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Vol. 52, Issue 6, 1071-1080, 1997
2-Adrenergic
Receptor Subtypes in the Developing Mouse Embryo Suggests a Role for
the 2A Subtype in Apoptosis
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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2-Adrenergic receptors (2-ARs) respond to
norepinephrine and epinephrine to mediate diverse physiological
effects. Using in situ hybridization, the expression
pattern of the mRNA encoding the three 2-AR subtypes
(2A, 2B, and 2C) was
examined in the mouse embryo. The mRNA encoding the three subtypes was
first detected at stage 9.5 days postcoitus (d.p.c.) for the
2A-AR (coincident with norepinephrine availability),
11.5 d.p.c. for the 2B-AR, and 14.5 d.p.c. for
the 2C-AR subtype. The mRNA encoding the 2A-AR subtype shows both the earliest and the most
widespread expression pattern, including developing stomach and cecum,
many craniofacial regions and areas in the central nervous system. Strikingly, the 2A-AR mRNA is expressed in the
interdigital mesenchyme between stage 12.5 and 14.5 d.p.c. in
parallel with digit separation, raising the possibility that the
2A-AR might contribute to the apoptotic events
underlying this process. To test whether 2A-AR can
signal apoptotic events, the 2A-AR subtype was
introduced into two mouse mesenchymal cell lines,
C3H/10t1/2 and NIH-3T3; expression of the
2A-AR correlated with accelerated apoptosis, as detected
both by the TUNEL assay and the loss of cell viability. In contrast to
the wide distribution of mRNA encoding the 2A-AR subtype, the 2B-AR mRNA was detected only in the
developing liver and was most readily detectable between 11.5 and
14.5 d.p.c., when the liver is the principal site of
hematopoiesis. The 2C-AR mRNA is detected in the nasal
cavity and cerebellar primordium only at 
14.5 d.p.c. These studies
represent the first characterization of the temporal and spatial
expressions of the 2A-AR, 2B-AR, and
2C-AR subtypes during embryogenesis and provide
important insights concerning the loci and possible roles of
2-AR-mediated regulation of physiological processes
during the developmental program.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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2-ARs
in adult animals and humans respond to EPI and NE to modulate metabolic
effects in adipose, transepithelial Na+ and water
transport in renal and intestinal epithelial cells, suppression of
insulin release from 
cells of the pancreas, and attenuation of
neurotransmitter release in the central and peripheral nervous systems
(1). Consequences of activation of 2-AR in the
central nervous system include lowering of blood pressure, sedation,
enhanced anesthesia, suppression of pain perception, and suppression of
epileptogenesis (2).
Recently, 2-ARs have been demonstrated to
represent a family of three subtypes based on pharmacological (3) and
molecular cloning (4-7) strategies. Northern and in situ
hybridization analyses have identified mRNA expression of all three
2-AR subtypes in a wide variety of tissues and
organs that correspond to loci of known 2A-AR
regulated physiological functions, such as the central nervous system
(8, 9), adrenal gland, cardiovascular system, and intestine (3). In
contrast, virtually nothing is known about the localization and
physiological roles of the AR subtypes in early development.
Recent findings from Thomas et al. (10) and Zhou et
al. (11) indicate that the enzymes responsible for NE synthesis
are moderately detectable at 8.5 d.p.c. (the earliest time point
examined). The catecholamines dopamine and NE are first detectable at
days 9.5 and 10.5 d.p.c., respectively, and EPI is consistently
detected in fetuses older than 13.5 d.p.c. (10). NE and EPI
activate 2-ARs. Because the lack of NE
synthesis leads to embryonic lethality (10), the possibility arises
that particular 2-AR subtypes may play
important roles during development.
As a first step in exploring potential functions for
2-AR in early embryonic development, we
identified the distribution of the mRNA encoding
2A-AR, 2B-AR, and
2C-AR subtypes in the early developing mouse
embryo through the use of in situ hybridization techniques.
In addition, when we observed that the distribution of mRNA encoding
the 2A-AR subtype coordinated temporally and spatially with the programmed cell death of mesenchymal cells during
the digit formation, we tested the hypothesis that the 2A-AR is capable of inducing or accelerating
apoptosis in mesenchymal cells. The ability of the
2A-AR to accelerate apoptosis in these cells
reveals a heretofore unappreciated regulatory consequence of
2A-AR activation in mammalian systems.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Embryo collection and fixation.
