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INTRODUCTION |
The cAMP-dependent protein kinase
(PKA)1 is a multisubstrate
serine/threonine protein kinase responsible for modulating a vast number of cell physiological processes ranging from the maintenance of
basal transcription of specific genes (1, 2) to rapid reorganization of
the cytoskeleton (3, 4). The mechanism by which PKA modulates cellular
physiology is by covalent modification of polypeptide substrates via
reversible enzymatic transfer of a phosphate moiety to a consensus
phosphorylation motif in the target protein (5). The number and range
of protein substrates phosphorylated by PKA is as varied as the cell
physiological processes which it is known to regulate. These substrates
occur in all cellular compartments, including the nucleus, cytoplasm,
and plasma membrane (6).
The PKA holoenzyme is a heterotetramer composed of two N-terminally
dimerized regulatory (R) subunits combined with two catalytic (C)
subunits (5). PKA is unique in that the regulatory domain of the enzyme
is encoded by separate genes located on different chromosomes. PKA is
also unique in that the mechanism of activation involves simple subunit
dissociation upon binding of cAMP to the regulatory subunits, thereby
liberating catalytically active C subunits that diffuse throughout the
cell, phosphorylating protein substrates containing appropriate
consensus phosphorylation motifs.
Four regulatory (RI
, RII, RI
, and RII) and two catalytic
(C and C) isoform genes have been described in the mouse (7). Messenger RNA and protein for the isoforms of PKA (RI, RII, and C) are found ubiquitously in the mouse and are expressed during
early embryogenesis, whereas the isoforms (RI, RII, and C)
have more tissue-specific patterns of expression. RI expression is
restricted to neurons, whereas RII expression is highest in brain
and brown and white adipose tissues (8, 9). Expression of C is
highest in brain with lower levels in all tissues (10). The pattern of
expression of R and C isoforms has been examined at day 14 of
embryogenesis in the mouse (11). However, a more detailed study of the
expression and function of PKA isoforms during early stages of
mammalian development is lacking.
The cAMP/PKA signaling system is likely to play an important role in
early development, and several lines of experimentation have suggested
important pathways that are modified by PKA activity. A potential
requirement for PKA activity in the regulation of zygotic gene
activation in the preimplantation mouse embryo has been documented (12,
13). Studies of Drosophila imaginal disks demonstrate that
imaginal disk cells lacking PKA activity behave as if they have
received an excessive sonic hedgehog signal, resulting in
dramatic pattern re-specifications (14-18). These observations led to
the hypothesis that PKA acts to antagonize sonic hedgehog signaling and thus repress hedgehog-responsive genes such as
decapentaplegic, patched, and
wingless. By using constitutively active and dominant negative forms of PKA, two independent studies have shown that PKA acts
as a common negative regulator of sonic hedgehog signaling in the zebrafish embryo (19-21). Apart from the role of PKA as a
general negative regulator of sonic hedgehog signaling,
research to date has not uncovered any other specific roles for PKA in vertebrate development. One recent report (22) has implicated PKA in
even earlier stages of vertebrate embryogenesis. In this study it was
demonstrated that dissociated zebrafish blastula cells required PKA
activity for activin induction of the early mesoderm marker genes
goosecoid and no tail. However, only mild effects
were observed in vivo by injection of a dominant negative regulatory subunit of PKA on goosecoid and no
tail expression. Furthermore, when Joore et al. (22)
injected 2-4 cell stage zebrafish embryos with a constitutively active
form of the catalytic subunit of PKA, it had no effect. This is in
direct contrast to injections of a similar construct into 2-4 cell
stage zebrafish embryos made by Hammerschmidt and McMahon (20), who
observed that blastula stage zebrafish embryos were exquisitely
sensitive to a constitutively active form of the catalytic subunit of
PKA, resulting in failure of a large fraction of these embryos. Recent evidence in our laboratory has confirmed the sensitivity of vertebrate embryos to a constitutively active form of PKA catalytic
subunit.2 In this study we
present evidence that increased basal PKA activity resulting from
targeted disruption of the RI isoform of PKA affects signaling in
the primitive streak, causing profound deficits in the production of
all mesoderm derivatives including the heart.
