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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 917-924
HEMATOPOIESIS
Expression of connexin 43 (Cx43) is critical for normal
hematopoiesis
Encarnacion Montecino-Rodriguez,
Hyosuk Leathers, and
Kenneth Dorshkind
From the Department of Pathology and Laboratory Medicine and the
Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los
Angeles, CA.
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Abstract |
Gap junctions are intercellular channels, formed by individual
structural units known as connexins (Cx), that allow the intercellular exchange of various messenger molecules. The finding that numbers of
Cx43-type gap junctions in bone marrow are elevated during establishment and regeneration of the hematopoietic system has led to
the hypothesis that expression of Cx43 is critical during the
initiation of blood cell formation. To test this hypothesis, lymphoid
and myeloid development were examined in mice with a targeted
disruption of the gene encoding Cx43. Because Cx43 / mice die
perinatally, initial analyses were performed on Cx43/, Cx43+/, and Cx43+/+ embryos and newborns. The data
indicate that lack of Cx43 expression during embryogenesis compromises
the terminal stages of primary T and B lymphopoiesis. Cx43/
embryos and neonates had a reduced frequency of CD4+ and
T-cell receptor-expressing thymocytes and surface IgM+
cells compared to their Cx43+/+ littermates. Surprisingly,
Cx43+/ embryos/neonates also showed defects in B- and T-cell
development similar to those observed in Cx43/ littermates, but
their hematopoietic system was normal at 4 weeks of age. However, the
regeneration of lymphoid and myeloid cells was severely impaired in the
Cx43+/ mice after cytoablative treatment. Taken together, these
data indicate that loss of a single Cx43 allele can affect blood cell formation. Finally, the results of reciprocal bone marrow transplants between Cx43+/+ and Cx43+/ mice and examination of
hematopoietic progenitors and stromal cells in vitro indicates that the
primary effects of Cx43 are mediated through its expression in the
hematopoietic microenvironment.
(Blood. 2000;96:917-924)
© 2000 by The American Society of Hematology.
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Introduction |
Gap junctions allow the direct exchange of ions,
metabolites, and other messenger molecules between cells. These
intercellular channels are formed by individual structural units known
as connexins (Cx), which are encoded by a multigene family estimated to
consist of approximately 20 members in vertebrates.1-7 Cx43
is among the most widely distributed and has been shown to be the
predominant Cx expressed in the bone marrow, thymus, spleen, and other
lymphoid tissues.8-14 That Cx43 protein in these tissues
assembles into functional gap junctions has been demonstrated by the
passage of low molecular weight dye or electronic current between
stromal cells in the thymus9,10,15 and bone
marrow.8,13,16 It has been suggested that gap
junction-mediated intercellular communication between hematopoietic and
stromal cells also occurs.10,12 However, the latter
heterologous junctions are rare. Although most stromal cells in contact
are coupled via gap junctions,8 only 0.1% of hematopoietic
cells form gap junctions with the stroma.11
Considerable evidence suggests that Cx43 expression might be of
particular importance during establishment or regeneration of the
hematopoietic system. Rosendaal and colleagues have demonstrated that
gap junction numbers are elevated at the sites of regeneration following cytoablative treatments and in the epiphyseal marrow of
growing animals.11-13 For example, the number of gap
junctions in the periosteal regions in growing mice was 80-fold greater than in adults in which steady-state hematopoiesis had
established.12,13 Comparable increases in the number of gap
junctions in the bone marrow of mice recovering from treatment with
5-fluorouracil (5-FU) or irradiation11 were also observed.
Consistent with the interpretation that gap junctions are associated
with active hematopoiesis are findings that during adipocyte
differentiation of stromal cells, which is believed to result in the
formation of yellow, inactive marrow, the number of gap junctions
decreases.17 Taken together, these data have led to the
hypothesis that expression of Cx43-type gap junctions in blood-forming
tissues is critical during periods of intense hematopoietic growth and differentiation.
The present study tested this hypothesis by examining hematopoiesis in
a recently described strain of mice with a targeted disruption of the
gene encoding Cx43.18 Cx43/ homozygotes die
immediately after birth as a result of a ventricular malformation, indicating that Cx43 expression is required for normal cardiac development, even though multiple Cxs are expressed in the
heart.18 By analogy, if Cx43 expression played an essential
role during the most active periods of hematopoiesis, defects in blood
cell formation should be apparent in Cx43/ mice.
The results of this study clearly demonstrate a role for Cx43 during
embryonic blood cell formation and during regeneration of the
hematopoietic system and further indicate, based on findings of
hematopoietic defects in Cx43+/ heterozygotes, a Cx43 gene dosage effect. Further characterization of the Cx43-deficient mice
suggested that at least some hematopoietic effects of Cx43 are manifest
via its expression in the hematopoietic microenvironment.
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Materials and methods |
Mice
Heterozygous male and female Gja mice, obtained from the Jackson
Laboratory, Bar Harbor, ME, were bred in the vivaria of the Division of
Laboratory Animal Medicine at UCLA or the University of Pennsylvania to
generate Cx43+/+, Cx43+/, and Cx43/ mice. As a
result of a cardiac malformation, mice die within hours of birth.
Therefore, Cx43/ animals were analyzed during late
embryogenesis (days 17-19 of gestation) or immediately after birth. All
heterozygote breeders and their progeny appeared healthy.
Genotyping of mice
The Cx43 mutation was generated by inserting a neomycin
(neo) resistance gene into exon 2 of the Cx43
gene.18 A polymerase chain reaction (PCR) protocol was
developed to assess the Cx43 genotype of the mice being analyzed by
incubating DNA aliquots with primers specific for Cx43 exon 2 and neo
sequences. The primer sequences were as follows: Cx43: 5'-CTG TAC
TTG GCT CAC GTG TTC TAT-3' and 5'-CGT GGG AGT TGG AGA TGG
TGC-3'13; neo: 5'-TGA CTG GGC ACA ACA GAC AAT
CGG C-3' and 5'-GTA GCC AAC GCT ATG TCC TGA TAG C-3'.
