1 Centre For Modeling Human Disease, Samuel Lunenfeld Research Institute, Mount
Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
2 Department of Medicine, Medical Sciences Building, 1 King's College Circle,
University of Toronto, Toronto, Ontario M5S 1A8, Canada
3 Department of Molecular and Medical Genetics, Medical Sciences Building, 1
King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8,
Canada
4 Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8,
Canada
5 Institute of Biomaterials and Biomedical Engineering, University of Toronto,
Toronto, Ontario M5G 1X8, Canada
6 Department of Physiology and Pharmacology, University of Western Ontario,
Dental Science Building, London, Ontario N6A 5C1, Canada
7 Department of Anatomy and Cell Biology, University of Western Ontario, Dental
Science Building, London, Ontario N6A 5C1, Canada
8 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario
M5G 1X8, Canada
9 Mouse Imaging Centre, The Hospital for Sick Children, 555 University Avenue
Toronto, Ontario M5G 1X8, Canada
10 Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S
1A8, Canada
11 Cardiovascular Research, The Hospital for Sick Children, Toronto, Ontario M5S
1A8, Canada
12 Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
13 Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
14 Department of Medicine and Centre for Bone and Periodontal Research, McGill
University, 740 Avenue Dr Penfield, Montreal, Quebec H3A 1A4, Canada
15 Integrative Biology Research Program, The Hospital for Sick Children, Toronto,
Ontario M5S 1A8, Canada
16 Department of Obstetrics and Gynecology, University of Toronto, Toronto,
Ontario M5S 1A8, Canada
Author for correspondence (e-mail:
rossant{at}mshri.on.ca)
Accepted 26 July 2005
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SUMMARY |
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Key words: Oculodentodigital dysplasia, Connexin 43, Missense mutation, Mouse model
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Introduction |
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Recently, mutations in the gap junction protein alpha 1 gene
(GJA1) encoding connexin 43 (Cx43) have been found in families with
ODDD (Paznekas et al., 2003).
In humans, Cx43 belongs to a large family of 21 proteins whose structure
consists of an intracellular N terminus, four transmembrane domains with one
intracellular loop and two extracellular loops, ending with an intracellular C
terminus. Six connexin proteins form a ring with a central pore, collectively
known as a connexon or hemichannel. An intercellular gap junction or channel
is formed when a hemichannel from one cell docks with a hemichannel from an
apposing cell. Gap junctions provide an intercellular pathway for the passage
of small ions and molecules involved in cell to cell communication that are
integral to many developmental and physiological processes
(Sohl and Willecke, 2004
).
Mutations in human GJA1 are predicted to perturb the formation of
functional gap junctions. To date, there are 27 reported mutations in the
GJA1 gene that are linked to ODDD but, in most cases, the mechanism
of action of these mutations remains unclear
(Kjaer et al., 2004
;
Paznekas et al., 2003
;
Richardson et al., 2004
;
van Steensel et al., 2005
;
Vitiello et al., 2005
).
Recently, it was shown that the G21R and G138R mutations result in
loss-of-function Cx43 and these mutants have dominant properties on wild-type
Cx43 (Roscoe et al., 2005
). In
these studies, however, the mutants were expressed in excess of wild type Cx43
leading to concerns that this did not adequately represent the human disease
condition.
In the course of performing an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice, we identified a dominant mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1, leading to the substitution of a highly conserved amino acid (G60S) in Cx43. The availability of this mouse model system allowed us to undertake a histological and functional analysis of gap junctions in order to determine their role in the ODDD phenotype. Interestingly, we found a dramatic reduction in total Cx43 protein, in gap junctional intercellular coupling and in the number of gap junction plaques, indicating that this mutation is not simply a loss-of-function mutation but rather a dominant-negative mutation. In addition, we have found alterations in bone properties and in the hematopoietic system that have not yet been reported for individuals with ODDD but which are consistent with the known importance of gap junction function in these tissues.
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Materials and methods |
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Genetic mapping
DNA was extracted from tail tissue using standard procedures followed by
PCR amplification of individual microsatellite markers using fluorescently
tagged primers (IDT, Coralville, IA). Cycles were performed as follows:
94°C for 3 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 30
seconds and 72°C for 30 seconds; and a final extension of 72°C for 5
minutes. The labeled products were then multiplexed and analyzed on a
BaseStation automated sequencer (MJ Research, Waltham, MA) to determine
whether, for any given marker, an allele from the mutagenized strain
(C57BL/6J) had been inherited.
In-life screening of mutants
The appearance and behavior screening was performed using a modified SHIRPA
protocol (Rogers et al., 1997)
with details at
www.CMHD.ca We
used a 20 kHz Clickbox (MRC Institute of Hearing, Nottingham, UK) to elicit
the Preyer reflex indicative of normal hearing. Eyes were scanned for
abnormalities using a pen light to reveal opacities and to assess pupillary
light reflex. Extended observation and handling was used to detect gait
abnormalities and/or limb weakness.
