just above Cx38. To assess the effect of antisense treatment on GJIC, oligonucleotide-injected oocytes were paired with oocytes injected with rat Cx43 cRNA. We chose to make heterotypic pairs for this evaluation because Cx38 has been shown to form gap junctions more avidly with Cx43 then with itself (Swenson et al., 1989Go). Junctional conductance was measured 48 hours after pairing using dualcell voltage clamp (Spray et al., 1981Go). A significant reduction of GJIC was observed in all antisense injected pairs (Fig. 2) as compared with sense and uninjected controls. A dose-response was determined for antisense oligo-1 (data not shown) that showed that 0.2-0.4 ng was sufficient to eliminate >96% of the conductance resulting from endogenous Cx38 pairing with Cx43.



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Fig. 2. Antisense oligonucleotides deplete endogenous maternal Cx38 and depress GJIC in oocytes. Antisense (oligos 1-4) or sense oligonucleotides were injected into defolliculated oocytes. After 24 hours, total RNA was extracted from a subset of oocytes and northern blotted. Specific degradation of Cx38 RNA was seen for all four oligos. Vg-1 probe was used as a loading control. The remaining oocytes were paired with Cx43 cRNA-injected oocytes, and the development of electrical conductance was monitored [mean conductance levels in microsiemens (µS), SD and number of pairs in parenthesis are below the blot]. All antisense oligonucleotides tested reduced conductance to background levels.

 

Cx38 is not essential for normal Xenopus development or for GJIC in the early embryo
We used the host-transfer technique (Heasman et al., 1991Go) to assess the influence of Cx38 on early development. In order to maximize the amount of oligonucleotide that could be injected without producing non-specific toxicity, sense and antisense versions of oligo-1 were HPLC purified. Oocytes were then injected with 0.8 ng of HPLC-purified oligo-1, a dosage two to four times the amount needed to eliminate coupling in the oocyte pair assay (see above). Following a 60 hour incubation, the oocytes were capacitated by host-transfer and fertilized. The majority of fertilized eggs cleaved normally and developed into tadpoles indistinguishable from controls (Oligo-1: 75/90 (83%), Sense: 77/82 (94%) see Fig. 3A), suggesting that Cx38 is not essential for normal development of the Xenopus embryo.



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Fig. 3. Cx38 was not essential for either normal development or GJIC in the early embryo. Oocytes were injected with sense or antisense oligo-1, capacitated by host-transfer and fertilized. Dye transfer was assessed at the 32-64 cell stage. (A) Both sense and antisense injected oocytes developed into grossly normal swimming tadpoles. (B) Northern blotting confirmed the elimination of Cx38 mRNA from the embryos by oligo-1. (C-J) A mixture of neurobiotin (red) and fluorescein-dextran (green) was injected into one animal cell at the 32-64 cell stage. After fixation and sectioning, GJIC is evident (stars) in blastomeres that contain neurobiotin but not fluorescein-dextran. Bar (C), 200 µm.

 

In order to test whether elimination of Cx38 abolished GJIC between blastomeres in the early embryo, the host transfer experiments were repeated and GJIC assessed using neurobiotin. Oocytes were injected with 2 ng of HPLC purified antisense oligo-1, the highest level that had no effect on early cleavages, and the embryos were produced using host-transfer as described above. At the 32-64 cell stage, the embryos were divided into two groups. The first group was used for northern analysis, which revealed a complete elimination of Cx38 mRNA in antisense-injected embryos (Fig. 3B). Note that in embryos, Cx38 transcript is completely eliminated and no cleavage products, as observed in oocytes, remain (compare Fig. 3B with Fig. 2). The second group of embryos was used for measurement of GJIC by dye transfer as previously described (Landesman et al., 2000Go). A single animal blastomere was injected with a mixture of neurobiotin, which can permeate gap junctional channels, and fluorescein-dextran, which is channel impermeable. The neurobiotin demonstrated the presence of GJIC whereas the dextran marked the injected cell and controlled for intercellular bridges. Ten minutes after injection, the embryos were fixed, sectioned and analyzed by fluorescence microscopy (Fig. 3C-J). Green fluorescence marked the injected cell and any cells connected by cytoplasmic bridges. Transfer of neurobiotin through gap junctions was indicated by red fluorescence in cells that did not contain fluorescein-dextran. Surprisingly, both sense (Fig. 3G-J) and antisense (Fig. 3C-F) injected embryos showed dye transfer. Thus, both GJIC and development in the early embryo were independent of Cx38. These data strongly indicated that additional maternal connexins were active in the early embryo and were capable of inducing GJIC.

