* Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Developmental Biology Program,
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912-2640; and § Department of
Cell Biology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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Previous studies showed that conotruncal
heart malformations can arise with the increase or decrease in 1 connexin function in neural crest cells. To
elucidate the possible basis for the quantitative requirement for
1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from
CMV43 transgenic embryos overexpressing
1 connexins, and from
1 connexin knockout (KO) mice and FC
transgenic mice expressing a dominant-negative
1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the
CMV43 embryos, but decreased in the FC transgenic
and
1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage
in the homozygous vs. hemizygous
1 connexin KO and
CMV43 embryos, respectively. Dye coupling analysis in
neural crest cells in the outgrowth cultures and also in
the living embryos showed an elevation of gap junction
communication in the CMV43 transgenic mice, while a
reduction was observed in the FC transgenic and
1
connexin KO mice. Further analysis using oleamide to
downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent
method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of
crest migration. To determine the possible relevance of
these findings to neural crest migration in vivo, a lacZ
transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies
showed more lacZ-positive cells in the outflow septum
in the CMV43 transgenic mice, while a reduction was
observed in the
1 connexin KO mice. Surprisingly, this
was accompanied by cell proliferation changes, not in
the cardiac neural crest cells, but in the myocardium
an elevation in the CMV43 mice vs. a reduction in the
1 connexin KO mice. The latter observation suggests
that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest-
derived tissues. Overall, these findings suggest that gap junction communication mediated by
1 connexins
plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural
crest perturbation is the likely underlying cause for
heart defects in mice with the gain or loss of
1 connexin function.
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Introduction |
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GAP junctions provide membrane channels, which allow the passage of ions and small molecules between cells. These channels are made up of a hexameric array of proteins encoded by a multigene family
known as the connexins (Willecke et al., 1991; Kumar and Gilula, 1992
). Most cells or tissues express multiple connexin genes and show functional coupling via gap junctions (Bennett et al., 1991
; Bruzzone et al., 1996
). Given
the prevalence of gap junctions, it is likely that cell-cell
communication via gap junctions mediates cell signaling
involved in the maintenance of tissue homeostasis and in
regulating growth and proliferation (Loewenstein, 1979
).
Because gap junctions are found from very early stages of
embryogenesis, it has been suggested that they mediate
cell-cell signaling events involved in patterning, differentiation, and development (Guthrie and Gilula, 1989
; Warner,
1992
; Lo, 1996
).
The 1 connexin gap junction gene (also referred to as
Cx43) is expressed in a developmentally regulated manner
in many different regions of the mouse embryo (Ruangvoravat and Lo, 1992; Yancey et al., 1992
). Studies of
the
1 connexin knockout (KO)1 mice have shown a role
for this connexin protein in heart morphogenesis, since
mice deficient in
1 connexin gap junctions die soon after birth because of conotruncal heart malformations and pulmonary outflow obstruction (Reaume et al., 1995
). However, since very little
1 connexin is normally found in this
region of the developing heart (Gourdie et al., 1992
), the
phenotype of the KO mice has not been very informative
as to the role of
1 connexin gap junctions in heart morphogenesis.
Some insights into this question have since come from
the analysis of transgenic mice (referred to as CMV43) in
which 1 connexin is overexpressed in subpopulations of
neural crest cells and their progenitors in the dorsal neural
tube. These transgenic animals also exhibited conotruncal
heart malformations and outflow tract obstructions (Ewart
et al., 1997
; Huang et al., 1998
). Together, the findings
from the CMV43 and
1 connexin KO mice would suggest that the precise level of
1 connexin function is of critical importance in conotruncal heart development, and that
this may involve the modulation of cardiac neural crest activity. Further evidence consistent with this possibility has
come from recent studies of transgenic mice (FC) expressing a dominant-negative
1 connexin fusion protein (Sullivan and Lo, 1995
; Sullivan et al., 1998
). Such mice also
exhibited conotruncal heart malformations and right ventricular outflow tract obstructions. Of importance to note
is the fact that expression of the fusion protein was localized to similar regions of the embryo, that is populations of
neural crest cells and their progenitors in the dorsal neural tube (Sullivan et al., 1998
).
Neural crest cells are migratory cells that originate from
the dorsolateral margins of the neural tube. Along with
participating in heart outflow tract morphogenesis, neural
crest cells contribute to the development of a diverse array
of tissues in the embryo, such as the peripheral nervous
system, the thymus, and the craniofacial mesenchyme.