Pathology
Mice were sacrificed using a combination of CO2 and
O2 and tissues collected and fixed in 10% neutral buffered formalin
or Bouin's fixative. Tissue sections (4 µm) were prepared and stained with
Hematoxylin and Eosin.
Limb and tooth analysis
External images of the limbs and teeth were taken using a Sony DSC-S50
Cyber-shot digital camera. X-ray imaging was performed using a Faxitron
Specimen Radiography System, Model MX-20. Freshly dissected teeth from an
11-week-old Gja1Jrt/+ mouse and unaffected littermate were
embedded in epoxy. By use of a Buehler grinder/polisher, teeth were ground and
polished with 1 µm diamond grit in sagittal section. Imaging was performed
on an FEI XL-30 scanning electron microscope at 20 kV with a back-scatter
detector.
MRI and micro-CT
Five Gja1Jrt/+ mice and five control mice (ranging in
age from 52 to 60 weeks of age) were analyzed using a 7 Tesla MRI (Varian
Instruments, Pala Alto, CA) modified for parallel imaging. T2-weighted 3D
datasets with 120 µm resolution were acquired
(Nieman et al., 2004) and
reviewed visually for intensity differences. Following MRI, the animals were
sacrificed, perfused with formalin and decapitated in preparation for scanning
using a micro-computed tomography unit (GE Medical Systems, London, ON). The
resulting 60 µm resolution 3D datasets were used to assess differences in
skull shape between animals using an automated technique for detecting shape
differences based on estimating the non-linear deformation needed to bring
individual images into alignment
(Kovacevic et al., 2005
). The
deformation representing the between-group shape differences was assessed by a
Hotelling T2 test (Gaser et al.,
1999
) with overall significance established by permutation testing
(Holmes et al., 1996
).
Analyses of the heart
Results are presented as mean±s.e.m. for n (number of
mice). Unpaired t-tests were performed for group comparisons.
Ultrasound
The rectal temperature of isoflurane-anesthetized mice was maintained at
36-38°C and the heart rate was monitored via transcutaneous electrodes
(ECG/heat Pad; Indus Instruments, Houston TX). A 20 MHz transcutaneous pulsed
Doppler instrument was used to measure the blood velocity in the ascending
aorta of five Gja1Jrt/+ mice and five wild-type
littermates at 11-14 weeks, and nine Gja1Jrt/+ mice and
seven controls at 50-67 weeks. Peak velocity, acceleration, pre-ejection time,
ejection time and stroke distance were measured from a representative waveform
in a 3 second file (DSPW; Indus Instruments). The same Doppler instrument and
the same mice were used to obtain the blood velocity waveform in the left
ventricular (LV) chamber. Peak E, peak A, isovolumetric relaxation (IVRT) and
contraction (IVCT) times, and ejection time (ET) were measured and the
Myocardial Performance Index (MPI) calculated ((IVRT + IVCT)/ET)
(Broberg et al., 2003). A 30
MHz ultrasound biomicroscope (Vevo 660; VisualSonics, Toronto, Canada) was
used to perform an echocardiographic exam on five
Gja1Jrt/+ mice and five wild-type littermates at 8-11
weeks, and four Gja1Jrt/+ mice and three controls at 50-67
weeks, using published methods (Zhou et
al., 2004
). LV and right ventricular (RV) inner chamber dimensions
(ID) and wall thicknesses (WT) at end-systole and end-diastole were measured
and fractional shortening (FS) [(IDd-IDs)/IDd
x 100] and relative wall thickness (wall thickness/inner dimension) were
calculated. Aortic and pulmonary artery diameters and peak blood velocities,
and right atrial chamber dimension were also measured. Dimension
weight0.33 was used to correct dimension and diameter measurements
for body size. The same ultrasound tests were also performed on five
Gja1Jrt/+ x FVB mice and five wild-type littermates
at 7 weeks of age.
Acute ECG
A 1-minute recording of ECG was obtained acutely from nine
isoflurane-anesthetized Gja1Jrt/+ mice and eight controls
at 50-67 weeks using subcutaneous pin electrodes, while rectal temperature was
maintained at 36-38°C. Heart rate, P duration, PR interval, QRS duration
and QTmax were measured from a signal-averaged ECG waveform obtained from a
relatively noise-free section of the file (averaged over 130 cycles;
SAECG Chart 4, ADInstruments).