At least four maternal connexins are expressed in the Xenopus embryo
To identify additional maternal connexins, we screened a fertilized egg cDNA library at low stringency. A mixture of cDNA fragments from all previously cloned Xenopus connexins (Cx30, Cx38, Cx41 and Cx43) was randomly labeled with 32P and used as a probe. Twenty-two clones were isolated, which represented three connexins. Xenopus Cx38 was recovered together with two new Xenopus maternal connexins. One had 68-69% identity to mouse (accession NP_032152), rat (NP_062113) and human (NP_076872) Cx31 and was designated Xenopus Cx31 (accession AY057997, Fig. 4A). The other had 58-59% identity to chicken (accession P18861), human (NP_005488), mouse (NP_032148) and dog (P28228) Cx45, and 69% identity to zebrafish Cx43.4 (Q92052). Therefore, it was designated Xenopus Cx43.4 (accession AY057998, Fig. 4B).



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Fig. 4. Xenopus Cx31 and Cx43.4 are highly related to other connexins. (A) Xenopus Cx31 (accession AY057997) has 68-69% similarity to mouse, rat and human Cx31. (B) Xenopus Cx43.4 (accession AY057998) is most related to zebrafish Cx43.4 at 69% similarity but also shows 58-59% similarity to chicken, human, mouse and dog Cx45. Characteristic features of connexins, such as four predicted transmembrane domains (red bars) and triplets of cysteine residues (yellow C), are evident.

 

To confirm maternal expression of the new connexins and re-assess temporal expression patterns, we analyzed total RNA isolated from defolliculated oocytes and embryos by RT-PCR (Fig. 5A,B) and northern blotting (Fig. 5C). As previously reported (Ebihara et al., 1989Go; Gimlich et al., 1988Go; Gimlich et al., 1990Go), zygotic expression of Cx30 (Fig. 5A) and maternal expression of Cx38 (Fig. 5A,C) were observed. Cx31 and Cx43.4 were expressed both maternally (see oocyte and two-cell stage lanes in Fig. 5A) and zygotically (see stage 15 and stage 30 lanes in Fig. 5A). Expression of Cx41 and Cx43 were examined using samples with a broader range of developmental stages (Fig. 5B), revealing Cx43 (Gimlich et al., 1990Go) expression in fertilized eggs and 64-cell stage embryos. Cx41 was not detected at those two developmental stages (Fig. 5B). This result confirmed earlier studies localizing Cx41 to ovarian somatic cells, but not oocytes (Bruzzone et al., 1995Go). We conclude that at least four maternal connexins, Cx31, Cx38, Cx43 and Cx43.4, were present in oocytes and early embryos.



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Fig. 5. Four connexins were maternally expressed in Xenopus embryos. (A) RT-PCR from ovary, oocytes and early embryos distinguished between maternally and zygotically expressed Xenopus connexins. Cx30 was solely zygotic and Cx38 solely maternal. Cx31 and Cx43.4 were both maternal and zygotic. Histone H4 was used as an internal control. (B) RT-PCR analysis revealed that Cx43 RNA, but not Cx41, was expressed maternally in fertilized eggs and 64-cell stage embryos. RT-minus controls were utilized for all cDNA samples in A and B, but only one example is shown in each panel. (C) Steady-state levels of Cx38 and Cx43.4 RNA are compared by northern blots. Fibronectin (FN) levels are provided as a loading control. In all three panels oocytes samples were obtained from defolliculated oocytes and ovary samples from pieces of ovary containing follicular oocytes and ovarian tissue.

 

Northern blotting was used to assess changes in levels of Cx38 and Cx43.4 during development (Fig. 5C). Cx38 transcript levels were high in the mature oocyte (stage 6 oocyte) but sharply declined after the initiation of zygotic transcription at embryonic stage 8.5. By contrast, the levels of Cx43.4 transcript increased from stage 8.5 onwards (Fig. 5C). Fibronectin (FN), which displays a characteristic pattern of accumulation after mid-blastula transition (MBT) (Krieg and Melton, 1985Go) was used as a loading control (Fig. 5C).