Neural crest cells express an abundance of 1 connexins and are functionally well coupled via gap junctions (Lo et al., 1997
). Although the involvement of gap junctions in the
development of this migratory cell population is unexpected, neural crest cells have been observed to migrate in
groups or sheets (Bancroft and Bellairs, 1976
; Davis and
Trinkaus, 1981
). Moreover, gap junctions are dynamic
structures that turn over rapidly (Bruzzone et al., 1996
).
The importance of direct cell-cell interactions in neural crest cell populations has also been intimated by a number
of previous studies (Kapur et al., 1995
; Kimura et al., 1995
;
Nakagawa and Takeichi, 1995
; Raible and Eisen, 1996
).
However, the role of gap junctions in cardiac neural crest
cells, the subpopulation of crest cells that migrates to the
heart and participates in outflow tract remodeling, remains unknown. Gap junctions are not likely involved in
the maintenance of neural crest cell viability, since the
cardiac phenotype of the
1 connexin KO mice, and the CMV43 and FC transgenic mice do not resemble the classic cardiac crest ablation phenotype (Kirby, 1993
; Kirby
and Waldo, 1995
).
In this study, we have undertaken an analysis of the migration and proliferation of neural crest cells with the gain
or loss of 1 connexin function. This has included an examination of cardiac crest cells from the CMV43 transgenic
mice exhibiting overexpression of
1 connexins, and crest
cells from the
1 connexin KO and the FC transgenic mice
with the deletion or inhibition of
1 connexin function, respectively. Using a neural tube explant culture system, we
showed that changes in the level of gap junctional communication were associated in parallel with changes in the
rate of neural crest migration. Thus, an increase in
1 connexin function increased the rate of crest migration, while
the dominant or recessive loss of
1 connexin function
slowed neural crest migration. In contrast, neural crest cell
proliferation was not affected. These results indicate a role
for gap junction communication in the modulation of neural crest cell migration. The in vivo analysis of neural crest
cells using a lacZ reporter transgene suggested that there were changes in neural crest abundance in the outflow
tract. In the CMV43 transgenic mice, lacZ-positive cells
were increased, while in the KO mice they were decreased. This was accompanied by changes in cell proliferation, not in neural crest cells, but in the ventricular myocardium. Myocyte cell proliferation was increased in the
CMV43 mice, while it was decreased in the KO mice.
These observations in conjunction with previous studies confirm the involvement of cardiac neural crest perturbation in the conotruncal heart defects seen with the perturbation of
1 connexin function. They suggest that gap junction communication plays an important role in modulating
cardiac neural crest migration, and through this process,
affects events in cardiac development.
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Materials and Method |
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Mouse Breeding and Genotyping
To examine the role of 1 connexin gap junctions in crest migration and
proliferation, we used three different mouse lines and the appropriate
breeding schemes to obtain embryos with different
1 connexin gene or
transgene dosages. For CMV43, a transgenic mouse line in which Cx43 is
overexpressed in neural crest cells, both hemizygous and homozygous
embryos could be obtained since these transgenic mice are homozygous
viable (Ewart et al., 1997
). Homozygous embryos were obtained by intercrossing the homozygous CMV43 mice, while hemizygous CMV43 embryos were obtained by breeding hemizygous CMV43 males with CD1 females. From the latter cross, both transgenic and control nontransgenic
embryos were obtained. It should be noted that the CMV43 line has been
outcrossed into CD1 mice and are maintained in a CD1 background (see
Ewart et al., 1997
). For the FC transgenic mice expressing a dominant-negative
1 connexin/lacZ fusion protein (Sullivan and Lo, 1995
; Sullivan et al., 1998
), breeding of hemizygous FC males with nontransgenic CD1
females provided hemizygous transgenic and wild-type nontransgenic control embryos. Because homozygous FC mice are inviable (Sullivan et al.,
1998
), it was not possible to obtain homozygous embryos for analysis. It
should be noted that hemizygous FC intercrosses were not used to obtain
homozygous FC embryos, given that PCR analysis cannot distinguish between the homozygous vs. hemizygous genotypes. For the
1 connexin KO
mice, interbreeding of the heterozygous KO mice provided wild-type, heterozygous, and homozygous
1 connexin KO mouse embryos.
