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
Author for correspondence (e-mail:
dpaul{at}hms.harvard.edu)
Accepted 19 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Gap junctions, Dye transfer, Xenopus connexins, Cx30, Cx31, Cx38, Cx41, Cx43, Cx43.4
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four Xenopus connexins have been identified to date. Cx43 and Cx30
transcripts appear at gastrula and tailbud stages, respectively
(Gimlich et al., 1988;
Gimlich et al., 1990
). Cx41 is
not detected until adulthood, where it is restricted to ovarian follicular
cells (Bruzzone et al., 1995
).
Cx38 is present in oocytes and eggs and persists in early embryos until
neurulation (Ebihara et al.,
1989
; Gimlich et al.,
1990
). Since zygotic transcription in Xenopus does not
initiate until midblastula stage (Newport
and Kirschner, 1982
), 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,
1995
; Guthrie et al.,
1988
; Guthrie,
1984
; Levin and Mercola,
1998
; Nagajski et al.,
1989
; Olson et al.,
1991
; Olson and Moon,
1992
; Warner et al.,
1984
). In addition, the number of communicating cells in the
Xenopus embryo increases rapidly during early cleavage stages
(Landesman et al., 2000
). 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., 1991). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.,
1991). 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., 2000
).
RT-PCR oligonucleotides
All sequences are listed in 5' to 3' orientation:
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., 1999).
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., 1999).
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.,
1989), 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,
1991
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.,
1989). Junctional conductance was measured 48 hours after pairing
using dualcell voltage clamp (Spray et
al., 1981
). 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.
|
Cx38 is not essential for normal Xenopus development or for
GJIC in the early embryo
We used the host-transfer technique
(Heasman et al., 1991) 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.
|
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.,
2000). 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).
|
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., 1989;
Gimlich et al., 1988
;
Gimlich et al., 1990
), 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., 1990
)
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., 1995
). We
conclude that at least four maternal connexins, Cx31, Cx38, Cx43 and Cx43.4,
were present in oocytes and early embryos.
|
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, 1985) 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., 1997). 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%).
|
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., 2002). 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).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1997). Another characteristic feature of intercellular
channels containing Cx45 is that they are not permeable to the fluorescent
molecule Lucifer Yellow (LY) (Cao et al.,
1998
; Koval et al.,
1995
; Steinberg et al.,
1994
; Veenstra et al.,
1994
). Moreover, expression of Cx45 can inhibit other connexins
from forming LY permeated gap junctions
(Koval et al., 1995
).
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.,
2000
).
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,
1984; Seydoux,
1996
; Taylor et al.,
1985
). 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., 1995
). Another
possibility is that the activity of channels consisting of the other connexins
was inhibited by post-translational modification as phosphorylation
(Giepmans et al., 2001
;
Lin et al., 2001
;
Swenson et al., 1990
). These
notions are supported by two observations. First, antisense ablation of
endogenous Cx38 mRNA eliminates all background conductance between oocyte
pairs (Barrio et al., 1991
).
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., 1989
; Werner et al.,
1985
) (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.,
1990) 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.,
1997
). Since follicular cells do not express Cx38
(Bruzzone et al., 1995
),
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., 1989
).
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., 1996). 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., 1993
).
A test of this hypothesis was attempted using Cx43-deficient embryos
(Reaume et al., 1995
) but GJIC
is not eliminated in these embryos and compaction occurs normally
(De Sousa et al., 1997
). 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,
1967). 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,
1967
), 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., 1996). 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., 2000
). For
example, the sole neuron-specific mammalian connexin, Cx36, has two
close orthologs in teleost, Cx35 and Cx34.7
(O'Brien et al., 1998
).
![]() |
Acknowledgments |
---|
![]() |
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.
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.
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.
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.
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.
Landesman, Y., Goodenough, D. A. and Paul, D. L.
(2000). Gap junctional communication in the early
Xenopus embryo. J. Cell Biol.
150,929
-936.
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.
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.
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.
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.
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.