The hybrid offspring between
129/SvEv and C57Black mice were used as mating pairs. Other embryos,
derived from mating of 129/SvEv purebred mice, B6D2 F1 hybrids, and ICR
mice, also were evaluated for comparison of mRNA expression patterns.
The pattern of the 2A-AR subtype expression
was indistinguishable in these varying genetic backgrounds. Embryos
harvested at 8.5 and 9.5 d.p.c. were fixed with 4%
paraformaldehyde at 4° for 2 hr. Embryos harvested at 12.5, 13.5, 14.5, 15.5, and 16.5 d.p.c. were perfused with cold 4%
paraformaldehyde, fixed overnight, transferred to 100% methanol, and
stored at 
20°.
Whole-mount in situ hybridization.
Digoxigenin-labeled cRNA probes encoding antisense (signal) and sense
(control) templates were synthesized from templates representing the
nearly full-length coding sequence of 2A-AR, 2B-AR, and 2C-AR
genes, as described previously (9). Whole-mount in situ
hybridization was performed as described previously (12) with minor
modifications of hybridization and washing temperatures as outlined
briefly below. After overnight hybridization at 70°, the
hybridization temperature was slowly reduced to 55° and maintained at
55° for 2 hr. The washing was performed at 55° before RNase A
treatment and 50° after RNase treatment. After whole-mount
hybridization, the embryos were washed with PBS (1× = 2.68 mM KCl, 1.47 mM KH2PO4, 136.9 mM NaCl, 8.06 mM
Na2HPO4·7 H2O, and 5.6 mM glucose, pH 7.4) and transferred into PBS containing
20% sucrose. Some embryos were sectioned after the hybridization and
wash steps.
2A-AR, 2B-AR, and
2C-AR subtypes were injected into the brain
cavity of early embryos to confirm the ability of the mRNA probes to
penetrate and identify the relevant mRNA in a receptor subtype-specific manner. These control studies both validated the selectivity of the
probes and ensured us that the lack of detectable endogenous mRNA
expression for a given 2-AR subtype at certain
embryonic stages was not a result of technical limitations in mRNA
detection.
Sectioning and evaluation of apoptosis in the embryo.
After
whole-mount in situ hybridization, the tissue of interest
was postfixed with 4% paraformaldehyde for 1 hr and sectioned with a
frozen microtome to the thickness of 60 µm. A hallmark of apoptotic
cells is DNA fragmentation, leading to the appearance of high
concentrations of 3
-OH ends of single- and double-stranded DNA; these
ends are detected by terminal deoxynucleotidyl transferase-catalyzed digoxigenin-dUTP end labeling of these 3-OH ends (TUNEL assay). Digoxigenin labeling is revealed using a fluorescein-conjugated anti-digoxigenin antibody fragment that lacks the Fc portion of the
antibody (Apop Tag Plus Kit; Oncor, Gaithersburg, MD). The TUNEL assay
was performed according to the instructions of the manufacturer, except
for the following modifications: (a) sections were rinsed twice with
0.2% Triton X-100 in PBS for 15 min before the reaction, (b) the
reaction was performed in 54 µl of working-strength terminal
deoxynucleotidyl transferase enzyme at 37° overnight, (c) sections
were rinsed twice with STOP/WASH buffer at 37° for 20 min twice and
with 2× standard saline citrate for 30 min three times, and (d) the
anti-digoxigenin-peroxidase incubation was prolonged to 2-4 hr and
washed four times for 30 min each.
Heterologous receptor expression and evaluation of apoptotic
signals in cultured cells.