The genotypes of the mice and embryos were determined by PCR analysis of tail or yolk sac DNA, respectively. For the FC transgenic mice,
PCR analysis was carried out using primers that detected the lacZ portion
of the 1 connexin/lacZ fusion protein construct (Echelard et al., 1994
).
For the CMV43 transgenic mice, PCR analysis was carried out using primers to the 3' untranslated sequence tag included in the construct (Ewart
et al., 1997
). For the
1 connexin KO mice, PCR analysis using primers to
the wild-type
1 connexin gene and neo insert in the
1 connexin knockout
allele (Reaume et al., 1995
) allowed the identification of wild-type, heterozygous, or homozygous
1 connexin KO embryos. In addition to these
three mouse lines exhibiting the perturbation of
1 connexin function, we
also used a transgenic mouse line expressing a lacZ reporter gene driven
by the
1 connexin promoter (see Lo et al., 1997
). This transgene was bred
into the CMV43 and
1 connexin KO mice to facilitate the identification
of presumptive crest cells in the CMV43 and
1 connexin KO mouse heart.
Neural Crest Outgrowth Cultures
To obtain embryos for neural crest outgrowth studies, mice were mated,
and the day the vaginal plug was found was considered E0.5. Embryos
from the transgenic or KO mouse lines were collected on E8.5, and the
yolk sac from each embryo was harvested for genotyping by PCR analysis.
The method used to establish the neural crest outgrowth cultures were
adapted from those described by Murphy et al. (1991) and Moiseiwitsch
and Lauder (1995). Each embryo was treated with 0.5 mg/ml of collagenase/dispase (Sigma Chemical Co., St. Louis, MO) in PBS for 10-15 min
and then bisected longitudinally to expose the hindbrain neural folds.
Each half was further transected to provide two fragments spanning the
pre- and postotic regions of the hindbrain neural folds. The dorsal ridge of
the neuroepithelium was surgically removed from the surrounding tissues
and cultured in a 35-mm, fibronectin-coated Petri dish containing DME
with high glucose and 10% fetal bovine serum. The cultures were maintained for 24-48 h at 37°C with 5% CO2. Explant cultures were generated from both the preotic and postotic hindbrain neural folds. Because these
two populations of neural crest cells exhibited the same migration and
proliferation rates in all three mouse models, the data were subsequently
pooled. Lineage studies in mouse embryos have confirmed that neural
crest cells from the postotic hindbrain neural fold give rise to the cardiac
crest cells as has been found in chick embryos (Fukiishi and Morriss-Kay,
1992
; for review see Kirby, 1993
).
Quantitation of Neural Crest Migration and Proliferation
Digital images of the outgrowth cultures at 24-48 h were acquired by video microscopy. Then NIH Image software was used to measure the area of the outgrowth. This was obtained by taking the area of the entire explant and subtracting from it the area of the dense central neural tube mass. Since the neural tube fragment is attached to the substratum predominantly via its edges where neural crest cells are emerging, we controlled for differences in migration area resulting from random variations in the shape of the explant by dividing the outgrowth area (mm2) by the perimeter (mm) of the explant. This normalized number (in mm) is referred to as the migration index and provides an estimate of the migration rate of the neural crest cells. For monitoring cell proliferation, 48-h explant cultures were incubated with bromodeoxyuridine (BrdU) (10 µM/ml) for 2 h, fixed, and processed for immunodetection using an anti-BrdU antibody and a streptavidin peroxidase detection system (Calbiochem, San Diego, CA) used in conjunction with the True BlueTM peroxidase substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD). Labeled cells were counted with the aid of NIH Image. To determine the total cell number in the outgrowth after BrdU immunostaining, the cultures were further stained with hematoxylin. This permitted a total cell count to be obtained using NIH Image. The proliferation rate was then calculated as the percent of BrdU-labeled cells in the outgrowth.