Xenopus Cx43.4 is a functional homolog of Cx45
Intercellular channels formed from mouse, zebrafish or chicken Cx45 are distinguished by their characteristic high sensitivity to transjunctional voltage; the highest of any connexin studied to date (Barrio et al., 1997Go). Thus, if Xenopus Cx43.4 is an amphibian ortholog of Cx45, it should display similar properties. To examine this, we transiently expressed it in N2A cells and performed dual whole-cell voltage clamping to characterize conductance-voltage relationships.

Junctional currents in N2A pairs expressing Cx43.4 displayed time-dependent inactivation in response to transjunctional potentials (Vj). In Fig. 6A, the junctional currents obtained in response to potentials of -100 to +100 mV are superimposed. Higher Vj produced instantaneous currents that decayed mono-exponentially to a steady-state level. This general behavior is characteristic of many connexins but the speed of inactivation and sensitivity to Vj is typical for Cx45. A quantitative analysis of macroscopic junctional currents is presented in Fig. 6B. The plot of the normalized conductance versus Vj closely matched a double Boltzmann distribution (N=7). Half-maximal inactivation of the conductance (V0) occurred at a transjunctional voltage of approximately 20 mV. Steady-state conductance (Gmin) at 100 mV, the highest transjunctional potential tested, was 5% of the maximal value. The Boltzmann parameters for Cx43.4 agree well with those published for Cx45 (V0=15 mV; Gmin=5%).



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Fig. 6. Properties of intercellular channels formed from Cx43.4 are similar to Cx45. N2A cells were transiently transfected with Xenopus Cx43.4 and analyzed by dual whole-cell voltage clamping. (A) Time-dependent inactivation of junctional currents in response to transjunctional potentials; 5 seconds long, 20 mV steps between -100 and +100 mV are shown. (B) Normalized steady-state conductance plotted against transjunctional voltage closely matches a double Boltzman distribution (N=7; V0=~20 mV; Gmin/Gmax=0.05). (C) Single channel recordings display 35 pS transitions.

 

A representative recording of single channel activity at a Vj of 80 mV is displayed in Fig. 6C where 35 pS transitions are clearly resolved (arrows). Gating to substates may also be evident (arrowheads) although poorly resolved. For Cx45, gating transitions between fully open and either sub-conductance states or fully closed states have been reported (Bukauskas et al., 2002Go). Thus, the single channel conductance and kinetics of Cx43.4 are at least consistent with those reported for Cx45. Together, our data indicate that Xenopus Cx43.4 resembles Cx45 in both structure and function.

Cx43.4 is localized to future dorsal structures in early zygotic embryos
Since Cx43.4 was expressed at high levels and forms active channels, we examined the distribution of the connexin using in situ hybridization. In the egg, maternal Cx43.4 mRNA was equally distributed along the animal-vegetal axis (data not shown), similar to Cx38 mRNA (see Fig. 1). However, accumulation of Cx43.4 in dorsal structures became evident early in neurulation (data not shown) and was easily detected from late neurulation (stage 19) onwards (Fig. 7). At stage 19, signal was detected in both rostral and caudal portions of the developing neural tube. At this stage, the anterior neural folds have not yet approximated to form the tube while in the caudal portions, the neural folds are already touching each other (Fig. 7A,B). The Cx43.4 signal was also detected in the eye vesicles as well as in the tail blastema (Fig. 7A-C). At stage 25, well after the neural tube fusion and formation of the neural central canal, high levels of Cx43.4 were noted along the fused neural tube (Fig. 7D) and in head structures as eyes and branchial arches (Fig. 7E,F). Cx43.4 signals in the head and tail continued to intensify at least until stage 33, which was the latest stage examined (Fig. 7G).