Chronic ECG by radio-telemetry
ECG telemetry devices (DSI) were implanted subcutaneously under anesthesia
in nine Gja1Jrt/+ x FVB mice and seven wild-type
littermates at 11-13 weeks of age. Mice were allowed to recover for 72 hours
before obtaining a continuous 48 hour recording. Measurements (P wave height
and width, PQ interval, QRS width, QT interval, heart rate) were made from 20
ECG waveforms obtained over 24 hours and averaged for each animal. Entire 48
hour recordings were examined manually by a trained observer blind to genotype
for sporadic events.
Micro-CT of femurs and vertebrae
The distal metaphysis of the left femurs and 4th lumbar vertebrae were
scanned with a Skyscan 1072 micro-CT instrument (Skyscan, Belgium) at the
Centre for Bone and Periodontal Research
(www.bone.mcgill.ca)
as described (Valverde-Franco et al.,
2004). Two-dimensional images were used to generate 3D
reconstructions of the bones from four Gja1Jrt/+ mice and
four wild-type littermates, ranging in age from 6-12 weeks. Morphometric
parameters, including percent bone, trabecular thickness distribution,
trabecular connectivity, structure model index and cortical thickness, were
calculated with 3D Creator software supplied with the instrument.
Bone mineral density
Dual energy x-ray absorptiometry (PIXImus, Lunar Corp., Madison, WI) was
used to measure bone mineral content (BMC), bone area and bone mineral density
(BMD) of femurs in five Gja1Jrt/+ x FVB mice and six
wild-type littermates (22-week-old males).
Mechanical testing
Destructive three-point bending was performed on femurs of five
Gja1Jrt/+ x FVB mice and six wild-type littermates
(22-week-old males) using a screw-driven mechanical testing machine (Instron
model 1011, Canton, MA). Each bone was placed on two supports spaced 6.0 mm
apart, and a load was applied to the bone midway between the supports at a
deformation rate of 1 mm/minute. From the load displacement curve, the maximum
load (ultimate load) and maximum displacement (failure displacement) were
measured, and the stiffness was determined from a linear regression of the
initial region of the curve. The toughness was determined by measuring the
area under the load deformation curve.
Whole-mount Alcian Blue-Alizarin Red staining
Three- and 8-day-old Gja1Jrt/+ mice and wild-type
littermates were stained with Alcian Blue-Alizarin Red S as described
previously (McLeod, 1980).
Hematopoietic analyses
Flow cytometric analysis of bone marrow, splenic and thymocyte
subpopulations was performed on four Gja1Jrt/+ mice and
four controls (15-18 weeks), and two Gja1Jrt/+ mice and
two controls (57-62 weeks) using standard procedures and a panel of
commercially available antibodies (anti-CD3, anti-CD4, anti-CD8,
anti-CD11b, anti-CD41, anti-CD61, anti-B220, anti-Ly6G, and anti-TER-119; BD
PharMingen, San Diego, CA) as previously described
(Ito et al., 2003
). The
appropriate conjugated rat anti-mouse mAbs were used as negative controls.
Side population analysis by Hoechst dye (Sigma H-6024) exclusion was performed
on two Gja1Jrt/+ mice and two controls (15 weeks), and two
Gja1Jrt/+ mice and two controls (57-62 weeks) according to
published protocols by the Goodell laboratory and posted online
(http://www.bcm.edu/genetherapy/goodell/new_site/index2.html).
Flow cytometry was performed using a MoFlow cell sorter (Dako-Cytomation).
Clonogenic assays were performed using commercially available methylcellulose
containing IL3, IL6, SLF, and EPO (M3434, Stem Cell Technologies, Vancouver,
BC) as previously described (Ito et al.,
2003
). Colony forming units-erythroid (CFU-E) were assayed after 2
days by staining in situ with benzidine (Sigma) to detect hemoglobin. BFU-E
(burst forming units-erythroid) and CFU-C (CFU-GEMM, colony forming
units-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM, colony
forming units-granulocyte, macrophage; CFU-M, colony forming units-macrophage;
CFU-G, colony forming units-granulocyte) were counted after 7-10 days by
colony morphology.
Analysis of gap junctions and intercellular coupling among mutant granulosa cells
Paraffin sections were prepared from Bouin's-fixed ovaries as described
(Roscoe et al., 2001). They
were immunostained using an affinity purified rabbit polyclonal antibody
raised against residues 360-382 of rat Cx43
(Mitchell et al., 2003
) and
Alexa Fluor-conjugated goat anti-rabbit IgG (Molecular Probes). The final wash
contained Hoechst 33342 (Molecular Probes) to stain nuclei.
Preantral follicles were isolated from ovaries of
Gja1Jrt/+ females and wild-type littermates at 6-8 weeks
and from Gja1Jrt/+ x FVB females and wild-type
littermates at 12 weeks and cultured on cover slips in modified Waymouth MB
752/1 medium for dye transfer experiments as previously described
(Gittens et al., 2003).