Dye Coupling In Vitro and In Vivo
To quantitate dye coupling in the neural crest outgrowths, the DME medium was replaced with phosphate-buffered L-15 medium containing 10% FBS, and the culture dish was placed on a heated microscope stage maintained at 37°C. Individual cells in the outgrowth cultures (24-h cultures) were impaled with microelectrodes filled with 5% carboxyfluorescein. This is followed by iontophoretic injection for 2 min using hyperpolarizing current pulses of 0.5 s duration at a frequency of once per second. The number of dye-filled cells found at the termination of dye injection was recorded. This count included the impaled cell. To examine dye coupling in vivo, E8.5 embryos were harvested and incubated in DME with 10% fetal bovine serum for up to 4 h. Just before microelectrode impalement for the analysis of dye coupling, the embryo was opened along the hindbrain neural fold and immobilized in a thin bed of 0.8% agarose, to which L15 medium was added. For these studies, microelectrode impalement and dye injection into the dorsolateral region of the postotic hindbrain were carried out, and the number of dye-filled cells was recorded as described above.
LacZ Expression and Immunohistochemical Analysis of Mouse Embryos
For detection of -galactosidase activity, embryos were fixed in 2%
paraformaldehyde for 30-45 min and then washed for 30 min twice in
PBS. X-gal staining was carried out overnight at room temperature.
Stained embryos were postfixed in 3.7% formaldehyde and then examined and photographed as whole-mount specimens. Some samples were
further processed for paraffin embedding and sectioning for histological
analysis. The tissue sections were either stained with hematoxylin/eosin or
further processed for immunohistochemistry with cardiac myosin heavy
chain, neurofilament, or proliferating cell nuclear antigen (PCNA) antibodies. For immunostaining, slides with 7-µm sections were treated for 30 min in 3% hydrogen peroxide in methanol and washed for 10 min in tap
water. Cardiac myosin antibody was used at a dilution of 1:1,000 (Accurate Antibodies, San Diego, CA), and PCNA antibody was used undiluted
(Zymed Laboratories, San Francisco, CA). For staining with cardiac myosin antibody, sections were treated for 15 min in 10 µg/ml of proteinase K
and washed briefly in water, then 5 min in 0.1 M Tris, pH 7.4, followed by
5 min in Tris with 2% fetal bovine serum. Immunodetection was carried
out using mouse ABC Elite Kit (Vector Laboratories, Burlingame, CA),
with the signal visualized with the VIP substrate (Vector Laboratories).
Slides were then dehydrated, cleared in xylene, and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ).
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Results |
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Modulation of Neural Crest Migration
Neural tube explant cultures were established from the
hindbrain neural folds of E8.5 embryos, spanning the region from which cardiac crest cells are derived. These explants were cultured for a period of 48 h to monitor neural
crest outgrowths. After 24 h, a ring of neural crest cells
was observed around the neural tube explant, and by 48 h,
this ring of cells was enlarged (Fig. 1). To obtain an estimate of the rate of neural crest migration during this culture period, a migration index was determined by measuring the area of the neural crest outgrowth (see Materials and Methods). In the CMV43 transgenic embryos, the migration index was elevated in a transgene dosage-dependent manner. The increase was greatest in the homozygous embryos, while neural crest cells in the hemizygous
embryos exhibited a migration index intermediate to that
of the homozygous and nontransgenic embryos (Table I). These migration changes can be detected at 24 and 48 h of
culture. In contrast to these results, with either the recessive or dominant loss of 1 connexin function, crest migration was inhibited. In the
1 connexin KO mice, the inhibition of migration occurred in step with
1 connexin gene
dosage. Thus, the migration index of the heterozygous KO
embryos was intermediate to that of the nontransgenic
and homozygous KO embryos. Similarly, crest cells from
the hemizygous FC transgenic mice exhibited a reduced
migration index. Note that the migration differences seen
in the nontransgenic outgrowths obtained from the different transgenic and KO crosses are highly reproducible and
reflect strain background effects on crest migration (Table
I). Overall, these results suggest that changes in the apparent rate of neural crest migration are correlated with
changes in
1 connexin gene or transgene dosage. They
suggest that
1 connexin gap junctions play a role in modulating neural crest migration.
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Neural Crest Cell Proliferation
In parallel to the analysis of neural crest migration, cell
proliferation in the outgrowth was also monitored. For this
analysis, BrdU incorporation was examined by immunostaining with a BrdU antibody (Fig. 2 A), after which the
cultures were stained with hematoxylin for the determination of total cell numbers in the crest outgrowth (Fig. 2 B).
From this analysis, the percentage of BrdU-labeled cells
was determined. In contrast to the results of the migration
study, we found that all of the transgenic/KO lines exhibited the same level of BrdU incorporation, including the
homozygous KO and homozygous CMV43 transgenic embryos. These results indicate that changes in the level of 1
connexin function do not affect neural crest cell proliferation.
