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First published online 19 April 2006
doi: 10.1242/dev.02374
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1 Leon H. Charney Division of Cardiology, Department of Medicine, New York
University School of Medicine, New York, NY 10016, USA.
2 Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA 19104,
USA.
3 Department of Cell Biology, New York University School of Medicine, New York,
NY 10016, USA.
* Author for correspondence (e-mail: david.gutstein{at}nyumc.org)
Accepted 20 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Connexin 43 (Gja1), Cardiac neural crest, Neural tube, Outflow tract, Heart defect, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Gap
junctions may play an important role in development by mediating the
cell-to-cell transmission of signaling molecules involved in normal embryonic
patterning (Lo and Wessels,
1998). Connexin 43 (Cx43; Gja1 - Mouse Genome Informatics) is the
predominant cardiac connexin subtype in the latter stages of embryogenesis and
postnatally (Gros and Jongsma,
1996; Severs et al.,
1996). Cx43 has been implicated in human developmental disorders
(Paznekas et al., 2003),
although its role in congenital cardiac disease is controversial
(Britz-Cunningham et al., 1995;
Casey and Ballabio, 1995;
Debrus et al., 1997;
Splitt et al., 1995). Germline
knockout (KO) of Cx43 in the mouse results in abnormal cardiac morphogenesis,
including right ventricular outflow tract (OFT) abnormalities and perinatal
death (Reaume et al.,
1995).
We have previously observed that loss of Cx43 specifically in
cardiomyocytes during embryogenesis is not associated with gross cardiac
morphological defects at birth (Gutstein
et al., 2001b). Although cardiomyocyte-restricted loss of Cx43
does not influence heart morphogenesis, affected mice had slowed
interventricular electrical impulse conduction and were prone to spontaneous
and inducible lethal ventricular arrhythmias
(Danik et al., 2004;
Gutstein et al., 2001b).
Others have predicted that Cx43 expression may be required specifically in the
cardiac neural crest (CNC) cells for normal heart morphogenesis
(Ewart et al., 1997;
Huang et al., 1998;
Sullivan et al., 1998). The
CNC is of vital importance in the development of the ventricular OFT and the
great vessels. CNC cells migrate from the caudal pharyngeal arches into the
common OFT of the developing heart tube, where they contribute to
aorticopulmonary septation and other nearby structures
(Hutson and Kirby, 2003). The
importance of the CNC in cardiac morphogenesis is underscored by avian NC
ablation, which produces profound morphological abnormalities of the aortic
arch and ventricular OFT, as well as myocardial dysfunction
(Kirby et al., 1983;
Waldo et al., 1999).
Studies employing transgenic rescue and dominant-negative approaches have
suggested that Cx43 expression in the NC may indeed influence cardiac
morphogenesis (Ewart et al.,
1997; Sullivan et al.,
1998). However, patterns of activity of both the CMV-IE and
EF-1
promoters used in these studies are not restricted to the NC cell
lineage (Kim et al., 1990;
Koedood et al., 1995;
Kothary et al., 1991;
Song et al., 1998).
Furthermore, the Cx43-null mutant phenotype, characterized by infundibular
bulging without septation defects, differs from other murine genetic models
that affect CNC function. Genetic models including those with mutations in or
knockouts of Pax3, neurotrophin 3/TrkC, TGFß receptor type II, BMP4, BMP
receptor IA, endothelin 1 and combinations of retinoic acid receptors
(Choudhary et al., 2006;
Donovan et al., 1996;
Epstein et al., 2000;
Kurihara et al., 1995;
Liu et al., 2004;
Mendelsohn et al., 1994;
Stottmann et al., 2004;
Youn et al., 2003) commonly
cause OFT septation defects similar to those resulting from chick NC ablation.
Thus, during development, Cx43 may be required in tissues that contribute to
heart formation other than or in addition to the CNC
(Li et al., 2002;
Walker et al., 2005).
To test the hypothesis that Cx43 expression in the CNC is crucial to normal
heart development, we used parallel conditional knockout (CKO) strategies
mediated by Wnt1-Cre and P3pro-Cre (driven by a proximal region of the Pax3
promoter) to generate lines of mice with CNC-restricted CKO of Cx43
(Epstein et al., 2000;
Jiang et al., 2000;
Li et al., 2000).