Granulosa cells were microinjected with 5% Lucifer yellow (Sigma-Aldrich) in
double distilled H2O. Images were captured 2 minutes after
injection and the number of cells receiving dye was scored. Some follicles
were fixed in 80% methanol/20% acetone for 15 minutes at 4°C for Cx43
immunostaining as described above.
For conductance measurements, granulosa cells were constantly perfused with
a solution containing (mM) NaCl (140.0), KCl (5.4), MgCl2 (1.0),
CaCl2 (1.8), and HEPES (10.0) (pH 7.4). Whole-cell voltage clamp
(VH -60 mV) was applied to a single granulosa cell at room
temperature. Current signals, low-pass filtered at 10 kHz, were recorded using
an Axopatch 200B amplifier and digitized at 100 kHz sampling rate. The
resulting capacitative current transient was analyzed to obtain the peak
current Ip and the steady-state current Iss. The gap
junctional conductance between the patched cell and the surrounding rings of
cells (G01x) was calculated according to the equation
G01x=Iss*Gser/(Ip-Iss),
where Gser is the series conductance
(Gser=Ip/10 mV) (de
Roos et al., 1996). The resistance of the patch pipette was 2-4
M
when filled with a solution containing (mM) KCl (130.0), NaCl (10.0),
EGTA (2.0), MgCl2 (4.0), HEPES (10.0) and TEA (5.0), pH 7.3.
|
Analysis of Cx43G60S localization and function in transfected cell lines
The Cx43G60S mutation (G to A at position 177) was constructed
using the Quick-Change Site Directed Mutagenesis Kit (Stratagene, La Jolla,
California) as directed. The wild-type and mutant cDNAs were fused with a GFP
tag at the C terminus and cloned into the pEGFP-N1 vector (BD Biosciences,
Clontech, La Jolla, California). Normal rat kidney (NRK), mouse neuroblastoma
(N2A) and human cervical carcinoma (HeLa) cells were transfected and
immunolabeled using established procedures
(Laird et al., 1995;
Roscoe et al., 2005
;
Thomas et al., 2004
). Dual
patch clamp recording was used to measure gap junctional coupling between
pairs of transfected N2A cells as previously described
(Roscoe et al., 2005
).
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Results |
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Limb and dental characteristics of Gja1Jrt/+ mutant mice
The phenotype of our syndactyly mutant showed striking similarities to that
of individuals with ODDD. As well as simple fusion of the digits, the middle
phalange on the last digit of both the forelimb and hindlimb was absent
(Fig. 1B,D) which is consistent
with ODDD (Loddenkemper et al.,
2002; Paznekas et al.,
2003
). In addition, digit 1 (pollex) on the forelimb consisted of
a thickened, malformed bone that resulted possibly from abnormal growth or a
fusion of the phalanges (Fig.
1B). Mutant mice had small, white upper and lower incisors that
were prone to breakage, instead of the normal yellow, enamel covered teeth
(Fig. 1E). Further analysis by
back-scatter scanning electron microscopy revealed a very thin, porous enamel
layer that was almost non-existent in some areas
(Fig. 1F). The majority of
individuals with ODDD also have abnormal dentition, with enamel hypoplasia,
microdontia, multiple caries and early tooth loss
(Loddenkemper et al., 2002
;
Paznekas et al., 2003
).
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Cardiac disturbances in Gja1Jrt/+ and FVB x Gja1Jrt/+ mutant mice
Documented heart dysfunction in ODDD includes atrioseptal defects and
arrhythmias such as ventricular tachycardia and atrioventricular (AV) block
(Loddenkemper et al., 2002;
Paznekas et al., 2003
).
Gja1Jrt/+ mice also exhibited abnormalities in heart
morphology and electrophysiological function. Immunofluorescence showed a
pronounced reduction in myocardial gap junctions
(Fig. 3A,B) and a patent
foramen ovale was observed in two out of five mutants examined
(Fig. 3C,D). Small, multifocal
lesions of myocardial mineralization, mild fibrosis and inflammation were also
observed in Gja1Jrt/+ mutants but not controls (see Fig.
S4 in the supplementary material and data not shown). Two out of nine mutants
exhibited highly abnormal cardiac conduction and/or contraction defects: the
QRS duration was prolonged and premature ventricular contractions (ectopic
beats) occurred during the 1-minute ECG recording session in one mutant; in
the other, the PR interval was prolonged and the `myocardial performance
index' was elevated (Broberg et al.,
2003
), suggesting poor global cardiac function. Variables were
more than two standard deviations from the mean of the controls, although
group means for these variables were not significantly affected. As a group,
older mutants (50-67 weeks) exhibited significantly reduced right ventricular
fractional shortening and diastolic wall thickness (expressed relative to
chamber dimension), suggesting the development of right ventricular failure
with aging (Fig. 3E). Left
ventricular structure and function were also significantly affected in
mutants; pre-ejection and ejection times were elevated, diastolic chamber
dimension (expressed relative to body weight0.33) was increased,
and relative diastolic wall thickness was reduced in young (8-14 weeks) and/or
old (50-67 weeks) mutants (Fig.