To test the hypothesis that
2A-AR are capable of inducing or accelerating
apoptosis in mesenchymal cells, we introduced the 2A-AR into two cultured cell lines derived
from the mouse embryonic mesenchyme
(C3H/10t1/2 and NlH 3T3) through
electroporation of a cDNA encoding the epitope-tagged
2A-AR subtype (13). Cells were cultured in
Eagle's modified medium plus 10% fetal calf serum. For
electroporation, 1.5 × 107 cells in 0.4 ml
of OPTI-MEM (GIBCO BRL, Gaithersburg, MD) were incubated with 10 µg
of 2A-AR cDNA and/or 10 µg of pGREEN LANTERN cDNA (GIBCO BRL) and exposed to 280 V/975 µF in a Gene Pulser II
(BioRad, Hercules, CA). Cells were then incubated in Eagle's modified
medium plus 10% fetal calf serum for various times (see figure
legends) in the absence (control) or presence of 10 µM UK
14304, an 2-AR agonist, or yohimbine, an
2-AR antagonist. Drug-containing and control
media were changed every 12 hr. Control experiments comparing the
expression of epitope-tagged 2AAR, identified
immunochemically, and GFP, detected by microscopy with 610-nm
illumination, confirmed that there is a 100% coincidence of
coexpression of these two proteins when cotransfected via the procedures used in this study. Thus, after cotransfection of cDNAs encoding GFP and 2AAR, detection of expression
of GFP can be used as a marker for cells expressing the cDNA encoding
the 2AAR.
20°. The expression of epitope-tagged 2A-AR was demonstrated by immunofluorescence
microscopy after incubation with the 12CA5 monoclonal antibody directed
against the hemagglutinin epitope engineered into the amino terminus of the 2A-AR and a Cy3-conjugated donkey
anti-mouse secondary antibody, as described in detail previously (14).
Apoptotic events were measured in control versus
2A-AR-expressing cells using the TUNEL assay
as described above. Two alternative strategies for cell handling were
used, with comparable findings obtained. Cells were either plated onto
coverslips and examined immunochemically for
2A-AR expression and then via the TUNEL assay
for apoptosis or they were harvested, fixed, and then examined in
suspension for 2A-AR expression and apoptosis,
after which the cells were applied to microscope slides and sealed
under coverslips using Permamount.
Photography. Photography for the whole-mount embryos was performed under an Olympus SZH10 dissecting microscope. Photography for cultured cells and sectioned embryos was performed under a Zeiss Axioplan microscope. All photographs were made using Kodak Ektachrome 160 Tungsten film.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Using whole-mount in situ hybridization strategies with
digoxigenin-labeled cRNA probes, the expression pattern of the distinct mRNAs encoding the 2A-AR,
2B-AR, and 2C-AR
subtypes was examined in mouse embryonic stages 8.5-16.5 d.p.c. At
later stages of development, some of the hybridized embryos also were
sectioned using a cryostat to permit more detailed evaluation of the
pattern of expression of mRNA encoding the
2A-AR subtypes.
Temporal Expression
Fig. 1 summarizes, via a schematic
time-line, the onset of the expression of the mRNA for each
2-AR subtype during embryogenesis. For
comparison, the temporal expression of mRNAs encoding TH and DH,
enzymes responsible for the synthesis of NE, an endogenous ligand for
2-AR, also are shown (10, 11). The mRNA
encoding the 2A-AR subtype was detected
earliest and could be identified readily in the 9.5 d.p.c. embryo
of the mouse, a time that parallels the detection of catecholamines in
the brain cavity of embryonic mice (10). The mRNA encoding the
2B-AR subtype was detected only over a narrow
time frame during development (11.5-14.5 d.p.c.). The mRNA encoding
the 2C-AR subtype was not detected until
14.5 d.p.c. and was still evident at 16.5 d.p.c.
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Spatial Expression
2A-AR subtype.
The expression of mRNA encoding
the 2A-AR, first detected at 9.5 d.p.c.,
was distributed in the developing somites and in scattered cells
covering the neural tube. The segmental expression of the
2A-AR in the somites lasts until 14.5 d.p.c. but is no longer detected in 15.5 d.p.c. embryos (Fig.
2). However, after 10.5 d.p.c., the
2A-AR mRNA can be detected in many regions, including a variety of craniofacial areas and developing limbs. The
regional distribution of the 2A-AR through
development is given (see Figs. 2, 3, 4, 5). Fig.
2 provides a whole-embryo view at days
9.5 (Fig. 2A) and 10.5 (Fig. 2B) d.p.c. and a view of bisected embryos
at 14.5 d.p.c. (Fig. 2, D- F). Also shown is the regional
expression of mRNAs encoding the 2A-AR subtype
at 11.5-14.5 d.p.c. (see Figs. 3, 4, 5).