PCR reactions were performed in 100 µL containing 100 mmol/L Tris-HCl
(pH 8.3), 2.5 mmol/L MgCl2, 200 mmol/L each of dATP, dTTP,
dGTP, dCTP, 2 mmol/L each of Cx43 and neo primers, 50 mmol/L KCl, and
2.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT). Thirty
cycles (1 minute denaturation at 94°C, 1 minute annealing at
65°C, and 1 minute polymerization at 72°C) were performed. The
final polymerization step was extended an additional 15 minutes. A
Perkin-Elmer Thermal Cycler was used for all reactions. Products were
run on a 1.2% agarose gel and the PCR fragments were detected by
ethidium bromide staining. A single 800-bp fragment, the 800-bp product
and a 600-bp fragment, or a single 600-bp product identifies Cx43+/+,
Cx43+/, and Cx43/ mice, respectively (Figure
1).
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| Fig 1.
Representative blot showing identification of Cx43+/+,
Cx43+/, and Cx43/mice by PCR.
Liver DNA from mice was amplified using neomycin and Cx43 primers as
described in "Materials and methods."
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Preparation of cell suspensions
Single cell suspensions of thymus and spleen from embryos were
prepared by gently pushing tissues through a 35-mm strainer fitted in
the cap of 12 × 75-mm tubes (Falcon, Oxnard, CA) into  -Minimal Essential Medium (MEM; GIBCO, Grand Island, NY)
supplemented with 5% fetal calf serum (FCS; Atlanta Biologicals,
Atlanta, GA). Bone marrow cells were flushed from femurs and tibiae
using a syringe fitted with a 30-gauge needle. Adult splenocyte
suspensions were prepared by gently pressing the organs through a fine
mesh screen. Cells were counted with a hemacytometer and cell viability was determined by eosin dye exclusion. In some experiments, testes were
harvested for DNA preparation. All bone marrow data are expressed as
number of white blood cells per 2 femurs and 2 tibiae.
Immunofluorescence analysis
Expression of specific cell surface determinants was detected by
labeling cells with the following phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, or biotin-conjugated antibodies: anti-CD11b (Mac-1, clone M1/70), FITC-conjugated antiheat-stable antigen (HSA,
clone M1/69), biotin-conjugated anti-CD43 (clone S7), PE-conjugated anti-CD45R (B220, clone RA3-6B2), T-cell receptor (TCR)  (clone H57-597), CD4 (clone RM4-5), and CD8 (clone 53-6.7), all from Pharmingen, La Jolla, CA, or FITC-conjugated anti-IgM (Southern Biotechnology, Birmingham, AL). Prior to staining, cell suspensions were treated with NH4Cl to lyse erythrocytes and incubated
with an antibody to Fc II and III receptors (CD16/32, clone
2.4G2, Pharmingen) to reduce nonspecific labeling of cells.
All staining protocols were conducted in calcium, magnesium-free
phosphate-buffered saline (PBS) at 4°C. Dead cells were excluded based on their SSC versus FSC profile or their staining
with propidium iodide, added to a final concentration of 0.5 µg/mL
per sample. Cell analysis was performed on a Becton Dickinson FACScan
(Becton Dickinson, San Jose, CA). Gates were set on the basis of
staining with either an isotype control antibody conjugated
to the same fluorochrome or the secondary reagent alone.
Treatment with 5-FU
The cytostatic drug, 5-FU (Sigma St Louis, MO) has been shown to
induce an extensive phase of hematopoietic progenitor proliferation prior to cell replenishment of lymphoid organs.19,20
Cx43+/ and Cx43+/+ mice received an intravenous injection of
either 125 mg/kg of 5-FU in 0.9 saline or saline alone. Nine days
after injection, mice were killed and the extent of recovery of their hematopoietic tissues assessed. This was done by calculating a ratio of
reconstitution (ratio 5-FU/saline), obtained by dividing the organ
cellularity (or population frequency) in mice treated with 5-FU by the
corresponding organ cellularity (or population frequency) in the
saline-treated mice for each lymphoid organ (or lymphoid cell
population) considered.
Long-term bone marrow cultures
Long-term myeloid bone marrow cultures were established as
described.21 Three weeks after recharge, cultures were
switched to B lymphoid-permissive conditions.22,23 Cells
were harvested from 8 cultures per group at weekly intervals and
analyzed for the emergence of CD45R+,
sIgM , and CD45R+IgM+ cells
by immunofluorescence.
In some experiments purified stromal cells from long-term bone marrow
cultures were analyzed for Cx43 gene expression. Purified stromal cell cultures were generated as described by treating established myeloid long-term bone marrow cultures with mycophenolic acid.24 This procedure eliminates hematopoietic cells from
the cultures but retains intact, functional stromal cells.
Bone marrow transplantation
Mice were preconditioned before transplantation with 900 R delivered
from a 60Cobalt irradiator (41.28 R/min). Five hours later,
recipient mice received an intravenous injection of
3 × 106 bone marrow cells resuspended in 0.9%
saline. Hematopoietic tissue reconstitution was assessed 17 days later.