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Dye Coupling Level Changes in Step
with 1 Connexin Transgene/Gene Dosage
The correlation of 1 connexin gene or transgene dosage
with the rate of neural crest cell migration suggests that
gap junction communication may have a direct role in the
modulation of crest migration. To examine this possibility,
microelectrode impalements and dye injections were carried out to monitor the level of dye coupling in the neural
crest outgrowths. These studies showed that dye coupling
was elevated in the CMV43 neural crest cells as compared
with neural crest cells derived from the nontransgenic embryos (Table II). In the homozygous CMV43 embryos,
coupling was increased to a higher level than in the hemizygous CMV43 embryos, thus indicating that coupling levels changed in step with CMV43 transgene dosage (Table
II). Analysis of the CMV43 neural crest outgrowths by
immunostaining with an
1 connexin antibody showed a
marked increase in the abundance of
1 connexin gap
junction plaques in regions of cell-cell contact (Fig. 3). In
contrast to these results, analysis of outgrowth cultures
from the FC and
1 connexin KO mice showed reductions
in dye coupling (Table II). In neural crest cells derived
from the KO mice, those that were homozygous null mutants showed a lower level of dye coupling than neural
crest cells derived from the heterozygous KO embryos
(Table II).
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To determine whether the dye coupling changes observed in vitro are indicative of the level of dye coupling in
vivo, we carried out microelectrode impalements and dye
injections directly into presumptive neural crest cells situated dorsolateral to the dorsal neural fold. We had previously shown with such in vivo dye coupling analysis, an
elevation of gap junction communication in presumptive crest cells of hemizygous CMV43 embryos (Ewart et al.,
1997). Here, we further examined and compared dye coupling levels in presumptive neural crest cells of both
hemizygous and homozygous CMV43 transgenic embryos.
These studies showed that dye coupling was elevated in
step with transgene dosage, with the highest level of dye
coupling exhibited in the homozygous embryos (Table II). Similar analysis of the FC and
1 connexin KO mice
showed decreases in coupling, and this occurred in step
with
1 connexin gene dosage in the KO embryos.
Oleamide Inhibits Gap Junctional Communication and also Crest Migration
The above results show that gap junction communication
modulates neural crest migration, with enhanced migration elicited by increased gap junction communication,
while decreased migration resulted from reductions in gap
junction communication. To further examine whether gap
junctions have a direct role in modulating neural crest migration, we examined the effects of oleamide, a compound
previously shown to be a potent inhibitor of gap junctional communication (Guan et al., 1997; Boger et al., 1998
). For
these studies, neural crest outgrowth cultures from nontransgenic embryos were plated in the presence of 50 µM
oleamide. At 24 h, the migration index was recorded, and
dye coupling was examined. As expected, gap junction
communication was decreased by oleamide treatment. More importantly, this was correlated with an inhibition in
neural crest migration (Table III). To control for the specificity of the effects seen with oleamide, we further analyzed the effects of treatment with trans-oleamide (trans-9-octadecenamide), a chemical analogue of oleamide
previously shown not to have any effects on gap junctional
communication. Treatment with trans-oleamide showed
no effects on coupling and no effects on migration. We also examined the effects of treatment with trifluoromethylketone, a compound that is known to block the fatty acid
amide hydrolase, which inactivates oleamide (Patterson
et al., 1996
). In contrast to the results obtained with trans-oleamide, treatment with trifluoromethylketone inhibited
coupling and crest migration (Table III). A parallel analysis of BrdU incorporation indicated that none of these
compounds had any effects on the rate of neural crest cell
proliferation. These results are in agreement with those
obtained above with the analysis of the transgenic and KO
mice and further indicate that gap junction communication has a direct role in the modulation of neural crest migration.
|
Analysis of Neural Crest Cells in the Outflow Tract
Although the above studies showed that gap junction communication levels in presumptive neural crest cells in the
embryo are similar to that of neural crest cells in the outgrowths, an important question is whether the migration
differences detected in vitro are indicative of changes in
the migratory behavior of neural crest cells in vivo. To examine this question, we bred into the CMV43 and 1 connexin KO mice, a lacZ reporter transgene previously shown to label neural crest cells (Lo et al., 1997
). Using
this reporter gene, it was possible to examine the distribution of cardiac neural crest cells in the heart outflow tract
by simply X-gal staining the embryos.