Surprisingly, Wnt1-Cre-mediated CKO of Cx43 (Cx43-WCKO), which was limited to
the dorsal neural tube (NT) and migrating NC cells, resulted in normal OFT
development despite the presence of coronary anomalies. By contrast,
P3pro-Cre-mediated CKO of Cx43 (Cx43-PCKO), which involved a broader
dorsoventral domain of the thoracic NT, resulted in infundibular abnormalities
similar to the germline Cx43 mutant, as well as aberrant coronary development.
A late phase of delamination of Cx43-deficient P3pro-Cre-labeled cells was
evident and an increased abundance of labeled cells were detected outside the
NT in the Cx43-PCKO embryos. At E15.5, when OFT abnormalities were first noted
in the Cx43-PCKO embryos, abundant labeled cells were detected in the heart,
including the infundibulum. These data suggest that, in addition to effects on
coronary deployment via expression in the CNC, Cx43 also acts through the
non-crest neuroepithelium to suppress delamination of NT cells, which in the
absence of Cx43 migrate aberrantly to the OFT and disrupt heart
development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cx43-PCKO mice were generated by interbreeding the
Cx43 floxed mice with P3pro-Cre transgenic mice, which use the proximal 1.6 kb
of the Pax3 promoter to drive Cre recombinase expression
(Epstein et al., 2000;
Li et al., 2000). Cx43-WCKO
and -PCKO lines were in similar mixed genetic backgrounds consisting of a
mixture of 129/Sv, C57BL/6J and FVB strains. Mating strategies for Cx43-WCKO
and -PCKO mice are shown in Fig.
1B,C, respectively. Cx43-WCKO and -PCKO lines were compared with
control mice in the same strain background that carried two wild-type Cx43
alleles or had one or two floxed Cx43 alleles in the absence of Cre. The Cx43
floxed line generated in our laboratory has previously been shown to produce
equivalent amounts of Cx43 in the heart when compared with wild-type
littermates (Gutstein et al.,
2001a). In addition, Cx43-PCKO mice were compared with littermates
that were homozygous null (`floxed-out') for the Cx43 gene.
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Immunostaining for Cx43, Pax3 and Cre recombinase expression in CKO mice
For Cx43 immunostaining, embryos were fixed in 4% paraformaldehyde,
equilibrated in sucrose and embedded in Tissue Tek OCT compound (Sakura
Finetek USA, Torrance, CA) on dry ice. For the detection of Pax3 and Cre
recombinase, embryos were fixed in 4% paraformaldehyde, dehydrated in graded
alcohols and embedded in paraffin. Sections were blocked and incubated with
primary antibodies, followed by fluorescence-conjugated secondary antibodies
as described previously (Gutstein et al.,
2003). Primary antibodies included a polyclonal anti-Cx43 antibody
(Gutstein et al., 2003), a
monoclonal anti-Pax3 antibody (Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City, IA) and a polyclonal anti-Cre recombinase
antibody (Novagen, La Jolla, CA). Sections were mounted with Vectashield
mounting medium (Vector Laboratories, Burlingame, CA). Samples were visualized
with an Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) equipped
with appropriate filter blocks, which were chosen to avoid overlapping
emission spectra.
Morphologic evaluation of CKO hearts
Whole explanted neonatal hearts were imaged to compare gross external
appearance using a Leica MZ12.5 stereomicroscope equipped with a DEI-750D
video camera (Leica, Wetzlar, Germany) with computer interface. Hearts were
then washed in PBS and fixed in 4% paraformaldehyde. After ethanol dehydration
and embedding in paraffin blocks, samples were sectioned at 5 µm. Selected
sections were stained with Hematoxylin and Eosin using a Zeiss HMS Series
Programmable Slide Stainer.
Detecting Cre transgene expression patterns in CKO mice
The EYFP-fluorescence Cre reporter strain
(Srinivas et al., 2001)
(kindly provided by Dr F. Costantini, Columbia University) was crossed into
both the Cx43-PCKO and -WCKO lines. CKO embryos expressing the EYFP reporter
construct were sectioned and imaged by fluorescence microscopy at E11.5 and
E15.5.
In situ hybridization
Radioactive in situ hybridization for the detection of plexinA2 was
performed according to the procedure available at
http://www.uphs.upenn.edu/mcrc/histology/histologyhome.html,
with probes that have been characterized elsewhere
(Brown et al., 2001).
Statistics
Pup weights and cell density measurements (expressed as mean±s.d.)
were compared with two-tailed t-tests using Microsoft Excel software.