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Fig. 7. Spatial localization of Cx43.4 transcript in whole-mount neurula and tailbud stages. Whole-mount in situ hybridization revealed expression of Cx43.4 in dorsal structures extending from head to tail (A-G). By contrast, Cx30 was localized to ectoderm including the hatching gland and the endoderm (H-I). Embryos before clearing in benzyl benzoate/benzyl alcohol were photographed on a blue background. Cleared embryos are shown on yellow background. (A-C) The signal of Cx43.4 is strongly detected in the late neurula at stage 19. (A) Cx43.4 is in the neural folds and the eye vesicles, but not in the groove between the two neural folds (see white arrow). (B) The embryo from A was cleared to demonstrate localization of Cx43.4 in the posterior, already fused neural tube, as well in the anterior open tube, the brain and eyes. (C) A side view of the cleared embryo, anterior to the left, shows that Cx43.4 is restricted to dorsal neuroectoderm: head, spinal cord and tail. (D-F) Intensified accumulation of Cx43.4 in anterior structures of the tail bud embryo at stage 25. (D) One dark band of Cx43.4 expression in the anterior fused neural tube is marked with a white arrow. (E) A side view of two embryos (anterior to the left), demonstrate head, spinal cord and tail expression of Cx43.4 (F) A side view of a cleared embryo shows expansion of Cx43.4 expression in the head and brain structures as well in a branchial arch. Clear, but relatively lower expression is seen in the spinal cord and tail. (G) A side view of stage 33 tadpoles (anterior to the left) shows strong Cx43.4 expression in head and tail structures. As indicated earlier, expression is seen also in the spinal cord. (H) Whole mount staining shows Cx30 expression in the hatching glad and the anus. This superficial staining seems similar to the Cx43.4 staining seen in D. However, the side view of the cleared embryo in I (anterior to the left) shows that hatching glad staining is ectodermal staining and is different from the neural staining as seen for Cx43.4 (compare I to F and see the nueral staining in Fig. 8B). Another significant difference between the staining patterns of the two connexins is that, unlike Cx43.4, most of Cx30 expression is in the embryonic endoderm.

 



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Fig. 8. Spatial localization of Cx43.4 transcripts in sectioned wholemount tailbud embryos. (A) Transverse section through head reveals Cx43.4 in brain, eyes and head mesenchyme. (B) Similar section stained for N-CAM to confirm neural structures. (C,D) Posterior transverse sections show Cx43.4 and N-CAM in spinal cord. Bar (A), 200 µm.

 
Since Cx30 expression has been reported in early dorsal head structures (Levin and Mercola, 2000Go), we compared its distribution to that of Cx43.4 in stage 25 of embryonic development. Comparison of the superficial staining of the two connexins in the dorso-anterior regions showed a similar pattern (compare Fig. 7D to 7H). In order to reveal internal structures, embryos were cleared with benzyl benzoate and benzyl alcohol and photographed with a yellow background. A substantial difference in the distribution of Cx30 and Cx43.4 was then observed (compare Fig. 7F with I). Cx30 signal was restricted to the hatching gland, an ectodermal derivative, and to endoderm whereas Cx43.4 signal was neither ectodermal nor endodermal (confirmed by sections as described in Fig. 8). Finer details of Cx43.4 expression were provided in sections of the whole-mount stained embryos (Fig. 8). Here, signal was evident in brain, eye and head mesenchyme (Fig. 8A,B) as well as in spinal cord (Fig. 8C,D). N-CAM control staining was done as well to show neural tissue staining (Kintner and Melton, 1987Go).


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have shown that multiple connexins were present in Xenopus oocytes and blastomeres prior to the MBT and that more than one of them contributed to GJIC in the embryo. In addition to Cx38, at least three other connexin transcripts, Cx31, Cx43 and Cx43.4, were added to the maternal mRNA pool during oogenesis and persisted during early cleavage stages. The presence of these other connexins may explain why GJIC was still evident between blastomeres of Cx38-depleted embryos. It remains to be established which of the other maternal connexins were necessary for the persistence of GJIC following Cx38 depletion. Although all four connexins were maternal, they displayed different spatial and temporal patterns of expression. Cx38 RNA levels declined during the blastula stage and disappeared a few hours after the initiation of zygotic transcription. By contrast, Cx31, Cx43 and Cx43.4 increased during neurulation and tadpole stages, with Cx43.4 levels significantly more abundant than the others. Cx43.4 in the neurula was highly localized to future dorsal structures such as eyes, brain and spinal cord.