Such an analysis of E14.5 hearts from the CMV43 and 1
connexin KO mice revealed opposite changes in the abundance of lacZ-positive cells in the outflow tract. In comparison to the distribution seen in wild-type hearts (Fig. 4,
A and E), the homozygous CMV43 transgenic embryo exhibited an increase in the abundance of lacZ-expressing cells in the outflow tract (Fig. 4, B and F). In contrast, in the
1 connexin KO hearts, the abundance of lacZ-expressing cells was decreased (Fig. 4, C and G). This decrease
varied with the apparent severity of the heart phenotype,
such that only a very low level of X-gal staining was observed in the outflow tract of hearts showing the most severe conotruncal malformation (compare Fig. 4, C and G
vs. D and H; compare region denoted by black arrows showing thinning of the myocardium). Histological analysis showed that these lacZ-expressing cells were in regions
previously shown to be populated by cardiac neural crest
cells in the outflow septum (Fig. 5; Waldo et al., 1998
). It is
interesting to note that the aorta in the homozygous KO
hearts exhibited an unusual acute bend (Fig. 4, G and H,
white arrows), a phenotype consistent with the looping defect recently described for the
1 connexin KO mouse heart (Ya et al., 1998
).
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|
The examination of sections from these same fetal
hearts by immunohistochemistry using an antibody to localize cells expressing the cell proliferation nuclear antigen PCNA (Cox, 1997) showed no changes in the apparent
rate of proliferation in the lacZ-expressing presumptive
neural crest cells (data not shown). This is consistent with
the BrdU analyses of cell proliferation in the neural crest outgrowths. However, we unexpectedly observed marked
changes in cell proliferation in the ventricular myocardium. In the CMV43 transgenic mice, there was an increase in PCNA labeling in the myocardium, while a decrease was observed in the homozygous
1 connexin KO
mouse heart (Fig. 6). Quantitative analysis of cross sections from the mid-ventricular regions of these hearts
showed a doubling of PCNA labeling in the CMV43 hearts
(three CMV43 hearts examined showed mean of 52% vs.
two wild-type hearts with a mean of 24% PCNA-labeled
cells; student's t test show P < 0.0001), but no significant
change in PCNA labeling was detected in the homozygous
1 connexin KO hearts (two KO hearts examined with mean of 23%; P = 0.78). However, it should be noted that
in the KO hearts, we observed much more regional variability in the level of PCNA labeling. This is likely to account for the inability to detect significant differences in
the KO hearts.
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Discussion |
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Gap Junctions and Neural Crest Migration
Using a neural tube explant culture system, we showed
that the manipulation of 1 connexin function, whether
through reverse genetics or oleamide treatment, elicited
changes in neural crest migration in step with changes in
the level of gap junction communication. Thus, increased
gap junction communication mediated by overexpression of wild-type
1 connexin gap junctions increased the apparent rate of neural crest migration. In contrast, neural
crest migration was decreased with either the dominant-negative inhibition of gap junction communication, or the
reduction or loss of
1 connexin gap junctions. Treatment
of the explant cultures with oleamide, a potent inhibitor
of gap junction communication, provided similar results.
Thus, oleamide-mediated gap junction inhibition was associated with a reduction in the rate of neural crest migration. As the migration index used in this study represents
measurements of the crest outgrowth area, the migration
changes detected could reflect alterations in the rate of
cell locomotion and/or the directionality of cell movement.
To distinguish between these possibilities, the migratory
behavior of individual neural crest cells will need to be examined in real time.
The relevance of these in vitro findings to neural crest
migratory behavior in vivo is suggested by the analysis of
mouse embryos using a lacZ reporter gene previously
shown to be expressed in neural crest cells (Lo et al.,
1997). LacZ-expressing cells in the outflow septum were
increased in the CMV43 transgenic embryos, while they
were decreased in the same region in the
1 connexin KO
mouse embryos. These changes in lacZ expression are not
likely due to alterations in the amount of cell proliferation,
as we found no change in PCNA expression in presumptive lacZ-expressing neural crest cells. This result is in
agreement with the in vitro analyses of the neural crest
outgrowths, which showed no change in BrdU incorporation with either the up or down regulation of gap junction
communication. In addition, we also observed no obvious change in cell death in the neural crest outgrowths or in
the lacZ-expressing neural crest cells in vivo (Huang,
G.Y., C.W. Lo, K. Waldo, and M.L. Kirby, unpublished
observations). Together these findings suggest that the
modulation of
1 connexin function may perturb the migratory behavior of cardiac neural crest cells in vivo as
well as in vitro (also see discussion below).