Purification of hematopoietic populations
In some experiments CD43+CD45R+ pro-B cells
were purified on a Becton Dickinson FACStarplus cell sorter after
labeling cells as described above and as previously
described.25 In other experiments, populations containing
hematopoietic stem cells were purified as described.26
Briefly, lineage depleted (Lin) bone marrow cells
were isolated from femurs and tibiae of 5 to 7 BALB/c mice by
incubation in PBS with a cocktail of optimal concentrations of rat
antibodies against the lineage specific antigens CD2 (clone RM2-5), CD8
(clone 53-6.7), CD45R (clone RA3-6B2), Gr-1 (clone RB6-8C5), Mac-1
(clone M1/70), and TER-119 (all from Pharmingen). The washed cells were
then exposed to antirat Ig -conjugated MicroBeads (Miltenyl Biotec,
Auburn, CA) for 30 minutes at 4°C and subsequently passed through a
magnetic field using a MACS separation column. Lineage-positive cells
were retained on the column while the Lin cells were
recovered from the eluate. The Lin fraction was then
incubated with a tricolor (TC) conjugated antirat Ig for 20 minutes. After washing, TC antirat Ig-free binding sites were blocked
by incubation with normal rat Ig (1 mg/106 cells) prior to the addition
of PE-anti-c-kit (clone 2B8) and FITC- conjugated anti-Sca-1 (Ly6A/E,
clone E13-161.7). Then, Lin cells expressing Sca-1
and c-kit were purified using a FACSstar plus flow cytometer (Becton
Dickinson). Reanalysis showed that the purity of the sorted
Lin cells obtained was more than 97%.
Reverse transcriptase-PCR (RT-PCR)
Messenger RNA (mRNA) was prepared from FACS purified populations
according to the manufacturer's directions (Invitrogen, Carlsbad, CA)
and reverse transcribed into complementary DNA (cDNA). The cDNA was
amplified with primer sets to Cx43, described above, and the following
reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) primers:
5' CCATGGAGAAGGCTGGGG and 3' CAAAGTTGTCATGGATGACC. PCR
reactions were performed in a final volume of 100 µL containing 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 600 µmol/L
concentrations each of the dATP, dGTP, dUTP, and dTTP, 1 µmol/L
concentrations of the primers, 50 mmol/L KCl, and 2.5 U Taq polymerase
(Perkin-Elmer Cetus). Cycles, repeated 30 times, consisted of 1 minute
denaturation at 94°C, 1 minute annealing at 65°C, 1 minute
polymerization at 72°C, and a final extension for 15 minutes at
72°C. A Perkin-Elmer Thermal Cycler was used for all reactions. PCR
products were run on 1.5% agarose gels and stained with ethidium bromide.
Statistical analysis
Statistical analyses were performed using a Student t
test. Because all neonatal mice were analyzed immediately after
birth and were thus the same developmental age, data from multiple
litters were pooled. However, so as not to bias results, embryonic data were pooled only if animals were from the same litter or it could be
confirmed through breeding records that embryos were the same gestational age. All data are expressed as mean ± SD.
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Results |
T-cell development in Cx43-deficient embryos and neonates
Whether or not failure to express Cx43 at normal levels affected
embryonic T-cell development was assessed by examining thymopoiesis in
Cx43/, Cx43+/, and Cx43+/+ late gestation embryos
(day 17-19), a time at which T-cell production is well
established,27 and in neonates.
A consistent result was that thymic cellularity was on average higher
in Cx43/ and Cx43+/ embryos and neonates than in their Cx43+/+ littermates (Figure 2).
Phenotypic analysis revealed additional developmental defects in the
embryos and newborn mice. These were most demonstrable in the neonates
in which the frequency of CD4+CD8+
double-positive thymocytes, which generate CD4 and CD8 single-positive thymocytes, was higher and the frequency of CD4+
single-positive cells significantly lower in the Cx43/
and Cx43+/ mice as compared to their Cx43+/+ littermates. The
decrease in the CD4+ cells correlated with a decrease in
the frequency and absolute number of TCR- T cells in the
Cx43/ and Cx43+/ neonates (Table 1).
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| Fig 2.
Thymus cellularity.
(A) Late gestation Cx43+/+ (n = 4), CX43+/ (n = 12), and
Cx43/ (n = 6) mice. (B) Neonatal Cx43+/+ (n = 6),
Cx43+/ (n = 5), and Cx43/ (n = 3) mice. (C)
Young adult Cx43+/+ (n = 4) and Cx43+/ (n = 4) mice. The
level of statistical significance between different groups of mice is
indicated.
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Because Cx43/ mice die immediately after birth, further
analysis of Cx43/ adult animals was not possible.
However, in view of the defects observed in Cx43+/ embryos and
neonates, thymopoiesis was assessed in 12-week-old Cx43+/ mice
to determine whether the thymic deficiencies persisted. As shown
in Figures 2C and 3C, thymic
cellularity and the frequency of CD4 and CD8 thymic
subpopulations in Cx43+/ adult mice were normal.
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| Fig 3.
Frequency of
CD4CD8, CD4+
CD8+, CD4+, and CD8+
thymocyte subpopulations.
(A) Late gestation Cx43+/+ (n = 4), Cx43+/ (n = 12), and
Cx43/ (n = 6) embryos. (B) Neonatal Cx43+/+ (n = 6),
Cx43+/ (n = 5), and Cx43/ (n = 3) mice. (C)
Young adult Cx43+/+ (n = 4) and Cx43+/ (n = 4) mice. The
level of statistical significance for each thymocyte subpopulation
between different groups of mice is indicated.
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B-cell development in Cx43-deficient embryos and neonates
The B lineage cell production peaks at approximately day 16 of
gestation in the fetal liver; it initiates by the time of birth in the
bone marrow and continues in that tissue throughout the life of the
animal. In addition, B-cell progenitors can also be found in the spleen
during embryogenesis and for up to 3 weeks after
birth.28-30 Accordingly, B lymphopoiesis was analyzed by determining the frequency of B lineage cells in late-gestation fetal
liver, the bone marrow, and spleen of Cx43/,
Cx43+/, and Cx43+/+ neonates. Because of the low number of
hematopoietic cells that could be obtained from individual embryos and
neonates, myelopoiesis in these mice was not analyzed.