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2A-AR mRNA is prominently expressed in the
maxillary arch, hyoid arch (Figs. 2B and
3A), mesenchyme cells covering the
teleocephalon, brain mesoderm (Figs. 2A and
4A), and mesenchyme condensation forming
future falx cerebri (Figs. 3C and 4B). In embryos harvested from
10.5-14.5 d.p.c. embryos, 2A-AR mRNA is
detected in mesenchyme of the nasal septum next to the posterior naris
(Fig. 3, B, C, and F). From 13.5 to 16.5 d.p.c., this expression
of 2A-AR mRNA in the mesenchymal cells spreads
to all areas of the nasal cavity, which parallels, temporally, the
folding and expansion of this cavity. At the lower and central sides of
the anterior naris, expression of 2A-AR mRNA
is first detected at 11.5 d.p.c. (Fig. 3, C and F) and then
becomes very prominent in embryos harvested at 12.5 and 13.5 d.p.c. (Fig. 3, B-E).
The mRNA encoding the 2A-AR subtype also is
detected in other craniofacial areas, such as the external auditory
meatus and the condensation of perioptic mesenchyme (ectomeninx), which
subsequently differentiates to form the sclera in 11.5 and 12.5 d.p.c. embryos (Fig. 3E). Expression of 2A-AR
mRNA also is noted in the tissues covering the olfactory bulb from 10.5 and 16.5 d.p.c. (Fig. 3) and in the submandibular gland in embryos
between 14.5 to 16.5 d.p.c. (data not shown).
In addition to its craniofacial distribution, mRNA encoding the
2A-AR subtype is detected in mesoderm of the
back of the cervical region in embryos harvested between 10.5 and
12.5 d.p.c. (Fig. 4A). The expression of
2A-AR mRNA also is noted between 10.5 and
14.5 d.p.c. in the developing cecum (a limited area of midgut loop
within the physiological umbilical hernia; Figs. 4C and
5), in the stomach between 11.5 and
12.5 d.p.c. (Fig. 4C), and in the genital tubercle in embryos
harvested at 14.5 and 15.5 d.p.c. (data not shown).
A striking and unexpected finding of this study was the detection of
2A-AR mRNA in the mesenchyma of the
interdigital areas in the developing limbs between 12.5 and 14.5 d.p.c. of embryonic development (Fig. 6),
closely paralleling the apoptotic regression leading to digit
separation. This interdigital expression of
2A-AR mRNA is significantly intensified at the
developmental stage of 13.5 d.p.c. (Fig. 6B) in comparison to
12.5 d.p.c. (Fig. 6A). At 14.5 d.p.c.,
2A-AR mRNA is mainly detected at the base and remaining web of the nearly separated digits, as well as at newly forming finger joints (Fig. 6C). The 2A-AR
mRNA is no longer detected in the 15.5 d.p.c. limb, a time when
digit separation is fully completed (data not shown). We also observed
that 2A-AR expression in the forelimb (Fig. 6,
A and C) is always slightly more advanced than that of the hindlimb, as
can be seen in a comparison of 2A-AR mRNA
expression at stages 12.5 d.p.c. (Fig. 6A) and 14.5 d.p.c.
(Fig. 6C). This temporal pattern is, again, consistent with the time
course of limb morphogenesis (15-17). Sectioning of the limb after
in situ hybridization reveals that only interdigital mesenchyme expresses 2A-AR mRNA (Fig. 6, D and
E). After sectioning, a hollowed indentation is detected between
mesenchymal cells that have expanded as the embryo gets older,
presumably as a result of mesenchyme regression after apoptosis (Fig.
6, D and E). Evaluation of the apoptotic zone in limb sections via the
TUNEL assay demonstrates a close spatial correlation between cells
undergoing apoptosis and cells expressing
2A-AR mRNA (Fig.
7).
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2A-AR
mRNA and mesenchymal regression in digit separation suggested the
possibility that the 2A-AR induces apoptosis
in embryonic mesenchymal cells. To evaluate whether
2A-ARs are capable of evoking apoptotic
events, two mouse embryonic mesenchymal cell lines,
C3H/10t1/2 and NIH-3T3 cells, were used as
model systems to determine whether activation of
2A-AR can induce apoptosis. A cDNA encoding an epitope-tagged 2A-AR (13) was cotransfected
via electroporation into cells with a cDNA encoding GFP Lantern.