Gap Junctions and Cardiac Development
Our previous study showed that differentiation of the
conotruncal myocardium is altered in the CMV43 and 1
connexin KO mice (Ewart et al., 1997
; Huang et al., 1998
;
Lo and Wessels, 1998
). This indicated that gap junction
communication in cardiac neural crest cells may have a
role to play in myocardialization of the conus, a process by
which the muscular outflow septum is formed. Here, we
further show that proliferation of the ventricular myocardium was altered in these same animals. Thus, in the
CMV43 mice where gap junction communication in neural
crest cells is elevated, myocyte proliferation is increased.
In contrast, the converse situation was found for the KO
mice. It is important to note that the ventricular myocardium is a tissue in which the CMV43 transgene is not expressed (Ewart et al., 1997
). Hence the increased myocyte cell proliferation in the CMV43 mice cannot be attributed
to myocyte overexpression of
1 connexin gap junctions.
Rather these results suggest that cardiac neural crest cells
may have a role in the modulation of myocyte cell proliferation and contribute in some manner to the myocyte defects in these mice.
In light of these findings, an important question to consider is how myocyte differentiation and proliferation may
be perturbed by alterations in the migratory behavior of
neural crest cells. Previous ablation studies have indicated
a quantitative requirement for neural crest cells in cardiac
development (Nishibatake et al., 1987). It was found that
ablating different amounts of cardiac neural crest cells resulted in different cardiac phenotypes. Hence, if
1 connexin perturbation were to alter the efficiency with which
neural crest cells home into the heart, or disturb the temporal regulation of cardiac neural crest migration, then
cardiac crest abundance could be altered. This could account for the observed changes in lacZ expression in the
outflow septum of the CMV43 and
1 connexin KO mice.
However, we note that changes in lacZ expression also
could result from altered promoter activity associated with
the lacZ transgene. In this case, the cardiac defects may not be causally related to the migration perturbations, but
rather may arise from changes in neural crest cell activity.
At present, we have no evidence to support this interpretation. Yet another possibility to consider is that changes
in coronary vessel deployment may contribute to the cardiac defects. Cardiac neural crest derivatives are known to
play an important role in the patterning of the coronary vasculature (Hood and Rosenquist, 1992
; Waldo et al.,
1994
), and in fact coronary vessel abnormalities have been
observed in both the CMV43 (Ewart et al., 1997
) and
1
connexin KO mouse hearts (Waldo, K., M.L. Kirby, and
C.W. Lo, unpublished observations).
Resolving these various issues will require a better understanding of the role of neural crest cells in cardiac morphogenesis. Given that cardiac neural crest cells generate
very little of the tissue in the heart and outflow tract, their
role(s) in cardiac morphogenesis may be indirect. Perhaps
changes in neural crest abundance, migration timing, or
functional activity may perturb the balance of cytokines
required for normal myocyte differentiation and proliferationcytokines that are produced by crest and/or noncrest cells. For example, changes in neural crest abundance or migration could affect the local concentration of
platelet-derived growth factor or endothelins in the developing heart. Both are expressed by noncrest cells found
along the cardiac crest migratory pathway, while their respective receptors are expressed by cardiac neural crest
cells and also the cardiomyoctes (Soriano, 1997
; Clouthier
et al., 1998
).
Gap Junction Communication and Cell-Cell Signaling in Crest Cells
Although traditionally gap junctions are not considered important in the physiology of migratory cells, the results from our studies indicate that gap junction communication plays an important role in neural crest migration. We propose that during neural crest migration, signals received by cells at the migration front may be relayed via gap junctions to cells throughout the migratory stream, and in this manner, facilitate a coordinated response to migratory cues. This intracellular pathway of cell-cell communication may help increase the efficiency of an otherwise seemingly chaotic process by which neural crest cells find their way to distant target sites.