The data in Table 2 demonstrate that the
frequency of CD45R+, surface IgM (sIgM)
pro/pre-B cells in fetal liver of Cx43/ and Cx43+/ mice was significantly higher than in their Cx43+/+ littermates. The
sIgM+ B cells were either not detected or present at too
low a frequency for reliable analysis in fetal liver at the time mice
were examined (data not shown). Although there was no difference in the
frequency of pro/pre-B cells in the bone marrow or spleen of
Cx43/ and Cx43+/ mice when compared to their
Cx43+/+ littermates, the frequency of sIgM+ mature B cells
was significantly lower in these organs (Table 2). This resulted in a
significant reduction in absolute numbers of B cells in
Cx43/ and Cx43+/ mice as a result of the lower cellularity of their bone marrow and spleen (Table 2).
B lymphopoiesis was also assessed in 12-week-old Cx43+/ mice.
The adult Cx43+/ mice exhibited a slight reduction in the frequency of bone marrow CD45R+sIgM
pro-B cells that was limited to the
CD43+CD45+CD24 (HSA)+ pro-B cell
fraction as defined by Hardy and colleagues31
(Cx43+/ = 0.8 ± 0.3 and Cx43+/+ = 1.9 ± 0.5,
difference significant at P < .005).
Hematopoietic recovery following cytoablative treatment is deficient
in Cx43+/ mice
Although the above data suggest that Cx43 expression is critical
during periods of active hematopoiesis, the small number of
Cx43/ animals that could be generated made it imperative to conduct additional experiments in a system where clear-cut effects
would be apparent. If Cx43 expression is critical during periods of
active blood cell formation, a prediction is that Cx43-deficient mice
should exhibit defects during regeneration of the hematopoietic system.
This hypothesis was tested by treating Cx43+/ mice with 5-FU. Treatment with 5-FU eliminates cycling hematopoietic cells and the surviving immature hematopoietic progenitors are driven into
cell cycle to replenish the lymphoid and myeloid
compartments.19,20
Defects were clearly evident in Cx43+/ mice treated with 5-FU.
Recovery of bone marrow cellularity in Cx43+/ mice was not as
efficient as in Cx43+/+ animals. As shown in Figure
4A, bone marrow cellularity in Cx43+/+ mice
treated with 5-FU had recovered to 70% of that in saline-treated
Cx43+/+ mice. However, in Cx43+/ mice treated with 5-FU,
recovery was only 20% of that in saline-treated Cx43+/
controls. Further analysis revealed a significant defect in the
recovery of CD45R+ B lineage cells in the bone marrow
(Figure 5A).
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| Fig 4.
Cellularity in the bone marrow, thymus, and spleen.
(A) Bone marrow (BM) from 2 femurs and 2 tibiae); (B) thymus (THY); and
(C) spleen (SPL) from Cx43+/+ and Cx43/ mice 9 days after
treatment with 5-FU or saline. Three to 4 mice of each genotype
received the indicated treatment, and animals were processed
individually. The ratio of recovery calculated as described in
Materials and methods is indicated in parenthesis. The level of
statistical significance between the different experimental groups is
indicated. The results shown are from 1 of 2 identical experiments.
Mice were 5 weeks of age when treated with 5-FU.
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| Fig 5.
Phenotypes.
The phenotypes are shown for CD45R+ (A), TER-119+
(B), and CD11b+ (C) cells in the bone marrow of
Cx43+/+ and Cx43+/ mice 9 days after 5-FU or saline treatment.
Mice are the same as those described in the legend to Figure 4. The
level of statistical significance between the different experimental
groups is indicated.
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In addition, thymic cellularity in Cx43+/ mice was only 30% of
that in saline-treated Cx43+/ mice 9 days following the 5-FU treatment, whereas it had returned to normal in Cx43+/+
mice treated with 5-FU (Figure 4B). No difference in the
frequency of thymocyte subpopulations defined by CD4 or CD8 (or both)
expression was observed in the 5-FU- or saline-treated Cx43+/
and Cx43+/+ animals (data not shown).
Because sufficient cell numbers were recovered, it was also possible to
assess myelopoiesis and erythropoiesis in these mice. Figure 5 further
demonstrates that the recovery of these lineages in Cx43+/ mice
treated with 5-FU was not as efficient as in Cx43+/+ mice treated with
5-FU (Figure 5B,C). These phenotypic data were corroborated by results
of colony assays performed on bone marrow from the mice treated with
5-FU. In both saline-treated Cx43+/+ and Cx43+/ mice the
frequency of day 11 colonies generated in response to interleukin
(IL)-3, IL-6, and c-kit ligand was comparable (22 ± 6 and
30 ± 3 per 5 × 104 cells, respectively).
However, although the number of colonies in Cx43+/+ mice 9 days after
5-FU treatment rose to 149 ± 53 per 5 × 104
cells, the number of colonies generated in Cx43+/ mice treated with 5-FU was only 67 ± 21 per 5 × 104 cells.
Although recovery of splenic cellularity in Cx43+/ mice 9 days following 5-FU treatment was lower than in Cx43+/+
animals, the differences were not statistically significant
(Figure 4C).
Cx43 bone marrow chimeras exhibit thymus defects
The above data demonstrate a clear requirement for Cx43 in the
establishment/regeneration of the hematopoietic system but provide no
information regarding in which cells expression is critical. In an
attempt to distinguish between effects intrinsic to hematopoietic cells
versus those in nonhematopoietic populations, bone marrow chimeras were
generated by reciprocal bone marrow transplantation between
Cx43+/ and Cx43+/+ mice. Irradiated Cx43+/+ and Cx43+/
mice received a graft of 3 × 106 bone marrow from
either Cx43+/+ or Cx43+/ sex- and age-matched mice
(Figure 6A). The frequency of
c-kit+, Sca-1+, Lin cells in
the Cx43+/+ and Cx43+/ donor cells was determined and found to
be identical (data not shown), therefore ensuring that mice received
the same number of hematopoietic stem cells.