New maternal Xenopus connexins
In terms of primary sequence, both of the new maternal Xenopus connexins (Xenopus Cx31 and Cx43.4) are orthologous to well studied mammalian connexins (Cx31 and Cx45). When analyzed by dual voltage clamp in either pairs of oocytes injected with cRNA (data not shown) or transiently transfected N2A cells, Cx43.4 formed functional intercellular channels. Strikingly, the biophysical properties of Xenopus Cx43.4 were very similar to those reported for zebrafish, chicken, mouse and human Cx45, which are all relatively conserved (Barrio et al., 1997Go). Another characteristic feature of intercellular channels containing Cx45 is that they are not permeable to the fluorescent molecule Lucifer Yellow (LY) (Cao et al., 1998Go; Koval et al., 1995Go; Steinberg et al., 1994Go; Veenstra et al., 1994Go). Moreover, expression of Cx45 can inhibit other connexins from forming LY permeated gap junctions (Koval et al., 1995Go). Cx45-like permselectivity would provide an explanation for our ability to detect neurobiotin but not LY permeation of gap junctions in the early Xenopus embryo (Landesman et al., 2000Go).

Roles for Cx38
Our results indicate that Cx38 was neither essential for embryonic GJIC nor for normal embryonic development. Presumably, Cx38 formed active channels in the early embryo but this activity was supplied redundantly by other connexins. One puzzling finding was that, despite the presence of maternal mRNA encoding Cx31, Cx43 and Cx43.4, ablation of Cx38 alone was sufficient to eliminate GJIC from paired oocytes. It is possible that only Cx38 mRNA was actually translated prior to fertilization, whereas the other maternal connexins were subjected to translational regulation during oogenesis and early development (Richter and Smith, 1984Go; Seydoux, 1996Go; Taylor et al., 1985Go). A specific example is FGF receptor-1, which is stored in the immature oocyte as untranslated maternal mRNA and becomes translationally active only upon meiotic maturation (Robbie et al., 1995Go). Another possibility is that the activity of channels consisting of the other connexins was inhibited by post-translational modification as phosphorylation (Giepmans et al., 2001Go; Lin et al., 2001Go; Swenson et al., 1990Go). These notions are supported by two observations. First, antisense ablation of endogenous Cx38 mRNA eliminates all background conductance between oocyte pairs (Barrio et al., 1991Go). Second, voltage gating of intercellular channels in naive oocyte pairs and those injected with exogenous Cx38 cRNA was identical and thus was unlikely to involve a mixture of connexins (Ebihara et al., 1989Go; Werner et al., 1985Go) (R.L.G., unpublished).

We speculate that Cx38 has unique, non-redundant activities during oogenesis in adult animals. Cx38 RNA levels peak in oogenesis between stage I and stage VI (Gimlich et al., 1990Go) and thus Cx38 could be required for GJIC between each developing oocyte and its complement of follicular cells, a critical function in mammalian oogenesis (Simon et al., 1997Go). Since follicular cells do not express Cx38 (Bruzzone et al., 1995Go), oocyte-follicular GJIC would then probably involve heterotypic Cx38-Cx43 intercellular channels. In this regard it is interesting that homotypic channels solely comprising Cx38 are relatively inefficient whereas heterotypic channels, containing Cx38 and Cx43, show much higher levels of activity (Swenson et al., 1989Go). Additional studies are needed to define a role for Cx38 in oogenesis.

Multiple connexins in Xenopus and mouse embryos
Our data indicate that Cx38 does not strongly influence normal patterning of the Xenopus embryo. However, it is possible that subtle alterations in structure and/or function result from its deletion. Regardless, major effects on patterning may be averted by functional redundancy in connexin expression, leaving open possible influences of GJIC in development. A similar problem arises in studies of GJIC in mouse development, where at least six connexins accumulate in the embryo (Davies et al., 1996Go). It was proposed that since gap junctions containing Cx43 appear immediately prior to compaction, GJIC could be required for the correct timing of this event (De Sousa et al., 1993Go). A test of this hypothesis was attempted using Cx43-deficient embryos (Reaume et al., 1995Go) but GJIC is not eliminated in these embryos and compaction occurs normally (De Sousa et al., 1997Go). Thus, mice and frogs use multiple connexins to establish intercellular channels in early development, and functional tests of GJIC will require the targeted elimination of all expressed connexins in different combinations.