P values<0.05 were considered significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Therefore, we predicted that cells derived from the NC lineage in Cx43-WCKO
embryos would be deficient in Cx43. At E9.5, control embryos expressed
abundant levels of Cx43 in the NC-derived cells of the pharyngeal arches
(Fig. 2B,C). The distribution
of Cx43 in the pharyngeal arches was largely punctate, consistent with
patterns of Cx43 expression observed in cultured NC cells
(Boot et al., 2006;
Xu et al., 2001). As expected,
pharyngeal arches of the Cx43-WCKO embryos at E9.5, in contrast to those of
controls, were nearly devoid of Cx43 expression
(Fig. 2E,F). Despite deficient
Cx43 expression in the pharyngeal arches of Cx43-WCKO embryos, Cx43
immunosignal was preserved in the cardiac ventricles at E10.5
(Fig. 2G,H). Thus, Cx43 was
efficiently knocked out in the NC lineage of the Cx43-WCKO mice.
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; Walker et al.,
2005). We investigated whether targeted loss of Cx43 using the
Cx43-WCKO strategy might influence coronary patterning. In all eight control
hearts examined at birth, a typical pattern of coronary deployment was
observed. This pattern consisted of right and left coronary arteries
originating from right and left coronary sinuses, respectively. Each coronary
subsequently bifurcated into myocardial (including anterior descending and
circumflex arteries on the left) and septal branches
(Fig. 3A-D). By contrast,
coronary abnormalities were observed in three out of five Cx43-WCKO hearts.
These included separate ostia for right septal and myocardial coronary
tributaries (Fig. 3E,F,
respectively), an accessory coronary artery originating from the non-coronary
sinus (Fig. 3G), and tunneling
of the left coronary artery through the wall of the aorta
(Fig. 3H), all of which have
been described previously in germline Cx43-null mice
(Li et al., 2002). These data
suggest that expression of Cx43 in the NC is crucial for normal coronary
development.
Wnt1-Cre-mediated loss of Cx43 does not result in infundibular defects
Based on data suggesting that Cx43 expression in the CNC lineage may be
crucial for normal OFT development (Ewart
et al., 1997; Huang et al.,
1998; Sullivan et al.,
1998), we predicted that Cx43-WCKO hearts would demonstrate
infundibular abnormalities similar to the germline Cx43 mutant. Surprisingly,
none of the 15 Cx43-WCKO neonatal mice assayed had gross morphological or
histological OFT abnormalities (Fig.
4). Thus, although expression of Cx43 in the dorsal NT and NC
influences coronary deployment, Cx43 is not required in the Wnt1-Cre
expression domain for normal OFT development. This suggests that expression of
Cx43 in a cell population outside of the Wnt1-Cre expression domain is
required to ensure normal OFT morphogenesis.
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Cx43-PCKO mice have gross right ventricular morphologic abnormalities
The morphological phenotype of the heart in neonatal Cx43-PCKO pups,
consisting of a grossly mis-shapen RVOT, was remarkably similar to that of the
germline Cx43 KO. Unlike in wild-type control hearts
(Fig. 6E,F,L), large bulges
were seen bilaterally flanking the OFT (indicated by arrows in
Fig. 6A,C) in 20 out of 22
neonatal Cx43-PCKO hearts. Infundibular bulges characteristic of the Cx43-null
hearts were not seen in any of the 27 wild-type control littermates and were
observed in only one of 23 heterozygous Cx43 KO mice. Additionally, focal
bulging segments were occasionally seen at the apices of neonatal Cx43-PCKO
hearts (arrowhead in Fig. 6A),
although similar apical outpouchings were seen in a minority of control
littermates. Detailed examples of the infundibular bulges were highlighted by
the broken lines in Fig. 6B,D,
where the contours of the ventricular walls just under the pulmonary trunk
were seen to protrude prominently to either side. By contrast, the normal RVOT
region tapered to form a triangular shape as it coursed into the pulmonic
valve and pulmonary trunk. This pattern was seen in hearts from pups that were
either wild type (Fig. 6E) or
heterozygous null at the Cx43 locus (Fig.
6G). Higher magnification images demonstrated that the
infundibular bulges seen in the Cx43-PCKO hearts were not present in either
wild-type (Fig. 6F) or
heterozygous Cx43-null hearts (Fig.