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| Fig 6.
Thymus cellularity in Cx43+/+ or Cx43+/ mice after
bone marrow transplantation.
(A) The transplantation protocol. (B) Thymus celllularity in Cx43+/+ or
Cx43+/ recipients of Cx43+/+ or Cx43+/ bone marrow cells.
(C) PCR analysis of testes, bone marrow (BM), and thymus (THY) from
Cx43+/+ or Cx43+/ mice grafted with either Cx43+/+ or
Cx43+/ bone marrow cells as indicated. Testis DNA was used to
confirm the identity of the recipient mouse. Note that the genetic
composition of the thymus of Cx43+/ mice that received Cx43+/+
bone marrow is not completely donor derived. Only 2 of 3 to 4 mice from
each group are shown. The asterisk indicates values significantly
different at P < .05.
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Mice were analyzed at 17 days after transplantation based on the
rationale that progenitor cell proliferation is occurring at this time
to replenish the various hematopoietic lineages. At this time, no
differences in bone marrow cell recovery were observed between the 4 groups of mice (data not shown). However, as shown in Figure 6B, thymic
cellularity in Cx43+/ recipients was significantly lower,
regardless of the Cx43 genotype of the bone marrow donor.
On the other hand, no significant difference in thymic cellularity was
observed in Cx43+/+ mice that received either Cx43+/ or Cx43+/+
bone marrow cells. The frequency of thymocyte subpopulations defined by
CD4 and CD8 expression was comparable in all of the different chimeras
(data not shown).
Because the Gja/Cx43 knock-out strain has not been bred onto a genetic
background that would allow phenotypic identification of donor cells in
the recipients, the PCR protocol shown in Figure 1 was used to assess
engraftment of donor cells in the chimeras. As shown in Figure 6C, the
genotype of bone marrow cells in the chimeras was consistent with that
of the donor cells, even though the extent of reconstitution by
endogenous radioresistant populations could not be determined by this
technique. Surprisingly, as opposed to what was observed when Cx43+/+
or Cx43+/ donor cells were grafted into Cx43+/+ hosts, cells of
the Cx43+/ genotype were present in the thymus of Cx43+/
recipients of Cx43+/+ cells (Figure 7).
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| Fig 7.
B lymphopoiesis in long-term bone marrow cultures from
Cx43+/ and Cx43+/+ mice.
(A) After establishment of confluent adherent layers from Cx43+/+ or
Cx43+/ bone marrow cells, cultures were recharged from the same
pool of Cx43+/+ or Cx43+/ bone marrow, respectively, and
maintained under myeloid long-term culture conditions for 3 weeks
before transfer to B lymphoid culture conditions. Cells were harvested
from cultures at weekly intervals thereafter and the frequency of
CD45R+sIgM and
CD45R+sIgM+ cells determined. Groups different
at ** = P < .0005; * = P < .005. There were
no significant differences in cellularity between the cultures
established with Cx43+/+ and Cx43+/ bone marrow (data not
shown). Data are based on individual analysis of 8 cultures per
experimental group. (B) Expression of Cx43 mRNA in sorted
Lin, Sca-1+, c-kit+ stem
cells, sorted CD43+CD45R+ pro-B cells, and
heterogeneous bone marrow stroma depleted of hematopoietic
cells. RT-PCR was performed as described in "Materials and
methods."
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B lymphopoietic defects occur in the bone marrow of Cx43+/
mice
To determine whether any effects of Cx43 are directly mediated on
the bone marrow, B lymphopoiesis was examined in the long-term bone
marrow myeloid to lymphoid switch system using cultures established from Cx43+/ and Cx43+/+ mice. As described
previously,22 on transfer of established myeloid long-term
bone marrow cultures to B lymphoid permissive conditions, B-cell
development initiates and predominates in the cultures by 4 weeks
thereafter. Thus, these in vitro cultures provide a system to
examine the "regeneration" B lymphopoiesis from immature
hematopoietic precursors.
As shown in Figure 7A, by week 2 after the switch, a time by which
myelopoiesis has declined and B lymphopoiesis is rapidly establishing,
the frequency of CD45R+sIgM and
CD45R+sIgM+ B lineage cells was significantly
higher in Cx43+/+ cultures. However, by week 4 after the switch, a time
at which B lymphopoiesis has attained steady-state levels in the
cultures, the frequency of B lineage cells in Cx43+/ and Cx43+/+
cultures was comparable.
Development of B cells in the myeloid to lymphoid switch cultures could
initiate from pluripotential hematopoietic stem cells or more
committed hematopoietic precursors or both. In view of reports
suggesting that coupling occurs between immature hematopoieticcells and
the stroma, albeit rarely, whether or not FACS purified
Lin, c-kit+, Sca-1+ and
CD43+, CD45R+ cells expressed Cx43 was
analyzed. The former population is enriched in pluripotential
hematopoietic stem cells, whereas the latter phenotype includes the
earliest B-cell committed progenitors. As shown in Figure 7B, Cx43
message was not detected in either population. However, consistent with
previous results,8 Cx43 mRNA was present in bone marrow
stromal cells.
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Discussion |
Based on findings that the number of Cx43 type gap junctions is
up-regulated during establishment and regeneration of the hematopoietic
system, it has been hypothesized that expression of Cx43 is critically
important during periods of active blood cell formation (reviewed in
reference 13). The aim of this study was to test this hypothesis by
analyzing hematopoiesis in mice deficient in the expression of Cx43.