Cx43.4 in Xenopus embrogenesis
Cx43.4 is consistently present in the early Xenopus embryo. It is first deposited in the oocyte as a maternal message in oogenesis and later continues to accumulate in the embryo by zygotic transcription, after the mid-blastula transition (Fig. 5A,C). Levels of the transcripts can be detected, by the whole mount technique, starting from the neural development stages onwards (Fig. 7). Then, Cx43.4 distribution is highly restricted to early derivatives of the central nervous system: neural folds, brain and the eye vesicles (Fig. 7A-C). In stage 19, the neural plate has just segregated from dorsal ectoderm and neural folds initiate the folding process to convert the neural plate to form a closed neural tube (Nieuwkoop and Faber, 1967Go). The tube closes in a posterior to anterior direction. The accumulation of Cx43.4 along the neural tube before its fusion suggests roles for this connexin in the segregation of the neural plate from the ectoderm and in the formation of the neural tube. A compelling hypothesis is that Cx43.4 expression is required to induce a gap junctional communication among specified neural plate cells and isolate them from the rest of the ectoderm. Then, this intimate cell-cell communication is required among the neural plate cells during cellular re-shaping and tapering to form a closed neural tube. Cx43.4 role in embryonic development does not seem to be restricted to neural tube development. After tube fusion, which occurs at stage 21 (Nieuwkoop and Faber, 1967Go), Cx43.4 expression intensifies in the brain, head mesencyme and the branchial arches of the stage 25 embryos (Fig. 7F), suggesting involvement in brain development, head and its visceral skeleton formation as well as in tail development. The latter is suggested by the accumulation of Cx43.4 in the tip of the tails of stage 33 embryos (Fig. 7G).

The expression patterns of the zebrafish Cx43.4 ortholog to its Xenopus counterpart are different (Essner et al., 1996Go). Both connexins are expressed during oogenesis and their levels increase in gastrula and neurula stages. However, unlike Xenopus, zebrafish Cx43.4 levels decline during late somite stages, and transcription halts shortly after hatching. During early somite stages, zebrafish Cx43.4 is expressed in the notochord and paraxial mesoderm but not in the neural tube and brain as we observe in Xenopus. Zebrafish Cx43.4 is found in the anterior nervous system and eyes during late somite stages but not in the whole brain as we observe in Xenopus. One explanation for these differences could be that frogs and fish use GJIC in fundamentally different ways. Another explanation could be that the function of Cx43.4 in Xenopus is supplied by a different connexin in zebrafish. In this regard, teleosts often express two gene products in the place of one in other species, since large parts of the teleost genome have undergone duplication (Woods et al., 2000Go). For example, the sole neuron-specific mammalian connexin, Cx36, has two close orthologs in teleost, Cx35 and Cx34.7 (O'Brien et al., 1998Go).


    Acknowledgments
 
We thank Michael W. King for the fertilized egg cDNA library. This work was supported by NIH GM37751 to D.L.P. and GM18974 to D.A.G.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Barrio, L. C., Suchyna, T., Bargiello, T., Xu, L. X., Roginski, R. S., Bennett, M. V. L. and Nicholson, B. J. (1991). Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by voltage. Proc. Natl. Acad. Sci. USA 88,8410 -8414.[Abstract]

Barrio, L. C., Capel, J., Jarillo, J. A., Castro, C. and Revilla, A. (1997). Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes. Biophys. J. 73,757 -769.[Abstract]

Bruzzone, R., White, T. W., Yoshizaki, G., Patino, R. and Paul, D. L. (1995). Intercellular channels in teleosts: functional characterization of two connexins from Atlantic croaker. FEBS Lett. 358,301 -304.[CrossRef][Medline]

Bukauskas, F. F., Angele, A. B., Verselis, V. K. and Bennett, M. V. (2002). Coupling asymmetry of heterotypic connexin 45/ connexin 43-EGFP gap junctions: Properties of fast and slow gating mechanisms. Proc. Natl. Acad. Sci. USA 99,7113 -7118.[Abstract/Free Full Text]

Cao, F. L., Eckert, R., Elfgang, C., Nitsche, J. M., Snyder, S. A., Hülser, D. F., Willecke, K. and Nicholson, B. J. (1998). A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J. Cell Sci. 111, 31-43.[Abstract/Free Full Text]

Davies, T. C., Barr, K. J., Jones, D. H., Zhu, D. and Kidder, G. M. (1996). Multiple members of the connexin gene family participate in preimplantation development of the mouse. Dev. Genet. 18,234 -243.[CrossRef][Medline]

De Sousa, P. A., Juneja, S. C., Caveney, S., Houghton, D. F., Davies, T. C., Reaume, A. G., Rossant, J. and Kidder, G. M. (1997). Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J. Cell Sci. 110,1751 -1758.[Abstract/Free Full Text]