Control studies confirmed that immunodetection of heterologously
expressed 2A-AR always coincided with GFP
expression, so monitoring of GFP expression permitted detection of
2A-AR-expressing cells after electroporation. We observed (Fig. 8) that cells expressing
2A-AR disappeared more rapidly in response to
the 2-agonist UK 14304 compared with control
cells (non-GFP-expressing cells) cultured in the same dish, implying
that the 2A-AR-expressing cells were excluded from the cell population, likely via apoptosis, due to activation of
the transfected 2A-AR receptor. To test the
correlation between apoptotic events and 2A-AR
expression more directly,
C3H/10t1/2 (Fig. 9) or NIH-3T3 (not shown) cells
transiently expressing 2A-AR cells were
incubated in the presence of an 2A-AR agonist,
UK14304 (10 µM) or, alternatively, an
2A-AR antagonist, yohimbine, and 2A-AR expression, as detected
immunochemically, was correlated with apoptotic events detected in the
TUNEL assay (see Materials and Methods). As shown in Fig. 9, apoptotic
nuclei were detected in 2A-AR-expressing
cells, identified via the 12CA5 antibody directed against the
amino-terminal hemaglutinin epitope tag engineered into the
2A-AR (13). When findings from 500-1000 cells
examined in random fields by two investigators blinded to the cellular treatment were tabulated at each time point, it was observed that 2A-AR-expressing cells manifest an increased
appearance of apoptotic nuclei and fragmented cells compared with
nonexpressing cells in the same culture dish (Fig. 9C) or control cells
never transfected with the 2-AR cDNA (not
shown). Furthermore, treatment with the 2A-AR
agonist seemed to accelerate the apoptotic process. The fact that
antagonist treatment delayed but did not entirely prevent the
2A-AR-dependent increase in apoptotic events
is likely due to the known capability of the
2A-AR to undergo agonist-independent conformational changes that lead to productive receptor/G
protein/effector interactions in target cells (18).
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2B-AR and 2C-AR subtypes.
A
moderate amount of mRNA encoding the 2B-AR
subtype was detected only in the liver and only in embryos harvested
between 11.5 and 13.5 d.p.c.(Fig.
10).
|
2C-AR subtype
is detected only very late during development (see Fig. 1). At
14.5 d.p.c., mRNA encoding 2C-AR is
weakly detectable in the nasal cavity and a part of cerebellar
primordium; this distribution pattern remains the same until embryonic
stage 16.5 d.p.c.
We are confident that the lack of detection of mRNA encoding the
2B-AR and 2C-AR
subtypes during differing developmental stages is not due to technical
limitations because we readily detected a strong signal for
hybridization to mRNA encoding these two subtypes when
2B-AR- or
2C-AR-expressing cells were injected into the
embryonic cavity before fixation and processing in parallel control
experiments.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The current results have revealed that the mRNAs encoding three
2A-AR subtypes display distinctive temporal
and spatial distribution patterns during embryonic development of the
mouse. The temporal expression of the 2A-AR
mRNA coincides with the first detection of the catecholamines, NE and
EPI, in the mouse central nervous system (10) and occurs 1 day later
than expression of mRNA encoding two critical enzymes in catecholamine
biosynthesis, TH and DH (10, 11). The observation that regional
distribution of 2A-AR mRNA occurs in a variety
of embryonic areas, including stomach and developing cecum (Figs. 4C
and 5C), interdigital mesenchyme (Fig. 6) of developing limbs during
the interim of digit separation, many craniofacial areas (Figs. 3, 4, 5),
and limited regions in the brainstem (Fig. 5D) implies that effects of
NE during development in these areas might be mediated by the
2A-AR subtype. Interestingly, expression of
2A-AR corresponds to a number of regions that
undergo rapid cell proliferation, including stomach at 11.5 and
12.5 d.p.c. and cecum between 10.5 and 14.5. Because the
2A-AR has been demonstrated to be capable of
activating the mitogen-activated protein kinase pathway (19-22), it is
possible the 2A-AR modulates critical
proliferation events during development.