Analysis of lymphopoiesis in fetal and neonatal Cx43/
mice demonstrated that Cx43 expression is required for embryonic
hematopoiesis. Maturation of double-positive thymocytes into
single-positive cells expressing CD4 and CD8 is clearly dependent on
the expression of Cx43. For example, the frequency of
CD4+CD8+ double-positive cells was higher and
the frequency of mature cells that express CD4, CD8, or TCR was
lower in Cx43/ mice than in their Cx43+/+ littermates. A
similar effect on the terminal stages of B lymphopoiesis was observed
as evidenced by a reduction in the frequency of mature
sIgM+ cells in Cx43/ and Cx43+/
embryos and neonates. This trend was particularly evident in the
spleen, which functions as a hematopoietic organ during embryonic and
early neonatal life.32 Cx43 expression has been detected in
stromal-like cells in that organ.10
The Cx43+/ mice also exhibited lymphopoietic defects, further
revealing that failure to express Cx43 at normal levels is critical to
this process. There is precedent for proposing a Cx43 gene
dosage effect. Reaume and coworkers18 noted that the
frequency of lucifer yellow dye transfer between Cx43+/
fibroblasts was intermediate between values observed in Cx43+/+ and
Cx43/ cells. Additional studies have demonstrated that
precise levels of Cx43-mediated gap-junction communication are
necessary for processes such as neural crest cell migration and cardiac
development,33,34 and other analyses have reported that
ventricular contraction in Cx43+/ mice is 30% slower than in
wild-type littermates.35,36
Even though lymphopoietic defects were observed in embryonic and
neonatal Cx43+/ mice, hematopoietic defects in adult
Cx43+/ animals were subtle to nonexistent. This result alone
provided support for Rosendaal's hypothesis that expression of Cx43 at normal levels is most critical during the establishment of blood cell
formation but not for steady-state hematopoiesis.11,13 However, additional experiments were designed to test this possibility directly. Because recovery of hematopoiesis from cytoablative treatment
may recapitulate aspects of embryonic blood cell formation, lymphoid
and myeloid regeneration was measured in Cx43+/ adult mice
treated with 5-FU. The results of these experiments showed a highly
significant defect in the recovery of cellularity in the thymus and
bone marrow of Cx43+/ animals when compared to Cx43+/+ mice,
thus supporting the hypothesis that gap-junction expression is somehow
involved in the regulation of hematopoietic progenitor cell
proliferation.12 No difference in the frequency of
thymocyte subpopulations was observed in the mice treated with 5-FU,
but severe effects on recovery of most hematopoietic lineages were
observed in the bone marrow. In addition to a delay in the recovery of
marrow CD45R+ B lineage cells, the Cx43+/ mice also
exhibited a severe retardation in the recovery of myelopoiesis and
erythropoiesis. The major effects observed on the myeloid lineage are
consistent with published observations showing that myelopoiesis
recovers more slowly than B lymphopoiesis after 5-FU
treatment.19 Interestingly, the number of progenitors that
formed colonies in semisolid medium in response to IL-3, IL-6, and
c-kit ligand was also significantly lower in Cx43+/ mice treated
with 5-FU. Because this cytokine combination targets developmentally
immature hematopoietic populations, these data provide additional
evidence that failure to express normal Cx43 levels significantly
affects the growth of stem and early progenitor cell
populations.12
Although these data provide clear evidence that Cx43 expression at
optimal levels is critical for normal hematopoiesis, it is much more
difficult to establish precisely the particular cellular compartment in
which expression is critical. It is conceivable that the ventricular
defect somehow could impair circulation and affect the distribution of
hematopoietic cells. This might explain the defects in the
Cx43/ embryos and neonates but not in Cx43+/ mice.
Steady-state hematopoiesis in the heterozygotes is normal, and defects
are only observed on hematopoietic challenge. In an attempt to
distinguish between effects intrinsic to hematopoietic versus those in
nonhematopoietic populations, chimeras were generated by reciprocal
bone marrow transplantation between Cx43+/ and Cx43+/+ mice. No
effects on bone marrow cellularity were observed. A thorough kinetic
analysis would be needed to address whether differences might have been
apparent at earlier or later time points during reconstitution.
However, such experiments are difficult to perform due to the limited
availability of the Cx43+/ mice. This strain breeds poorly, and
the mothers often fail to nurse their litters after delivery
(unpublished observations).
On the other hand, clear differences were apparent in the thymus 17 days after transplantation. Thymus cellularity in Cx43+/+ recipient
mice was similar regardless of the Cx43 genotype of the bone marrow
donor cells. This result suggested that Cx43+/ hematopoietic
cells are able to develop in a normal environment. On the other hand,
Cx43+/ recipient mice exhibited a reduced thymic cellularity
whether they were transplanted with Cx43+/+ or Cx43+/ bone
marrow cells, supporting the hypothesis that the effects of Cx43
expression are manifest at a nonhematopoietic level. An unexpected
observation in these studies was that Cx43+/ message was
detected in the thymus of Cx43+/ recipients of Cx43+/+ cells.
This could be due to the fact that the ratio of stroma/thymocytes is
higher in Cx43+/ recipients and the PCR reaction is detecting radioresistant host stromal cells. In any case, the results of the bone
marrow transplantation studies strongly suggest that there is an
environmental defect in Cx43+/ mice localized to the thymus stroma.
Because the transplantation experiments did not reveal effects on the
bone marrow, the final set of experiments used the established myeloid
to lymphoid switch culture system to assess whether or not effects of
Cx43 occurred at that level. These "switch" cultures measure the
initiation of B-cell development from immature hematopoietic precursors
that do not yet express B lineage cell surface
determinants.22 Thus, this system allows the
"regeneration" of B-cell development to be assessed in vitro. The
results obtained in vitro paralleled what was observed in Cx43+/
mice in vivo. During the exponential phase in which B lymphopoiesis is
being "established," between day 0 and week 2 following the
transfer of cultures to B lymphoid permissive conditions, the frequency
of both CD45R+sIgM and
CD45R+sIgM+ B lineage cells was
significantly reduced in cultures from Cx43+/ mice. However, by
3 weeks after the switch, when B lymphopoiesis has established and the
cultures are producing B lineage cells at steady-state levels, no
differences in the frequency or number of CD45R-expressing
cells in Cx43+/+ or Cx43+/ cultures were observed.