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EXPERIMENTAL PROCEDURES |
Mapping the RI Gene--
The mouse RI genomic sequences
were isolated from a Charon 4A 
phage library containing partial
HaeIII/AluI fragments of BALB/c mouse
genomic DNA. The promoter sites for the mouse RI gene were
determined by primer extension and S1 nuclease mapping.
RIREC3 Targeting Vector--
In order to make a targeted
disruption in the RI gene, a 5.0-kb genomic HindIII
fragment was isolated from a 129svJ mouse phage library using
plaque hybridization screening with a 514-bp XhoI-EcoRI [32P]cDNA probe from
RI. A genomic fragment was isolated that contains a 5' 3.2-kb
intron, the 170-bp exon 3, and 1.5-kb of 3' sequence including exons 4 and 5. This genomic fragment was then subcloned into a pUC18 vector.
The vector was linearized at the XhoI site in exon 3 corresponding to amino acid 77 in the RI coding sequence. A neomycin
resistance cassette containing the SV40 promoter, the neomycin
phosphotransferase cDNA, and poly(A) sequence was blunt end-ligated
into the XhoI site. A thymidine kinase cassette containing the HSVtk promoter, the thymidine kinase cDNA, and poly(A) sequence was inserted 5' to the genomic sequence using EcoRI and
BamHI sites in the pUC18 polylinker. This replacement type
targeting vector was designated RIREC3 and was linearized at the
BamHI site before electroporation into ES cells.
Transfection of ES Cells and Generation of Germ Line
Chimeras--
The generation of germ line-competent ES cells in our
laboratory has been described (23). REK2 or REK3 ES cells were
electroporated with 25 µg of BamHI-linearized RIREC3
per 107 cells using a ProgenitorTM II PG200
electroporator (Hoefer Scientific Instruments) and subsequently grown
under double selection in G418 (280 µg/ml) and ganciclovir (2 µM). DNA from ES cell clones was analyzed by Southern
blot using a 1-kb SacI/HindIII RI genomic
probe located 3' to the targeting construct. Positively targeted clones
were expanded and injected into C57BL/6 blastocysts that were
subsequently implanted into BL6/CBAF1 pseudopregnant females. The
resulting male chimeras were mated with C57BL/6 females, and agouti
offspring were genotyped by Southern blot analysis. Both REK2- and
REK3-derived male chimeras passed the mutation through the germ line
resulting in heterozygous agouti offspring. Heterozygous agouti mice
were then interbred and thus maintained on the mixed (129svJ × C57BL/6) background for all subsequent analyses.
Generation of RI+/
C+/ Animals
for Interbreeding--
Targeting of the C gene and creation of C
null mutant mice are the subject of a separate study (24). In order to
generate RI+/C+/ mice, RI
heterozygous female mice were crossed to C heterozygous male mice,
which are both on the 129svJ-C57BL/6 mixed background. Genotyping of
offspring for RI was carried out using the Southern blot strategy
described above. Genotyping of offspring for C was carried
out using a Southern blot strategy in which a 315-bp HindIII/EcoRI probe located outside the C
targeting construct was hybridized to BamHI-digested genomic
tail DNA. The targeted C allele yields a 1.4-kb band, whereas the
wild type allele yields a 4.1-kb band.
PCR Analysis of Yolk Sac DNA--
Individual yolk sacs were
dissected away from the embryos in M2 media (Sigma) and transferred to
1× SET buffer (10 mM Tris, pH 7.4, 1 mM EDTA,
1% SDS) followed by overnight digestion with 300 µg/ml proteinase K
in a 45 °C water bath. Yolk sac DNA was then extracted twice with
phenol/chloroform and precipitated. DNA was resuspended in a total
volume of 25 and 1 µl of the resuspended yolk sac DNA was used for
PCR analysis with TaqDNA polymerase (PEC) using the
following oligos at 1 µM final concentration: (A) RI 5' exon 3, 5'-GAGGAGGCAAGACAGAT-3'; (B)
RI 3' exon 3, 5'-CTTTCTAACGTAGGAGG-3'; (C) Neo 5'-oligo,
5'-TCGCATGATTGAACAAG-3'; (D) Neo 3'-oligo,
5'-AAGCACGAGGAAGCGGT-3'. Reaction conditions for RI exon 3 oligos
were 94 °C for 1 min, 45 °C for 1 min, and 72 °C for 1.5 min
for 35 cycles. Reaction conditions for the neomycin oligos were
94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min for 35 cycles. Reaction conditions were optimized using the PCR
OptimizerTM Kit (Invitrogen). In order to genotype embryos
from RI+//C+/ × RI+//C+/ intercrosses, yolk sac DNA
was isolated as described above, and the following two sets of
C-specific primers were used to genotype for C: (A)
C 5' exon 6, 5'-CTGACCTTTGAGTATCTGCAC-3'; (B) C 3'
exon 7, 5'-GTCCCACACAAGGTCCAAGTA-3'; (C) Neo 5'-oligo,
5'-GTGGTTTGTCCAAACTCATCAATGT-3'; (D) C 3' intron 7, 5'-AGACTACTGCTCTATCACTGA-3'. Reaction conditions for the C
exon 6 and 7 oligos were 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s for 30 cycles. Reaction conditions for the Neo and C intron 7 oligos were 94 °C for 30 s, 62 °C
for 30 s, and 72 °C for 1 min for 30 cycles.