Because it has been postulated that tissues with high proliferative
activity may be more likely to undergo programmed cell death or
apoptosis (23), it was of particular interest to note that
2A-AR expression also occurs in some cells
that undergo programmed cell death (Fig.6, A and C), such as the
mesenchyme in the interdigital region of the limb bud from 12.5-14.5
d.p.c. (15-17, 23). Our results show that the onset and disappearance of 2A-AR mRNA expression are slightly advanced
in the forelimb compared with hindlimb buds (Fig. 6), a temporal
expression pattern that matches the time frame of forelimb and hindlimb
development (24). This temporal and spatial pattern of
2A-AR mRNA expression in the interdigital
areas suggested that 2A-AR may be related to
pattern formation of the limb (i.e., apoptotic events responsible for
digit separation), perhaps in response to available norepinephrine. Our
findings in C3H/10t1/2 and NIH-3T3 cells
demonstrate that 2A-AR can accelerate
apoptotic events, as demonstrated by accelerated cell lysis and
appearance of apoptotic nuclei (Figs. 9 and 10). These findings are the
first demonstration that mRNA encoding 2A-AR
is expressed in apoptotic mesenchyme cells and that
2A-AR can evoke apoptotic signals when
examined in a mesenchymal cell preparation after heterologous
expression.
Recently, Zou and Niswander (25) demonstrated that introduction of a
dominant negative form of BMP receptor, dnBMPR-1B, into developing
chick limb resulted in a reduction in apoptosis, truncation of digits,
and conversion of scales to feathers, suggesting that the BMP signaling
pathway is involved in apoptosis of digit separation. Interestingly,
BMP has been shown to be a critical factor in the development of
sympathetic (catecholamine-synthesizing) neurons and neuronal
regeneration (26, 27). Our findings that the
2A-AR facilitates apoptosis in embryonic
mesenchymal cells and that the expression of mRNA encoding the
2A-AR directly parallels interdigital
mesenchymal regression suggest that the sympathetic nervous system,
regulated in its development by BMP, may play a role in programmed cell
death essential for digit formation. Even if
2A-ARs do modulate the apoptotic events that
contribute to digit formation, however, these receptor-mediated events
cannot be solely responsible for digit formation because altered digits are not noted in mice expressing a mutant
2A-AR (D79N) that behaves as a functional
knockout for this receptor subtype (28). However, we do know that the
spatial and temporal expression of mRNA encoding all three
2-AR subtypes is indistinguishable in
wild-type and D79N 2-AR
mice.(null)2 Thus, functional redundancy mediated
by the 2B-AR or 2C-AR subtype in digit formation is unlikely; it is more likely is that the
2A-AR, although modulatory, is not essential
for this mesenchymal apoptotic event. The molecular signals modulated
by 2A-AR during this window of embryonic
development have yet to be clarified.
The temporal and spatial expression of the mRNAs encoding the
2B-AR and 2C-AR also
is of interest. For example, from the period of 11.5-14.5 d.p.c., the
2B-AR mRNA is detected in the liver, which
serves as a blood-forming organ in embryonic development. This profile
of 2B-AR mRNA expression suggests that the
2B-AR subtype may play a role in embryonic
hematopoiesis.
The current study represents the first comprehensive description of the
pattern of expression of 2A-AR,
2B-AR, and 2C-AR subtypes in developing mouse embryo. The subtype-specific temporal and
spatial patterns of expression suggest that the existence of
2-AR subtypes, all of which couple to the same
effectors and are comparable in their affinity for the endogenous
agonist ligands EPI and NE, may serve as a means to provide diversity
in the timing or localization of NE-responsive receptors (29);
presumably, the 5 and 3 regulatory regions of the intronless genes
for the 2-AR subtypes define subtype-specific
receptor patterning. Taken together, the correlation of
2A-AR mRNA distribution with mesoderm regression, the observation that 2A-AR can
facilitate apoptosis in cultured mesenchymal cells, and the ability of
particular G protein-coupled receptors to induce apoptosis in some
settings (29, 30) suggest that 2A-AR may
control apoptotic events, such as those leading to digit separation,
worthy of future exploration in other cellular settings. The patterns
of expression noted for all three subtypes may reveal
as-yet-unappreciated roles for all of these
2-AR subtypes during embryonic development.
| |
Acknowledgments |
|---|
We want to acknowledge the enthusiastic assistance of Carol Ann
Bonner in culturing 2-AR-expressing cell lines
for these experiments. We are also grateful to Dr. Brigid Hogan and her laboratory colleagues for advice concerning whole-mount in
situ hybridization and embryo sectioning. L. E. L. thanks Dr.
David Piston for advice on handling data files of morphological data and Dr. Tom Jetton for assistance in preparation of the figures.