6H). However, infundibular bulges seen in the Cx43-PCKO hearts
were comparable with those of the germline Cx43 KO hearts
(Fig. 6I,J). Oblique views of
Cx43-PCKO (Fig. 6K) and
wild-type hearts (Fig. 6L)
underscored the grossly altered contour of the mutant OFT when compared with
that of the wild type. Thus, in addition to premature death, Cx43-PCKOs also
share gross morphological features of their cardiac phenotype with germline
Cx43 KOs.
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Cell counts in the infundibulum at E15.5 revealed a trend towards increased
cell density in the Cx43-PCKO compared with controls. Cell density in the
infundibular regions of Cx43-PCKO hearts was 4.5±1.6 cells/10
µm2 (n=4), compared with a density of 3.5±0.7
cells/10 µm2 (n=5) in controls. Although this
difference was not statistically significant, in conjunction with upregulation
of genes specific for vascular smooth muscle cells, endothelial cells and
fibroblasts in Cx43 KO infundibular tissue
(Walker et al., 2005), these
data suggest an altered cellular composition of the mutant RVOTs. Infundibular
bulging may result from such a change in cellular composition, which makes the
RVOT unable to withstand increasing myocardial wall stress as development
progresses.
At E17.5 and at birth, the infundibular abnormalities in the Cx43-PCKO more closely reflected the germline Cx43 KO appearance than at earlier stages. Cx43-PCKO and Cx43fo/fo OFTs were grossly deformed, with the ventricular wall adjacent to the pulmonic valve bulging prominently above the valve plane (arrows in Fig. 7E,F,K,L). In comparison with controls (Fig. 7D,J), the Cx43-PCKO and Cx43fo/fo OFTs appeared dilated and more densely trabeculated, with regions of the infundibular wall demonstrating considerable thinning. Although the LVOT of the Cx43-PCKO and Cx43fo/fo hearts (Fig. 7H,I, respectively) appeared similar to those of control hearts (Fig. 7G), the RV free wall was slightly thinner in the Cx43-PCKO and Cx43fo/fo hearts (asterisks in Fig. 7H,I) than in controls. Thus, the bulging infundibular phenotype, which is seen in both Cx43-PCKO and Cx43fo/fo embryos by E15.5, is more apparent in the Cx43fo/fo embryos at that initial stage, but appears similar in both mutant lines by E17.5.
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), was
incidentally noted in an E17.5 Cx43-PCKO
(Fig. 8I-K).
Numerous coronary abnormalities have previously been described in
heterozygous Cx43-null mice (Li et al.,
2002). As the Cx43-PCKO mice are in a heterozygous Cx43-null
background, we examined Cx43fo/wt hearts for evidence of coronary
anomalies. In fact, two out of five Cx43fo/wt hearts demonstrated
myocardial and septal coronary arteries originating from the same ostium on
the aorta, rather than branching from a main coronary artery
(Fig. 8L), a finding previously
noted in heterozygous Cx43-null mice (Li
et al., 2002). In summary, Cx43-PCKOs share a number of phenotypic
features with germline Cx43 KO mice, including coronary anomalies,
infundibular bulging and perinatal death.
Late phase of cellular delamination from the neural tube in Cx43-PCKOs
Given the dramatic morphological differences between the Cx43-WCKO and
-PCKO lines, we investigated how the expression patterns of Cre recombinase in
the CKO embryos might impact on their phenotypes. Cells expressing Cre were
fluorescence labeled by crossing an EYFP Cre reporter strain
(Srinivas et al., 2001) into
the Cx43-PCKO and -WCKO lines. Cells derived from P3pro-Cre expressing
precursors were located throughout the thoracic NT
(Fig. 9A; EYFP is represented
by green fluorescence, propidium iodide to highlight cell nuclei is red and
their overlap results in yellow signal). By contrast, cells derived from
Wnt1-Cre expressing precursors were primarily limited to the dorsal NT
(Fig. 9J). Surprisingly,
P3pro-Cre-expressing cells were delaminating from the dorsal and lateral
aspects of the NT in the Cx43-PCKO embryos at E11.5
(Fig. 9E,F), a phenomenon also
observed in Cx43 germline KO (Cx43fo/fo) embryos at this stage
(Fig. 9G-I). Cellular
delamination from the NT was not seen in control littermates
(Fig. 9B,C,K,L) or in Cx43-WCKO
embryos (Fig. 9N,O) at this
stage. These data suggest that Cx43 is required in areas of the NT outside of
the Wnt1-Cre expression domain in order to maintain normal neuroepithelial
cell behavior.