3E).
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Novel phenotypes of bone and hematopoietic stem cells in Gja1Jrt/+ mutant mice
We also observed a number of phenotypes that have not been previously
reported in individuals with ODDD, but which are consistent with known
functions of Cx43. Bone mineral density (BMD), bone mineral content (BMC) and
mechanical strength were all significantly reduced in
Gja1Jrt/+ x FVB mice versus wild-type littermates
(+/+) at all ages tested (Fig.
5A; Table 1).
Whole-mount Alcian Blue-Alizarin Red staining revealed that craniofacial bones
originating from both mesoderm and neural crest cells displayed delayed
ossification, and were thin and porous with open foramena at 3 days and
beyond, suggestive of an osteogenic defect (see Fig. S2 in the supplementary
material). In adult mutant mice, all endochondral bones examined by micro-CT
[femurs (Fig. 5B) and vertebrae
(not shown)] and histological analysis [femurs
(Fig. 5C), tibiae and
sternebrae (not shown)] were osteopenic, but the phenotype was most marked in
the long bones.
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ODDD characteristics of variable penetrance not found in Gja1Jrt/+ mutant mice
Additional symptoms with variable penetrance have been described in
individuals with ODDD, including conductive hearing loss, and neurological
dysfunction such as ataxia and paraparesis, along with changes in cerebral
white matter and basal ganglia intensities on magnetic resonance imaging (MRI)
(Loddenkemper et al., 2002).
We did not detect any hearing abnormalities in five
Gja1Jrt/+ mutants at 10 weeks of age using the click box
test. Although we have not undertaken an extensive neurological analysis in
mutant mice, T2-weighted MRI analyses of the brains of
Gja1Jrt/+ mice (five mice, 52-60 weeks) did not show any
variations in intensity compared with control wild-type mice (five mice, 52-60
weeks) (data not shown), nor did we detect weakness of the limbs or an
abnormal gait as determined by prolonged observation and handling of the
affected mice. More-sensitive neurological tests and/or testing at later ages
may reveal more-subtle neurological deficits.
Localization and functional analysis of the Gja1Jrt mutant protein in vitro and in vivo
To assess whether Cx43G60S could be transported to the cell
surface and form gap junctions, we introduced an expression construct for a
Cx43G60S-GFP tagged protein into both communication competent (NRK)
and incompetent (HeLa, N2A) cells. In all cases, Cx43G60S-GFP was
transported to the cell surface and assembled into gap junction-like
structures (compare Fig. S3C,G,L with S3B,F,K in the supplementary material).
However, the ability of Cx43G60S to form functional gap junctions,
as measured by dual patch clamp analysis, was severely affected. Only one out
of 30 pairs of Cx43G60S transfected N2A cells was coupled with a
low level of junctional conductance (3.1 nS), compared with 31 out of 31 pairs
of N2A cell pairs expressing wild-type Cx43, which were coupled with an
average junctional conductance of 47±4.4 nS. This low percentage
coupling (1/30) was not significantly different from non-transfected N2A cell
pairs (data not shown).
To explore the effect of the Gja1Jrt mutation on gap
junctional intercellular communication in cells from the mutant mice, we chose
to examine ovarian granulosa cells. In granulosa cells of immature mouse
ovarian follicles, Cx43 is the sole connexin involved in cell-cell coupling,
providing an ideal cell type to monitor Cx43 function in
Gja1Jrt mutants
(Gittens et al., 2003;
Veitch et al., 2004
). In
Gja1Jrt/+ x FVB ovaries, only a few scattered gap
junction plaques were seen when compared with wild-type cells (+/+)
(Fig. 7A). This difference was
maintained in granulosa cells growing out from follicles cultured in vitro
(Fig. 7B), which were then
tested for gap junctional coupling by Lucifer yellow dye injection
(Fig. 7C-E) and by capacitative
current transient analysis (Fig.
7F,G). Whereas all wild-type granulosa cells were strongly dye
coupled, mutant cells were of two distinct types: those that were not
detectably coupled (17 of the 27 cells tested) and those that were weakly
coupled (10 out of 27; Fig.
7D). For those mutant granulosa cells that were coupled, the mean
number of cells receiving dye from an injected cell was 2.2 when compared with
32.9 for wild-type cells (Fig.