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Footnotes |
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Received June 27, 1997; Accepted August 11, 1997
1 Current affiliation: BC Cancer Research Center, Vancouver, British Columbia, Canada, V5V 1L3.
2 R.-X. Wang and L. E. Limbird, unpublished observations.
This work was supported by National Institutes of Health Grant HL43671.
Send reprint requests to: Lee E. Limbird, Ph.D., Department of Pharmacology, Vanderbilt University Medical Center, 476 MRB I, Nashville, Tennessee 37232-6600. E-mail: lee.limbird{at}mcmail.vanderbilt.edu
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Abbreviations |
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d.p.c., days postcoitus;
AR, adrenergic
receptor;
EPI, epinephrine;
NE, norepinephrine;
TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP-biotin end labeling;
TH, tyrosine hydroxylase;
DH, dopamine -hydroxylase;
PBS, phosphate-buffered saline;
GFP, green fluorescent protein;
BMP, bone
morphogenetic protein.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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|---|
| 1. | Limbird, L. E. Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms. FASEB J. 2:2686-2695 (1988)[Abstract]. |
| 2. |
Ruffolo, R. R., Jr.,
A. J. Nichols,
J. M. Stadel, and
J. P. Hieble.
Pharmacologic and therapeutic applications of 2-adrenoceptor subtypes.
Annu. Rev. Pharmacol. Toxicol.
32:243-279 (1993).
|
| 3. |
Bylund, D. B.
Subtypes of 1- and 2-adrenergic receptors.
FASEB J.
6:832-839 (1992)[Abstract].
|
| 4. | Chruscinski, A. J., R. E. Link, D. A. Daunt, G. S. Barsh, and B. K. Kobilka. Cloning and expression of the mouse homolog of the human alpha2-C2 adrenergic receptor. Biochem. Biophys. Res. Commun. 186:1280-1287 (1992)[Medline]. |
| 5. |
Fraser, C. M.,
S. Arakawa,
W. R. McCombie, and
J. C. Venter.
Cloning, sequence analysis, and permanent expression of a human 2-adrenergic receptor in Chinese hamster ovary cells: evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation.
J. Biol. Chem.
264:11754-11761 (1989) |
| 6. |
Guyer, C. A.,
D. A. Horstman,
A. L. Wilson,
J. D. Clark,
E. J. Cragoe, Jr., and
L. E. Limbird.
Cloning, sequencing, and expression of the gene encoding the porcine 2-adrenergic receptor: allosteric modulation by Na+, H+, and amiloride analogs.
J. Biol. Chem.
265:17307-17317 (1990) |
| 7. |
Kobilka, B. K.,
H. Matsui,
T. S. Kobilka,
T. L. Yang-Geng,
U. Franck,
M. G. Caron,
R. J. Lefkowitz, and
J. W. Regan.
Cloning, sequencing, and expression of the gene coding for the human platelet alpha2-adrenergic receptor.
Science (Washington D. C.)
238:650-656 (1987) |
| 8. |
Scheinin, M.,
J. W. Lomasney,
D. M. Hayden-Hixson,
U. B. Schambra,
M. G. Caron,
R. J. Lefkowitz, and
R. T. Fremeau, Jr.
Distribution of 2-adrenergic receptor subtype gene expression in rat brain.
Mol. Brain Res.
21:133-149 (1994).
[Medline] |
| 9. |
Wang, R.-X.,
L. B. MacMillan,
R. T. Fremeau, Jr.,
M. A. Magnuson,
J. Linder, and
L. E. Limbird.
Expression of 2-adrenergic receptor subtypes in the mouse brain: evaluation of spatial and temporal information imparted by 3 kb of 5 regulatory sequence for the 2AAR gene in transgenic animals.
Neuroscience
74:199-218 (1996)[Medline].
|
| 10. | Thomas, S. A., A. M. Matsumoto, and R. D. Palmiter. Noradrenaline is essential for mouse fetal development. Nature (Lond.) 374:643-646 (1995)[Medline]. |
| 11. | Zhou, Q. Y., C. J. Quaife, and R. D. Palmiter. Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development. Nature (Lond.) 374:640-643 (1995)[Medline]. |
| 12. | Wilkinson, D. G. Whole mount in situ hybridization of vertebrate embryos, in In Situ Hybridization: A Practical Approach (D. G. Wilkinson, ed.). Oxford University Press, Oxford, UK, 75-83 (1992). |
| 13. |
Keefer, J. and
L. E. Limbird.
The 2A-adrenergic receptor is targeted directly to the basolateral membrane domain of Madin-Darby canine kidney cells independent of coupling to pertussis toxin-sensitive GTP-binding proteins.