Increased abundance of migrating neuroepithelial cells in the Cx43-PCKO embryo
Loss of Cx43 was associated with an increased abundance of EYFP-labeled
cells outside the NT at E11.5 in the Cx43-PCKO embryos
(Fig. 10C) compared with
controls (Fig. 10A). In
addition to an increased abundance of labeled cells, the normal distribution
of labeled cells in the Cx43-PCKO embryos appeared disrupted. For example, in
control embryos at E11.5, the trachea at the level of the OFT was flanked by
discrete collections of labeled cells that appeared to be streaming into the
OFT (Fig. 10B), as previously
described (Epstein et al.,
2000). By contrast, labeled cells in the Cx43-PCKO embryo at this
stage were greatly increased in abundance and have extensively infiltrated the
paratracheal tissue in close proximity to the OFT
(Fig. 10D). The disordered
distribution of labeled cells observed in the Cx43-PCKO embryos was not seen
in Cx43-WCKOs (see Fig. 10F;
compare with Wnt1-Cre+ controls in
Fig. 10E).
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). In
control embryos, plexin A2 expression was detected by in situ hybridization in
the NT (arrowhead, Fig. 10G)
and in the cells entering the cardiac OFT (arrow). In Cx43-PCKO embryos,
expression of plexin A2 was more abundant, particularly in the NT (arrowhead,
Fig. 10H) and in cells
adjacent to the OFT (arrow). Increased expression of plexin A2 in the
Cx43-PCKO embryos correlated well with the increased abundance of EYFP-labeled
cells observed around the OFT at E11.5. Thus, neuroepithelial cells appear to
be migrating through the Cx43-PCKO embryo at E11.5 in substantially greater
numbers than in control embryos. To investigate whether altered P3pro-Cre transgene activity in Cx43-PCKO embryos might contribute to the increase in EYFP-labeled cells outside of the NT, we immunostained E11.5 specimens for both Pax3 and Cre recombinase. As demonstrated in Fig. 10I, the native Pax3 expression domain in controls is limited to the dorsal and medial regions of the NT at E11.5. This expression pattern is unchanged in Cx43-PCKO littermates, and there is no discernible increase in Pax3 expression outside of the NT in Cx43-PCKOs (Fig. 10J). Cre recombinase expression is absent in control specimens, as expected (Fig. 10K). In E11.5 Cx43-PCKOs, Cre immunosignal is primarily limited to a small region of the Pax3 expression domain at the dorsal aspect of the NT (Fig. 10L) with scattered and less intense expression in cells in the medial and ventral NT. These data suggest that increased EYFP-labeled cells outside of the NT in Cx43-PCKO embryos do not derive from ectopic P3pro-Cre transgene activity, but rather result from the loss of Cx43 in the non-crest neuroepithelium.
Extensive infiltration of EYFP-labeled cells in E15.5 Cx43-PCKO hearts
EYFP-labeled cells in control embryos expressing the P3pro-Cre transgene at
E15.5 were largely confined to the OFT region
(Fig. 11A,B). EYFP-labeling
mediated by Wnt1-Cre in control (Fig.
11E,F) and Cx43-WCKO E15.5 hearts
(Fig. 11G,H), like that seen
in the P3pro-Cre-expressing controls, demonstrated a limited presence of
labeled cells in the septal region of the OFT, adjacent to the aortic valve.
EYFP-positive cells were detected only rarely in the RV infundibulum of the
P3pro-Cre-expressing controls, Wnt1-Cre-expressing controls and Cx43-WCKOs
(arrows in Fig. 11A,B,E-H). By
contrast, abundant labeled cells in the Cx43-PCKO embryos at E15.5 have
infiltrated the infundibular myocardium and other areas of the heart
(Fig. 11C,D). Thus, in
contrast to controls and Cx43-WCKO embryos, Cx43-PCKO embryos had an increased
abundance of labeled cells, some of which appeared to have delaminated
aberrantly from the NT, migrated abnormally and ultimately incorporated into
the heart. These data strongly argue for a novel function of Cx43 on NT
regions outside of the Wnt1-Cre expression domain in regulating
neuroepithelial cell behavior.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chesnutt et al.,
2004; Cheung et al.,
2005; McKeown et al.,
2005; Testaz et al.,
2001). However, a direct role for connexin proteins in these
signaling pathways in the NT has not been reported previously. Alternatively,
Cx43 gap junctions may allow for the intercellular transfer of neuroepithelial
regulatory signals. In the absence of gap junctions, such signals may be less
able to diffuse through the NT as the embryo grows, leading to a late phase of
delamination.