Western Analysis and Kinase Assays--
For Western analysis
only, E8.5 embryos were isolated in M2 media, rinsed through several
changes of ice-cold PBS, and placed directly into 20 µl of 1×
Laemmli sample buffer and boiled for 5 min. Lysates were loaded
directly onto 10% SDS-PAGE gels and subsequently transferred to
nitrocellulose membranes. Blots were blocked overnight, probed with an
affinity-purified polyclonal antibody to RI, and visualized using
the Amersham Biosciences ECLTM system. Kinase assays were
performed as described previously (8). Briefly, E8.5 embryos were
isolated in M2 media, rinsed through several changes of ice-cold PBS,
and subsequently homogenized on ice in 150 µl of lysis buffer (20 mM Tris, pH 7.5, 250 mM sucrose, 0.1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 10 mM dithiothreitol, 1 µg/ml leupeptin, 3 µg/ml
aprotinin, 40 µg/ml soybean trypsin inhibitor, 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride) in a Dounce homogenizer
followed by sonication using a Branson Sonifier 250 (5 pulses: output
3, duty cycle 50%). Homogenates were then snap-frozen and stored at
80 °C. Homogenates were thawed on wet ice and kinase assays
performed in triplicate in the presence and absence of 5 µM cAMP with Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) as
the substrate. Assays were also performed in the presence of 40 µg/ml
protein kinase inhibitor peptide-(5-24) to demonstrate that the kinase
activity was PKA-specific (25). Bradford assays (Bio-Rad) were
performed to determine protein concentration, and kinase activity was
normalized to milligrams of protein.
HPLC Analysis--
HPLC analysis of RI wild type and knockout
E8.5 primary embryonic fibroblasts was performed as described (26).
Briefly, fibroblasts were grown on gelatin-coated 60-mm dishes in 10%
FBS/DMEM and incubated in a 5% CO2 incubator at 37 °C.
Cells were lysed in homogenization buffer and stored at 80 °C
until the day of the assay. The sample was diluted with homogenization
buffer to a final concentration of 1-2 mg/ml, loaded onto a DEAE/HPLC
column, and eluted using a linear salt gradient from 0 to 250 mM. Immediately following the HPLC separation, fractions
were collected and assayed for kinase activity in the presence and
absence of 5 µM cAMP with Kemptide as a substrate
(8).
Histology--
Embryos were isolated in M2 media and fixed in
Methacarnoys fixative (3:1.5:0.5 methanol/chloroform/glacial
acetic acid) at room temperature. After fixation, embryos were
processed through methanol (3 times for 15 min), methyl benzoate (1 times for 30 min), and xylenes (2 times for 15 min) and embedded in
paraffin. Paraffin blocks were sectioned at 8 µm on a Reichert-Jung
microtome, and histological sections were stained with hematoxylin and
eosin. Sections were subsequently dehydrated through graded ethanols into xylenes and coverslipped with PermountTM. Embryo
sections were photographed on a Nikon Microphot-FXA microscope.