J. Biol. Chem.
268:11340-11347 (1993) |
| 14. |
Wozniak, M. and
L. E. Limbird.
The three 2A-adrenergic receptor subtypes achieve basolateral localization in Madin-Darby canine kidney II cells via different targeting mechanisms.
J. Biol. Chem.
271:5017-5024 (1996) |
| 15. | Zakeri, Z. F., D. Quaglino, T. Latham, and R. A. Lockshin. Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J. 7:470-478 (1993)[Abstract]. |
| 16. | Zakeri, Z., D. Quaglino, and H. S. Ahuja. Apoptotic cell death in the mouse limb and its suppression in the hammertoe mutant. Dev. Biol. 165:294-297 (1994)[Medline]. |
| 17. | Mori, C., N. Nakamura, S. Kimura, H. Irie, T. Takigawa, and K. Shiota. Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation. Anat. Record 242:103-110 (1995)[Medline]. |
| 18. |
Neubig, R. R.,
R. D. Gantzos, and
W. J. Thomsen.
Mechanism of agonist and antagonist binding to 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex.
Biochemistry
27:2374-2384 (1988)[Medline].
|
| 19. |
Ablas, J.,
E. J. van Corven,
P. L. Hordiji,
G. Milligan, and
W. H. Moolenaar.
Gi- mediated activation of the p21ras-mitogen-activated protein kinase pathway by 2- adrenergic receptors expressed in fibroblasts.
J. Biol. Chem.
268:22235-22238 (1993) |
| 20. | Anderson, N. G. and G. Milligan. Regulation of p42 and p44MAP kinase isoforms in rat-1 fibroblasts stably transfected with alpha2-C10 adrenoceptors. Biochem. Biophys. Res. Commun. 200:1529-1535 (1994)[Medline]. |
| 21. |
Bouloumie, A.,
V. Planat,
J.-C. Devedjian,
P. Valet,
J.-S. Saulnier-Blache,
M. Record, and
M. Lafontan.
2-Adrenergic stimulation promotes preadipocyte proliferation.
J. Biol. Chem.
269:30254-30259 (1994) |
| 22. |
Faure, M.,
T. A. Voyno-Yasenetskaya, and
H. R. Bourne.
cAMP and subunits of  heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells.
J. Biol. Chem.
269:7851-7854 (1994) |
| 23. | Sulik, K. K., C. S. Cook, and W. S. Webster. Teratogen and craniofacial malformations: relationships to cell death. Dev. Suppl. 103:213-232 (1988). |
| 24. | Kaufmen, M. H. The Atlas of Mouse Development. Academic Press, London (1991). |
| 25. | Zou, H. and L. Niswander. Requirement for BMP signaling an interdigital apoptosis and scale formation. Science (Washington D. C.) 272:738-741 (1996)[Abstract]. |
| 26. | Lein, P., M. Johnson, X. Guo, D. Rueger, and D. Higgins. Osteogenic bone morphogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron 15:597-605 (1995)[Medline]. |
| 27. | Lein, P., X. Guo, A. M. Hedges, D. Rueger, M. Johnson, and D. Higgins. The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons. Int. J. Dev. Neurosci. 14:203-215 (1996)[Medline]. |
| 28. |
MacMillan, L. B.,
H. Lutz,
M. S. Smith,
M. T. Piascik, and
L. E. Limbird.
Central hypotensive effects of the 2a-adrenergic receptor subtype.
Science (Washington D. C.)
273:801-803 (1996)[Abstract].
|
| 29. |
Yamada, T.,
M. Horiuchi, and
V. J. Dzau.
Angiotensin II type 2 receptor mediates programmed cell death.
Proc. Natl. Acad. Sci. USA
93:156-160 (1996) |
| 30. | Yamatsuji, T., T. Matsui, T. Okamoto, K. Komatsuzaki, S. Takeda, H. Fukumoto, T. Iwatsubo, N. Suzuki, A. Asami-Odaka, S. Ireland, T. B. Kinane, U. Giambarella, and I. Nishimoto. G protein-mediated neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP. Science (Washington D. C.) 272:1349-1352 (1996)[Abstract]. |
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