As an initial step to help distinguish the lineage responsible for the
Cx43-null phenotype, we have previously used a targeted approach to delete
Cx43 expression specifically in cardiomyocytes with two separate
cardiac-specific Cre recombinase mouse lines, MLC-2v-Cre
(Chen et al., 1998) and
-MHC-Cre (Agah et al.,
1997). Both MLC-2v-Cre and -MHC-Cre are expressed in the
heart prior to E9 (Chen et al.,
1998; de Lange et al.,
2004). Despite an 85-95% reduction in cardiac Cx43 expression,
cardiac-specific Cx43 CKO mice are grossly and histologically
indistinguishable from their non-KO littermates and have normal ventricular
function by echocardiography. Although the cardiac-specific Cx43 CKO mice are
structurally normal, they demonstrate slowed intracardiac conduction and began
to die suddenly and spontaneously, starting at 2-3 weeks of age from
ventricular arrhythmias (Danik et al.,
2004; Gutstein et al.,
2001b). Thus, Cx43 expression in cardiomyocytes does not appear to
be necessary for normal cardiac morphogenesis.
Other studies using dominant-negative
(Sullivan et al., 1998) and
transgenic overexpression approaches
(Ewart et al., 1997;
Huang et al., 1998) have
suggested that Cx43 expression in the CNC is of crucial importance to OFT
development. However, important differences exist between the phenotypes of
the dominant-negative mutant and the germline Cx43 KO, which may result from
nonspecific effects of a dominant-negative approach. Transgenic rescue of the
germline Cx43-null mouse is incomplete and data from this approach are
necessarily obfuscated by the unknown effects on cell function of
overexpression of Cx43. In addition, patterns of activity of both the
EF-1 and CMV-IE promoters used for the dominant-negative and
overexpression studies, respectively, are not restricted to the NC cell
lineage (Guo et al., 1996;
Kim et al., 1990;
Koedood et al., 1995;
Kothary et al., 1991;
Song et al., 1998). Wnt1-Cre,
however, allowed for a much more precise evaluation of the role of the CNC in
the Cx43-null phenotype (Jiang et al.,
2000). Despite the near-complete loss of Cx43 expression in the
NC-derived pharyngeal arches of the Cx43-WCKO embryos, heart morphology was
normal, although coronary patterning was altered.
Previous work by others has established that Cx43 plays an important role
in coronary artery development (Li et al.,
2002; Walker et al.,
2005). Our study suggests that the effect of Cx43 on coronary
patterning is mediated through its expression in the NC. As NC cells invest
the proximal regions of murine coronary arteries
(Jiang et al., 2000), loss of
Cx43 may influence ostial coronary development by directly regulating crest
cell function. Alternatively, crest cells may exert an indirect effect on
coronary artery patterning through epicardial cells via a gap
junction-dependent mechanism
(Gittenberger-de Groot et al.,
2004; Walker et al.,
2005). Either way, the effect of Cx43 on NC cell biology
vis-à-vis coronary development may prove to be clinically relevant.
Isolated anomalies of the coronary arteries have been associated with sudden
death and exercise-related death, particularly in young athletes
(Frescura et al., 1998;
Maron et al., 1996;
Taylor et al., 1992).
Recently, other investigators have described a population of migratory
cells that emerge from the ventral NT and contribute widely to the formation
of visceral organs, vascular structures and connective tissue (reviewed by
Dickinson et al., 2004). These
ventrally emigrating NT (VENT) cells, the existence of which is highly
controversial (Boot et al.,
2003; Yaneza et al.,
2002), are thought to emerge from the ventral NT at sites of nerve
exit. As labeled NT cells in the Cx43-PCKO embryos appear to be delaminating
from the dorsal and lateral aspects of the NT, however, regulatory pathways
involving Cx43 probably differ from those of the VENT cells.
In conclusion, by comparing tissue-restricted CKOs of Cx43 using Wnt1-Cre and P3pro-Cre, we have found that Cx43 expression in the non-crest NT plays a key role in OFT morphogenesis in the embryonic mouse. Our findings suggest that the Cx43 gap junction protein acts through the non-crest neuroepithelium to regulate transformation, delamination and/or migration in NT cells, and indirectly influences heart development as a result.
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ACKNOWLEDGMENTS |
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