De Sousa, P. A., Valdimarsson, G., Nicholson, B. J. and Kidder, G. M. (1993). Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development 117,1355 -1367.[Abstract/Free Full Text]

Ebihara, L., Beyer, E. C., Swenson, K. I., Paul, D. L. and Goodenough, D. A. (1989). Cloning and expression of a Xenopus embryonic gap junction protein. Science 243,1194 -1195.[Medline]

Essner, J. J., Laing, J. G., Beyer, E. C., Johnson, R. G. and Hackett, P. B. (1996). Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos. Dev. Biol. 177,449 -462.[CrossRef][Medline]

Giepmans, B. N., Verlaan, I. and Moolenaar, W. H. (2001). Connexin-43 interactions with ZO-1 and alpha- and beta-tubulin. Cell Adhes. Commun. 8, 219-223.

Gimlich, R. L., Kumar, N. M. and Gilula, N. B. (1988). Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus. J. Cell Biol. 107,1065 -1073.[Abstract]

Gimlich, R. L., Kumar, N. M. and Gilula, N. B. (1990). Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J. Cell Biol. 110,597 -605.[Abstract]

Guger, K. A. and Gumbiner, B. M. (1995). ß-catenin has wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning. Dev. Biol. 172,115 -125.[CrossRef][Medline]

Guthrie, S. (1984). Patterns of junctional communication in the early amphibian embryo. Nature 311,149 -151.[Medline]

Guthrie, S. C., Turin, L. and Warner, A. E. (1988). Patterns of junctional communication during development of the early amphibian embryo. Development 103,769 -783.[Abstract]

Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]

Heasman, J., Holwill, S. and Wylie, C. C. (1991). Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules. In Xenopus laevis: Practical Uses in Cell and Molecular Biology, vol.36 (eds B. K., Kay and H. B. Peng), pp.213 -230. New York: Academic Press, Inc.

Kintner, C. R. and Melton, D. A. (1987). Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction. Development 99,311 -325.[Abstract]

Koval, M., Geist, S. T., Westphale, E. M., Kemendy, A. E., Civitelli, R., Beyer, E. C. and Steinberg, T. H. (1995). Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43. J. Cell Biol. 130,987 -995.[Abstract]

Krieg, P. A. and Melton, D. A. (1985). Developmental regulation of a gastrula-specific gene injected into fertilized Xenopus eggs. EMBO J. 4,3463 -3471.[Abstract]

Kumar, N. and Gilula, N. B. (1996). The gap junction communication channel. Cell 84,381 -388.[Medline]

Landesman, Y., White, T. W., Starich, T. A., Shaw, J. E., Goodenough, D. A. and Paul, D. L. (1999). Innexin-3 forms connexin-like intercellular channels. J. Cell Sci. 112,2391 -2396.[Abstract/Free Full Text]

Landesman, Y., Goodenough, D. A. and Paul, D. L. (2000). Gap junctional communication in the early Xenopus embryo. J. Cell Biol. 150,929 -936.[Abstract/Free Full Text]

Levin, M. and Mercola, M. (1998). Gap junctions are involved in the early generation of left-right asymmetry. Dev. Biol. 203,90 -105.[CrossRef][Medline]

Levin, M. and Mercola, M. (2000). Expression of connexin 30 in Xenopus embryos and its involvement in hatching gland function. Dev. Dyn. 219,96 -101.[Medline]

Lin, R., Warn-Cramer, B. J., Kurata, W. E. and Lau, A. F. (2001). v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication. J. Cell Biol. 154,815 -828.[Abstract/Free Full Text]

Nagajski, D. J., Guthrie, S. C., Ford, C. C. and Warner, A. E. (1989). The correlation between patterns of dye transfer through gap junctions and future developmental fate in Xenopus: the consequences of u.v. irradiation and lithium treatment. Development 105,747 -752.[Abstract]

Newport, J. and Kirschner, M. (1982). A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30,675 -686.[Medline]

Niehrs, C., Steinbeisser, H. and de Robertis, E. M. (1994). Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid. Science 263,817 -820.[Medline]

Nieuwkoop, P. and Faber, J. (1967). 2nd ed. Amsterdam: North-Holland Publishing Co.