Whole Mount in Situ Analysis--
Whole mount in situ
analysis was performed as described (27). The following cRNA probes
were generated as runoff transcripts using T3, T7, or SP6 RNA
polymerase to generate sense and antisense probes: (A)
Brachyury (gift from Virginia Papaioannou) linearized with BamHI (antisense) or HindIII (sense) and
(B) Pax3 (gift from Peter Gruss) linearized with
HindIII (antisense) or PstI (sense). Whole mounts
were photographed on a Nikon SMZ-U Dissecting Scope.
Generation of Primary Embryonic Fibroblasts--
The derivation
of primary embryonic fibroblasts has been described (28). Briefly, E8.5
mouse embryos were dissected out from the decidua, and all decidual
tissues were removed from the yolk sacs, including Reichardt's
membrane. Yolk sacs were removed from each individual embryo and kept
for PCR analysis, and the amniotic membrane and allantois were removed
and discarded. RI mutant E8.5 embryos were easily identified by
their morphology. Mutant or wild type embryos were pooled and placed in
the tip of a 1-ml syringe in PBS and then triturated several times with a 20-gauge needle into a 96-well plate containing 10% FBS/DMEM and
subsequently incubated in a 5% CO2 incubator at 37 °C.
Primary embryonic fibroblasts were expanded from 96- to 24-well to
60-mm dishes over several weeks. The genotype of RI mutant primary embryonic fibroblasts was confirmed by yolk sac PCR and Western analysis.
Cell Cycle Analysis Using Primary Embryonic
Fibroblasts--
Analysis of cell cycle using DAPI staining has been
described (29, 30). Briefly, subconfluent RI mutant or wild type primary embryonic fibroblasts growing in 10% FBS/DMEM in 5%
CO2 at 37 °C were gently trypsinized and subsequently
washed twice through PBS and resuspended in 200 µl of buffer
containing 4,6-diamino-2-phenylindole (10 µg/ml DAPI, 0.1% Nonidet
P-40, 20 mM Tris, pH 7.5, 150 mM NaCl). The
suspensions were triturated with a 26-gauge needle and analyzed using a
Coulter ELITE cytometer with ultraviolet excitation and DAPI emission
collected at >450 nm. DNA content and cell cycle were analyzed using
the software program Multicycle.
In Vitro Migration Assay Using Primary Embryonic
Fibroblasts--
Wild type and RI mutant primary embryonic
fibroblasts were seeded onto sterile glass coverslips in 12-well tissue
culture dishes in 10% FBS/DMEM and grown to confluency in a 5%
CO2 incubator at 37 °C. By using a p200 pipette tip, a
wound was incised across the central area of the coverslip, and a line
perpendicular to the incision was made on the bottom of the 12-well
plate to mark the location for image collection. Images were collected
at 4-h intervals using a Leica inverted scope interfaced with a Kodak 290 Digital camera and the Kodak Capture DC290 software.
Confocal Imaging of Fibroblasts--
For actin cytoskeletal
visualization, fibroblasts grown in 10% FBS/DMEM at 37 °C in a 5%
CO2 incubator were seeded onto 1% gelatin-coated
coverslips in 12-well plates. At the time of staining, cells were fixed
with 4% paraformaldehyde in PBS for 20 min, washed, delipidated with
PBS containing 1% Triton X-100 for 5 min, washed, blocked with 1%
bovine serum albumin in PBS for 20 min, and subsequently stained for 20 min with Texas Red phalloidin (Amersham Biosciences). After staining,
cells were washed again and coverslipped in phosphate-buffered glycerol
containing m-phenylenediamine (Sigma) to counterstain the
nuclei. Slides were imaged using a Bio-Rad MRC 600 confocal microscope
using standard excitation filters for Texas Red and fluorescein
isothiocyanate labels. For RI and C visualization, wild type and
RI knockout fibroblasts were seeded onto gelatin-coated coverslips
in 12-well plates as above. Cells were fixed in 4% paraformaldehyde at
room temperature and then blocked in a 2% biotin solution, a 2%
avidin solution, and 10% normal goat serum, Tris-buffered saline,
0.015% Triton X-100 at 4 °C overnight. The following day cells were
incubated in a 1:500 dilution of either rabbit polyclonal anti-C or
rabbit polyclonal anti-RI in 10% normal goat serum, Tris-buffered
saline, 0.015% Triton X-100 overnight at 4 °C. The next day cells
were washed, incubated in a 1:300 dilution of biotinylated goat
anti-rabbit antibody for 2 h, followed by a 1:300 dilution of
avidin-fluorescein for 2 h. Cells were washed a final time and
coverslipped with vectashield containing 1.5 µM propidium
iodide to visualize nuclei. Staining was visualized as mentioned above
using the Bio-Rad MRC600 confocal microscope using the Texas Red filter
for nuclei and the fluorescein isothiocyanate filter for C or
RI.