Stromal cells in situ and in long-term bone marrow cultures are well
coupled, suggesting that they are the main site at which effects of
Cx43 expression occurs. However, it has been reported that immature
hematopoietic cells and stromal cells couple at a frequency of
0.1%.11 Because this is the approximate frequency at which
stem cells are found in the bone marrow,37 the heterologous couplings may also be important in the regulation of stem cell growth
or differentiation.38 To determine where in the bone marrow
Cx43 expression is most critical, the expression of Cx43 in stromal and
hematopoietic cells was analyzed. The data herein do not provide
support for stromal cell-stem cell or for stromal cell-lymphocyte
coupling in the regulation B-cell development. Although stromal cell
expression of Cx43 was confirmed, no expression of Cx43 in stem cell
purified populations or pro-B cells was detected. These data are
consistent with previous results from this laboratory that consistently
failed to detect dye transfer between stromal and B lineage
cells.8 Thus, taken together with the thymus results, the
data in this paper support a role for expression of Cx43 in the marrow
and thymus microenvironments.
In addition to the need to investigate further how Cx43 might affect
microenvironmental function, several other issues remain to be
resolved. For example, the nature of the regulatory signals that are
transmitted via gap junctions has not been identified, but a
possibility is that they may ultimately regulate cytokines produced by
the hematopoietic microenvironment. Proposing that failure to express
gap junctions at normal levels compromises cytokine production by the
hematopoietic microenvironment is not unprecedented, based on the
finding that coupling of pancreatic secretory epithelia via gap
junctions plays a critical role in their biosynthesis, storage, and
release of specific secretory products.39 It is also not
clear that the manner in which gap junctions regulate hematopoiesis
during embryonic development is comparable to what occurs during
regeneration of the hematopoietic system. In this regard, clear effects
on the development of mature CD4+ and CD8+
thymocytes were observed in Cx43-deficient embryos and neonates, and
cellularity in the thymus of the mice was higher in Cx43/ and Cx43+/ mice than in their Cx43+/+ littermates. However,
opposite results were observed in adult Cx43+/ mice following
bone marrow transplantation or recovery from 5-FU treatment. In these
instances, defects in restoration of organ cellularity, rather than
maturation effects, were observed. Efforts to delineate Cx43 effects on
hematopoietic cell differentiation versus proliferation are needed in
this regard.
These issues aside, there are additional implications of the present
data that have demonstrated that a defect in the expression of Cx43 can
compromise blood cell production. Cx expression and channel formation
is sensitive to a variety of drugs.33,39 If one or more
chemotherapeutic agents used in various clinical scenarios also
interfere with gap-junction expression or function, this could have
significant adverse effects on the recovery of blood cell production in
treated individuals.
| |
Acknowledgments |
The authors thank A. Johnson and L. Collins for expert technical
assistance, Dr Cecelia Lo for critical comments and provision of
Cx43+/ mice, and Dr F. Zaera for statistical analysis.
| |
Footnotes |
Submitted January 12, 2000; accepted March 29, 2000.
Supported by grant HL60658 from the National Institutes of
Health. The flow cytometry core at UCLA is supported in part by grant
CA16042 from the National Institutes of Health.
Reprints: Kenneth Dorshkind, Department of Pathology 173216, UCLA School of Medicine,10833 Le Conte Avenue, Los Angeles, CA
90095-1732; e-mail: kdorshki{at}mednet.ucla.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Kumar NM, Gilula NB.
The gap junction communication channel.
Cell.
1996;84:381-388[Medline]
[Order article via Infotrieve].
2.
Wolburg H, Rohlmann A.
Structure:function relationships in gap junctions.
Int Rev Cytol.
1995;157:315-373[Medline]
[Order article via Infotrieve].
3.
Beyer EC.
Gap junctions.
Int Rev Cytol.
1993;137C:1-37.
4.
Bruzzone R, White TW, Paul DL.
Connections with connexins: the molecular basis of direct intercellular signaling.
Eur J Biochem.
1996;238:1-27[Medline]
[Order article via Infotrieve].
5.
Stagg RB, Fletcher WH.
The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation.
Endocrinol Rev.
1990;11:1-27.
6.
Lowenstein WR, Rose B.
The cell-cell channel in the control of growth.
Semin Cell Biol.
1992;3:59-79[Medline]
[Order article via Infotrieve].
7.
White TW, Paul DL.
Genetic diseases and gene knockouts reveal diverse connexin functions.
Annu Rev Physiol.
1999;61:283-310[Medline]
[Order article via Infotrieve].
8.
Dorshkind K, Green L, Godwin A, Fletcher WH.
Connexin-43-type gap junctions mediate communication between bone marrow stromal cells.
Blood.
1993;82:38-45[Abstract/Free Full Text].
9.
Alves LA, Campos de Carvalho AC, et al.
Functional gap junctions in thymic epithelial cells are formed by connexin 43.
Eur J Immunol.
1995;25:431-437[Medline]
[Order article via Infotrieve].
10.
Alves LA, Campos de Carvalho AC, Savino W.
Gap junctions: a novel route for direct cell-cell communication in the immune system.
Immunol Today.
1998;19:269-275[Medline]
[Order article via Infotrieve].
11.
Krenacs T, Rosendaal M.
Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias.
Am J Pathol.
1998;142:993-1004.
12.
Rosendaal M, Green CR, Rahman A, Morgan D.