O'Brien, J., Bruzzone, R., White, T. W., Al-Ubaidi, M. R. and Ripps, H. (1998). Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. J. Neurosci. 18,7625 -7637.[Abstract/Free Full Text]

Olson, D. J., Christian, J. L. and Moon, R. T. (1991). Effect of wnt-1 and related proteins on gap junctional communication in Xenopus embryos. Science 252,1173 -1176.[Medline]

Olson, D. J. and Moon, R. T. (1992). Distinct effects of ectopic expression of Wnt-1, Activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos. Dev. Biol. 151,204 -212.[Medline]

Reaume, A. G., de Sousa, P. A., Kulkarni, S., Langille, B. L., Zhu, D., Davies, T. C., Juneja, S. C., Kidder, G. M. and Rossant, J. (1995). Cardiac malformation in neonatal mice lacking connexin43. Science 267,1831 -1834.[Medline]

Richter, J. D. and Smith, L. D. (1984). Reversible inhibition of translation by Xenopus oocyte-specific proteins. Nature 309,378 -380.[Medline]

Robbie, E. P., Peterson, M., Amaya, E. and Musci, T. J. (1995). Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3' untranslated region. Development 121,1775 -1785.[Abstract/Free Full Text]

Seydoux, G. (1996). Mechanisms of translational control in early development. Curr. Opin. Genet. Dev. 6, 555-561.[CrossRef][Medline]

Simon, A. M., Goodenough, D. A., Li, E. and Paul, D. L. (1997). Female infertility in mice lacking connexin 37. Nature 385,525 -529.[CrossRef][Medline]

Spray, D. C., Harris, A. L. and Bennett, M. V. L. (1981). Equilibrium properties of a voltage-dependent junctional conductance. J. Gen. Physiol. 77, 75-94.

Srinivas, M., Rozental, R., Kojima, T., Dermietzel, R., Mehler, M., Condorelli, D. F., Kessler, J. A. and Spray, D. C. (1999). Functional properties of channels formed by the neuronal gap junction protein connexin36. J. Neurosci. 19,9848 -9855.[Abstract/Free Full Text]

Steinberg, T. H., Civitelli, R., Geist, S. T., Robertson, A. J., Hick, E., Veenstra, R. D., Wang, H. Z., Warlow, P. M., Westphale, E. M., Laing. J. G. et al. (1994). Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J. 13,744 -750.[Abstract]

Swenson, K. I., Jordan, J. R., Beyer, E. C. and Paul, D. L. (1989). Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell 57,145 -155.[Medline]

Swenson, K. I., Piwnica-Worms, H., McNamee, H. and Paul, D. L. (1990). Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul. 1,989 -1002.[Medline]

Taylor, M. A., Robinson, K. R. and Smith, L. D. (1985). Intracellular pH and ribosomal protein S6 phosphorylation: role in translational control in Xenopus oocytes. J. Embryol. Exp. Morphol. 89Suppl., 35-51.[Medline]

Veenstra, R. D., Wang, H. Z., Beyer, E. C. and Brink, P. R. (1994). Selective dye and ionic permeability of gap junction channels formed by connexin45. Circ. Res. 75,483 -490.[Abstract]

Warner, A. E., Guthrie, S. C. and Gilula, N. B. (1984). Antibodies to gap-junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311,127 -131.[Medline]

Weeks, D. L. and Melton, D. A. (1987). A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-ß. Cell 51,861 -867.[Medline]

Werner, R., Miller, T., Azarnia, R. and Dahl, G. (1985). Translation and functional expression of cell-cell channel mRNA in Xenopus oocytes. J. Membr. Biol. 87,253 -268.[Medline]

White, T. W. and Bruzzone, R. (2000). Intercellular communication in the eye: clarifying the need for connexin diversity. Brain Res. Rev. 32,130 -137.[Medline]

White, T. W., Bruzzone, R., Wolfram, S., Paul, D. L. and Goodenough, D. A. (1994). Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J. Cell Biol. 125,879 -892.[Abstract]

White, T. W. and Paul, D. L. (1999). Genetic diseases and gene knockouts reveal diverse connexin functions. Annu. Rev. Physiol. 61,283 -310.[CrossRef][Medline]

Woods, I. G., Kelly, P. D., Chu, F., Ngo-Hazelett, P., Yan, Y. L., Huang, H., Postlethwait, J. H. and Talbot, W. S. (2000). A comparative map of the zebrafish genome. Genome Res. 10,1903 -1914.[Abstract/Free Full Text]