Given that cardiac neural crest cells also give rise to the
enteric ganglia and the thymus capsule (LeDourain, 1973;
Bockman and Kirby, 1984; Kuratani and Kirby, 1991
; Epstein et al., 1994
; LeDourain and Teillet, 1994), one might
predict that there should be defects related to these structures with the perturbation of
1 connexin function. In
fact, recent studies have revealed enteric ganglionic deficiencies and thymus defects in the
1 connexin KO mice
(Lo, C.W., K. Waldo, M.L. Kirby, and L. Spain, unpublished observations). It is interesting to note that it was
previously reported that the enteric ganglionic deficiencies
in the piebald lethal mice arose from crest migration defects that were non-cell autonomous, that is defects that
are non-cell intrinsic (Kapur et al., 1995
). Given that this
mutation is a deletion of the endothelin receptor B gene, a
receptor which is normally expressed in cardiac neural
crest cells, the finding of non-cell autonomy was unexpected but entirely consistent with a role for gap junction
communication in mediating cell signaling events important in neural crest migration and development (Kapur et al.,
1995
).
As the 1 connexin gap junctions are also expressed in
noncardiac neural crest cells (Lo et al., 1997
), it is interesting to note that we have found abnormalities associated
with the trigeminal ganglia in the CMV43 (Ewart et al.,
1997
), and FC and
1 connexin KO mice (Sullivan et al.,
1998
). Formation of a portion of the trigeminal ganglia
and nerves is dependent on preotic crest cells (D'Amico-Martel and Noden, 1983
). Hence, the latter findings would suggest a role for
1 connexin gap junctions in development of the preotic hindbrain neural crest cells. This
is consistent with the outgrowth studies, which in fact
showed migration changes in the preotic crest cells that
were indistinguishable from those seen in the postotic
crest cells.
A Role for 1 Connexins in Other Migratory Cells?
In light of these observations on neural crest migration, it
is interesting to note that another migratory cell population is also affected by the loss of 1 connexin gap junctions, the primordial germ cells. Like neural crest cells,
germ cells must traverse long distances to reach their target organ. They also have been described as migrating in a
continuous stream, with all of the germ cells in direct cell-
cell contact with one another (Gomperts et al., 1994
). This
has led to the suggestion that cell-cell interactions may
play an important role in germ cell migration. In the
1
connexin KO mouse, both sexes show a severe reduction (90% or more) in germ cell numbers (Kidder, G., personal
communication). As this deficiency appears to arise very
early in development, it is possible that germ cell migration
may be perturbed by the loss of
1 connexin function.
Consistent with this possibility is our observation of sterility associated with heterozygous
1 connexin KO mice
maintained in certain genetic backgrounds (Lo, C.W., unpublished observations). This would indicate that the precise level of
1 connexin function also may be critically important for modulating germ cell migration. Of further
significance is that polymorphonuclear leukocytes and lymphoid tissue have been reported to express
1 connexin gap
junctions (Krenacs and Rosendaal, 1995
; Branes, M.C., J. Conteras, M.B. Bono, and J.C. Saez. 1997. Mol. Cell. Biol.
8:417a). Thus, perhaps gap junctional communication may
play a role in the migration and/or homing of cells in the immune system. Overall, these observations suggest the
possibility that
1 connexin gap junctions may have a general role in modulating the behavior of motile cells. The
future challenge is to identify the signal transduction pathways, and the gap junction-permeable cell signaling molecules involved in the modulation of cell migration. In addition, given that our studies showed a low level of gap junction communication persisting in neural crest cells of
homozygous
1 connexin KO mice, in future studies it will
be important to examine the possible role of other connexins in the modulation of neural crest migration and cell-
cell signaling. Perhaps such studies may reveal a role for
gap junctions in other neural crest populations.
![]() |
Footnotes |
---|
Address correspondence to C.W. Lo, Biology Department, University of Pennsylvania, Philadelphia, PA 19104. Tel.: (215) 898-8394. Fax: (215) 898-8780. E-mail: clo{at}mail.sas.upenn.edu
Received for publication 8 September 1998 and in revised form 26 October 1998.
This work was supported by grants from the Nation Science Foundation (IBN31544) and the National Institutes of Health (HD29573 to C.W. Lo; HL36059, HL51533, and HD17063 to M.L. Kirby, and GM37904 to N.B. Gilula).
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Abbreviations used in this paper |
---|
BrdU, bromodeoxyuridine; KO, knockout; PCNA, proliferating cell nuclear antigen.
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References |
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