Up-regulation of the connexin43+ gap junction network in haemopoietic tissue before the growth of stem cells.
J Cell Sci.
1994;107:29-37[Abstract].
13.
Rosendaal M.
Gap junctions in blood forming tissues.
Microscopy Res Tech.
1995;31:396-407.
14.
Krenacs T, Rosendaal M.
Immunohistological detection of gap junctions in human lymphoid tissue: connexin43 in follicular dendritic cells and lymphoendothelial cells.
J Histochem Cytochem.
1995;43:1125-1137[Abstract].
15.
Head GM, Mentlein R, Kranz A, Downing JE, Kendall MD.
Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion.
J Anat.
1996;191:355-365.
16.
Rosendaal M, Gregan A, Green CR.
Direct cell-cell communication in the blood-forming system.
Tissue Cell.
1991;23:457-470[Medline]
[Order article via Infotrieve].
17.
Umezawa A, Hata J.
Expression of gap-junctional protein (connexin 43 or 1 gap junction) is down-regulated at the transcriptional level during adipocyte differentiation of H-1/A marrow stromal cells.
Cell Structure Function.
1992;17:177-184[Medline]
[Order article via Infotrieve].
18.
Reaume AG, de Sousa PA, Kulkarn S, et al.
Cardiac malformation in neonatal mice lacking connexin43.
Science.
1995;267:1831-1834[Abstract/Free Full Text].
19.
Vetvicka V P, Kincade PW, Witte P.
Effects of 5-fluorouracil on B lymphocyte lineage cells.
J Immunol.
1986;137:2045-2410.
20.
Hodgson GS, Bradley TR.
Properties of haematopoietic stem cells surviving 5-fluoruracil treatment: evidence for a pre-CFU-S stem cell?
Nature.
1979;281:381-382[Medline]
[Order article via Infotrieve].
21.
Dexter TM, Allen TD, Lajtha LG.
Conditions controlling the proliferation of haemaopoietic stem cells in vitro.
J Cell Physiol.
1977;91:335-344[Medline]
[Order article via Infotrieve].
22.
Dorshkind K.
In vitro differentiation of B lymphocytes from immature hematopoietic precursors present in long-term bone marrow cultures.
J Immunol.
1986;136:422-429[Abstract].
23.
Whitlock CA, Witte ON.
Long-term culture of B lymphocytes and their precursors from murine bone marrow.
Proc Natl Acad Sci U S A.
1982;79:3608-3612[Abstract/Free Full Text].
24.
Dorshkind K, Johnson A, Collins L, Keller GM, Phillips RA.
Generation of purified stromal cell cultures that support lymphoid and myeloid precursors.
J Immunol Methods.
1986;89:37-47[Medline]
[Order article via Infotrieve].
25.
Foster MP, Montecino-Rodriguez E, Dorshkind K.
Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis.
J Immunol.
1999;163:5883-5890[Abstract/Free Full Text].
26.
Hashimoto Y, Dorshkind K, Montecino-Rodriguez E, Taguchi N, Schultz L, Gershwin ME.
NZB mice exhibit a primary T cell defect in fetal thymic organ culture.
J Immunol.
2000;164:1569-1575[Abstract/Free Full Text].
27.
Fowlkes BJ, Pardoll DM.
Molecular and cellular events of T cell development.
Adv Immunol.
1989;44:207-264[Medline]
[Order article via Infotrieve].
28.
Rolink A, Haasner D, Nishikawa SI, Melchers F.
Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice.
Blood.
1993;81:2290-2300[Abstract/Free Full Text].
29.
Strasser A, Rolink A, Melchers F.
One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.
J Exp Med.
1989;170:1973-1986[Abstract/Free Full Text].
30.
Velardi A, Cooper MD.
An immunofluorescence analysis of the ontogeny of myeloid, T and B lineage cells in mouse hemopoietic tissues.
J Immunol.
1984;133:672-677[Abstract].
31.
Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K.
Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.
J Exp Med.
1991;173:1213-1225[Abstract/Free Full Text].
32.
Metcalf D, Moore MAS.
Hematopoietic Cells. Frontiers of Biology Vol 24. London: North Holland; 1971.
33.
Huang GY, Cooper ES, Waldo K, Kirby ML, Gilula NB, Lo CW.
Gap junction-mediated cell-cell communication modulates mouse neural crest migration.
J Cell Biol.
1999;143:1725-1734[Abstract/Free Full Text].
34.
Ewart J, Cohen MF, Meyer RA, et al.
Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene.
Development.
1997;124:1281-1292[Abstract].
35.
Thomas SA, Schuessler RB, Berul CI, et al.
Disparate effects of deficient expression of connexin43 on atrial and ventricular conduction evidence for chamber specific molecular determinants of conduction.
Circulation.
1998;97:686-691[Abstract/Free Full Text].
36.
Guerrero PA, Schuessler RB, Davis LM, et al.
Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.
J Clin Invest.
1997;99:1991-1998[Medline]
[Order article via Infotrieve].
37.
Akashi K, Kondo M, Schlageter AM, Weissman IL.
T-cell development from hematopoietic stem cells. In:
Monroe JG,Rothenberg EV, eds.
Molecular Biology of B-Cell and T-Cell Development. Totowa, NJ: Humana Press; 1998:305.
38.
Rosendaal M, Mayers A, de Koning A, Dunina-Barkovskaya T, Krenacs T, Ploemacher R.
Does transmembrane communication through gap junctions enable stem cells to overcome stromal inhibition?
Leukemia.
1997;11:1281-1289[Medline]
[Order article via Infotrieve].
39.
Meda P.
The role of gap junction membrane channels in secretion and hormonal action.
J. Bioenergetics and Biomembranes.
1996;28:369-377[Medline]
[Order article via Infotrieve].
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Abstract
Introduction