7E), which was significantly different according to an unpaired
t-test (P< 0.05). Analysis of the capacitative current
transients obtained from granulosa cells of mutant follicles confirmed the
existence of distinct populations of weakly coupled and non-coupled cells. In
cultured wild-type follicles, the granulosa cells were well coupled, as
indicated by large steady-state currents and slow decay phases in response to
the voltage step (Fig. 7G). By
contrast, the steady-state currents and decay phases from Cx43 knockout
granulosa cells (Gja1-/Gja1-)
(Fig. 7G) were
indistinguishable from those of single completely isolated wild-type granulosa
cells (not shown), confirming that the knockout granulosa cells were not
electrically coupled. Whereas some (five out of 17)
Gja1Jrt/+ mutant granulosa cells were no better coupled
than Cx43 knockout granulosa cells, the remaining 12 displayed a weak
capacitative current, indicating the presence of limited gap junctional
coupling (Fig. 7G). The
difference in the strength of intercellular conductance between wild-type and
coupled mutant cells (Fig. 7F)
was significant (P<0.05) and similar to the difference in strength
of coupling revealed by dye transfer (Fig.
7E). Thus, gap junctional coupling among mutant granulosa cells is
both sporadic and weak, consistent with the paucity of gap junctions revealed
by immunostaining.
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Discussion |
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|
|
While examining the pleiotropic phenotypes presented by
Gja1Jrt/+ mice, we found additional abnormalities in
osteogenesis and hematopoiesis that are consistent with known functions of
Cx43. Craniofacial abnormalities with delayed ossification throughout the
skeleton, but essentially normal appendicular and axial skeletons at birth,
have previously been reported in homozygous Cx43-null mice
(Lecanda et al., 2000). We
also found delayed ossification in craniofacial bones, which may be the origin
of the craniofacial abnormalities detected by micro-CT in older
Gja1Jrt/+ mice. Neonatal lethality precluded determination
of whether the absence of Cx43 results in the reduced bone mass and mechanical
strength in adult animals as observed in our Gja1Jrt/+
mutant mice, although this might be predicted based on the osteoblast
dysfunction observed in homozygous Cx43-null calvarial cells in vitro
(Lecanda et al., 2000
). Thus
far, bone mineralization defects have not been reported in humans with ODDD;
however, these results suggest that individuals should be examined for
osteopenia, as it may pose a serious health risk for them, especially as they
age.
|
The Gja1Jrt/+ mice did not display a subset of the variably penetrant symptoms of ODDD, including conductive hearing loss and neurological disorders such as weakness of the lower extremities and abnormal gait. Although it may be necessary to perform more sensitive neurological tests to reveal subtle neurological deficits, it is also possible that mutations in specific domains of the Cx43 protein generate a variable spectrum of phenotypes as different domains are known to govern diverse properties of the gap junction channel such as conductance, permeability and protein interactions. Importantly, this animal model of ODDD allows for a thorough evaluation of Cx43 function under conditions where both the wild-type and mutant Cx43 are predicted to be expressed at equal levels. In addition, these mice provide new insights into potential defects or abnormalities that may have remained undetected or undiagnosed in individuals with ODDD, and, in future, will provide a useful model with which to develop and evaluate potential intervention strategies for the treatment of ODDD.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/19/4375/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Broberg, C. S., Pantely, G. A., Barber, B. J., Mack, G. K., Lee, K., Thigpen, T., Davis, L. E., Sahn, D. and Hohimer, A. R. (2003). Validation of the myocardial performance index by echocardiography in mice: a noninvasive measure of left ventricular function. J. Am. Soc. Echocardiog. 16,814 -823.[CrossRef][Medline]
Cancelas, J. A., Koevoet, W. L. M., de Konig, A. E., Mayen, A.
E. M., Rombouts, E. J. C. and Ploemacher, R. E. (2000).
Connexin43 gap junctions are involved in multiconnexin-expressing stromal
support of hemopoietic progenitors and stem cells.
Blood 96,498
-505.
de Roos, A. D., van Zoelen, E. J. and Theuvenet, A. P. (1996). Determination of gap junctional intercellular communication by capacitance measurements. Pflugers Arch. 431,556 -563.[CrossRef][Medline]
Dhein, S. (1998). Gap junction channels in the cardiovascular system: pharmacological and physiological modulation. Trends Pharmacol. Sci. 19,229 -241.[CrossRef][Medline]
Eckardt, D., Theis, M., Degen, J., Ott, T., van Rijen, H. V., Kirchhoff, S., Kim, J. S., de Bakker, J. M. and Willecke, K. (2004). Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion. J. Mol. Cell Cardiol. 36,101 -110.[CrossRef][Medline]
Foote, C. I., Zhou, L., Zhu, X. and Nicholson, B. J.
(1998). The pattern of disulfide linkages in the extracellular
loop regions of connexin32 suggests a model for the docking interface of gap
junctions. J. Cell Biol.
140,1187
-1197.
Gaser, C., Volz, H.-P., Kiebel, S., Riehemann, S. and Sauer, H. (1999). Detecting structural changes in whole brain based on nonlinear deformation-application to schizophrenia research. NeuroImage 10,107 -113.[CrossRef][Medline]
Giepmans, B. N. (2004). Gap junctions and connexin-interacting proteins. Cardiovasc. Res. 62,233 -245.[CrossRef][Medline]
Gittens, J. E. I., Mhawi, A. A., Lidington, D., Ouellette, Y.
and Kidder, G. M. (2003). Functional analysis of gap
junctions in ovarian granulosa cells: a distinct role for connexin43 in early
stages of folliculogenesis. Am. J. Physiol. Cell
Physiol. 284,C880
-C887.
Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. and
Mulligan, R. C. (1996). Isolation and functional properties
of murine hematopoietic stem cells that are replicating in vivo. J.
Exp. Med. 183,1797
-1806.
Gutstein, D. E., Morley, G. E., Tamaddon, H., Vaidya, D.,
Schneider, M. D., Chen, J., Chein, K. R., Stuhlmann, H. and Fishman, G. I.
(2001). Conduction slowing and sudden arrhythmic death in mice
with cardiac-restricted inactivation of connexin43. Circ.
Res. 88,333
-339.
Holmes, A. P., Blair, m. C., Watson, G. J. D. and Ford, I. (1996). Nonparametric analysis of statistic images from functional mapping experiments. J. Cereb. Blood Flow Met. 16,7 -22.[CrossRef][Medline]
Houghton, F. D., Thonnissen, E., Kidder, G. M., Naus, C. C. G., Willecke, K. and Winterhager, E. (1999). Doubly mutant mice, deficient in Connexin32 and -43, show normal prenatal development of organs where the two gap junction proteins are expressed in the same cells. Dev. Genet. 24,5 -12.[CrossRef][Medline]
Huang, G. Y., Cooper, E. S., Waldo, K., Kirby, M. L., Gilula, N.
B. and Lo, C. W. (1998a). Gap junction mediated cell-cell
communication modulates mouse neural crest migration. J. Cell
Biol. 143,1725
-1734.
Huang, G. Y., Wessels, A., Smith, B. R., Linask, K. K., Ewart, J. L. and Lo, C. W. (1998b). Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart development. Dev. Biol. 198,32 -44.[CrossRef][Medline]
Ito, C. Y., Li, C. Y. J., Bernstein, A., Dick, J. E. and
Stanford, W. L. (2003). Hematopoietic stem cell and
progenitor defects in Sca-1/Ly-6A null mice. Blood
101,517
-523.
Joao, S. M. A. and Arana-Chavez, V. E. (2003). Expression of connexin43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs. Histochem. Cell. Biol. 119,21 -26.[Medline]
Justice, M. J., Carpenter, D. A., Favor, J., Neuhauser-Klaus, A., Hrabe de Angelis, M., Soewarto, D., Moser, A., Cordes, S., Miller, D., Chapman, V. et al. (2000). Effects of ENU dosage on mouse strains. Mammal. Genome 11,484 -488.[CrossRef][Medline]
Kjaer, K. W., Hansen, L., Eiberg, H., Leicht, P., Opitz, J. M. and Tommerup, N. (2004). Novel connexin43 (GJA1) mutation causes oculo-dento-digital dysplasia with curly hair. Am. J. Med. Genet. 127A,152 -157.
Kovacevic, N., Henderson, J. T., Chan, E., Lifshitz, N., Bishop,
J., Evans, A. C., Henkelman, R. M. and Chen, X. J. (2005). A
three-dimensional MRI atlas of the mouse brain with estimates of the average
and variability. Cereb. Cortex
15,639
-645.
Krenacs, T., van Dartel, M., Lindhout, E. and Rosendaal, M. (1997). Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes. Eur. J. Immunol. 27,1489 -1497.[Medline]
Laird, D. W., Castillo, M. and Kasprzak, L. (1995). Gap junction turnover, intracellular trafficking and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells. J. Cell Biol. 131,1193 -1203.[Abstract]
Lecanda, F., Warlow, P. M., Sheikh, S., Furlan, F., Steinberg,
T. H. and Civitelli, R. (2000). Connexin43 deficiency causes
delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.
J. Cell Biol. 151,931
-943.
Lo, C. W., Waldo, K. L. and Kirby, M. L. (1999). Gap junction communication and the modulation of cardiac neural crest cells. Trends Cardiovasc. Med. 9, 63-69.[CrossRef][Medline]
Loddenkemper, T., Grote, K., Evers, S., Oelerich, M. and Stogbauer, F. (2002). Neurological manifestations of the oculodentodigital dysplasia syndrome. J. Neurol. 249,584 -595.[CrossRef][Medline]
McLeod, M. J. (1980). Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22,299 -301.[Medline]
Mitchell, J. A., Ting, T. C., Wong, S., Mitchell, B. F. and Lye,
S. J. (2003). Parathyroid hormone-related protein treatment
of pregnant rats delays the increase in connexin43 and oxytocin receptor
expression in the myometrium. Biol. Reprod.
69,556
-562.
Nieman, B. J., Bock, N. A., Bishop, J., Sled, J. G., Chen, X. J. and Henkelman, R. M. (2004). Fast spin-echo for multiple mouse MR phenotyping. Magn. Reson. Med. (in press).
Paznekas, W. A., Boyadjiev, S. A., Shapiro, R. E., Daniels, O., Wollnik, B., Keegan, C. E., Innis, J. W., Dinulos, M. B., Christian, C., Hannibal, M. C. et al. (2003). Connexin43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am. J. Hum. Genet. 72,408 -418.[CrossRef][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]
Richardson, R. R., Donnai, D., Meire, F. and Dixon, M. J.
(2004). Expression of Gja1 correlates with the phenotype observed
in oculodentodigital syndrome/type III syndactyly. J. Med.
Genet. 41,60
-67.
Rogers, D. C., Fisher, E. M., Brown, S. D., Peters, J., Hunter, A. J. and Martin, J. E. (1997). Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mammal. Genome 8, 711-713.[CrossRef][Medline]
Roscoe, W. A., Barr, K. J., Mhawi, A. A., Pomerantz, D. K. and
Kidder, G. M. (2001). Failure of spermatogenesis in mice
lacking connexin43. Biol. Reprod.
65,829
-838.
Roscoe, W. A., Veitch, G. I., Gong, X. Q., Pellegrino, E., Bai,
D., McLachlan, E., Shao, Q., Kidder, G. M. and Laird, D. W.
(2005). Oculodentodigital dysplasia-causing connexin43 mutants
are non-funtional and exhibit dominant effects on wild-type connexin43.
J. Biol. Chem. 280,11458
-11466.
Saez, J. C., Berthoud, V. M., Branes, M. C., Martinez, A. D. and
Beyer, E. C. (2003). Plasma membrane channels formed by
connexins: their regulation and functions. Physiol.
Rev. 83,1359
-1400.
Sohl, G. and Willecke, K. (2004). Gap junctions and the connexin protein family. Cardio. Res. 62,228 -232.[CrossRef]
Thomas, T., Telford, D. and Laird, D. W.
(2004). Functional domain mapping and selective trans-dominant
effects exhibited by Cx26 disease-causing mutations. J. Biol.
Chem. 279,19157
-19168.
Valverde-Franco, G., Liu, H., Davidson, D., Chai, S.,
Valderrama-Carvajal, H., Goltzman, D., Ornitz, D. M. and Henderson, J. E.
(2004). Defective bone mineralization and osteopenia in young
adult FGFR3-/- mice. Hum. Mol. Gen.
13,271
-284.
van Steensel, M. A. M., Spruijt, L., van der Burgt, I., Bladergroen, R. S., Vermeer, M., Steijlen, P. M. and van Geel, M. (2005). A 2-bp deletion in the GJA1 gene is associated with oculo-dento-digital dysplasia with palmoplantar keratoderma. Am. J. Med. Genet. 132A,171 -174.
Veitch, G. I., Gittens, J. E. I., Shao, Q., Laird, D. W. and
Kidder, G. M. (2004). Selective assembly of connexin37 into
heterocellular gap junctions at the oocyte/granulosa cell interface.
J. Cell Sci. 117,2699
-2707.
Vitiello, C., D'Adamo, P., Gentile, F., Vingolo, E. M., Gasparini, P. and Banfi, S. (2005). A novel GJA1 mutation causes Oculodentodigital Dysplasia without Syndactyly. Am. J. Med. Genet. 133A,58 -60.
Yu, Q., Shen, Y., Chatterjee, B., Seigfried, B. H., Leatherbury,
L., Rosenthal, J., Lucas, J. F., Wessels, A., Spurney, C. R., Wu, Y.-J. et
al. (2004). ENU induced mutations causing congenital
cardiovascular anomalies. Development
131,6211
-6223.
Zhou, Y. Q., Foster, F. S., Neiman, B. J., Davidson, L., Chen,
X. J. and Henkelman, R. M. (2004). Comprehensive
transthoracic cardiac imaging in mice using ultrasound biomicroscopy with
anatomical confirmation by magnetic resonance imaging. Physiol.
Genomics 18,232
-244.
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