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RESULTS |
Structure of the Mouse RI Gene--
Mapping and sequencing of
clones generated a map of the RI locus showing that the gene
contains 11 exons and spans about 18 kb in the mouse genome. The
position of intronic sequences within the coding region corresponds to
the genomic structure of the closely related mouse RI gene (31). A
previous mapping of the human RI gene showed only 10 exons (32), but
a recent report demonstrates 11 exons (33) indicating conservation
between the human and mouse RI genes. The promoter site for the
mouse RI gene was mapped by primer extension and S1 mapping and
revealed multiple start sites within a GC-rich 5'-flanking region with no recognizable TATA or CCAAT box homologies. This promoter and first
non-coding exon corresponds to promoter 1a as characterized for the
human RI gene. Other promoter regions are used in humans (32) but
all of these first exons are non-coding and splice to the first coding
exon, exon 2, to give identical protein products. Fig.
1A shows a diagram of the
mouse RI gene and the overlapping clones that were analyzed, and
Fig. 1B contains the sequence of the exons and exon/intron
boundaries as well as the sequence of the 1a promoter region.
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Fig. 1.
Structure of the RI
gene. A, genomic map of the RI gene locus
showing the location of the 11 exons and the Charon 4A phage clones
used for the analysis of the RI locus. B, sequence
of the RI promoter and exons with positions of introns and
intron/exon boundary sequences shown. The region boxed in from
nucleotides 171 to 223 contained multiple transcriptional start sites.
Poly(A) addition signals are underlined in the
3'-untranslated region.
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Targeting the RI Gene in Mice Results in Increased Basal PKA
Activity--
In order to address the role of the RI isoform of PKA
in mouse development, we generated a targeted disruption of the gene. The targeting vector, RIREK3, contained a neomycin resistance cassette inserted into exon 3 of a 5-kb 129svJ genomic fragment of the
RI gene (Fig. 2A) and a
thymidine kinase cassette flanking the RI genomic coding sequence.
This targeting vector was electroporated into REK2 and REK3 ES cell
lines (34), and Southern blot analysis identified clones from both REK2
and REK3 ES cells that had homologously recombined the replacement
vector sequence into the RI gene (Fig. 2B). The targeting
efficiency observed for the construct over several electroporations was
~1 in 30. Targeted REK2 and REK3 ES cell lines were injected into
C57BL/6 blastocysts, and the resulting male chimeric offspring were
bred to C57BL/6 females. Chimeras generated from the two independently
derived targeted ES cell lines transmitted the mutation through the
germ line. The analysis of RI mutant embryos in this study are
derived from mice generated from REK2- and REK3-targeted ES cell clones
on the mixed (129svJ × C57BL/6) background.
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Fig. 2.
Targeted disruption of the
RI gene. A, schematic
representation of the targeting scheme. The top line shows a
restriction map of a 5-kb segment of the RI genomic coding sequence
containing exons 3-5 that was subcloned into Bluescript
KS+. The middle line shows a schematic
representation of the targeting construct that has a neomycin
resistance cassette subcloned into the XhoI site of exon 3. No genomic coding sequence was removed in the targeting vector. A
Southern blot of EcoRV-digested genomic DNA using the 3'
SacI/HindIII genomic probe indicated on the
right of the targeting vector produces a 5.5-kb fragment for
the wild type allele and a 7.5-kb band for the targeted allele. RI
exon 3 oligos (black arrowheads) and neomycin oligos
(white arrowheads) used for PCR genotyping of yolk sac DNA
are indicated by arrowheads. A thymidine kinase
resistance cassette was subcloned into the multiple cloning site at the
5' end of the genomic sequence in the targeting vector to select
against random integrations. The bottom line shows the
targeted locus in the RI gene that contains the neomycin cassette
inserted into exon 3. B, EcoRV-digested
genomic tail blots of agouti offspring obtained from breeding high
percentage RI male chimeras to C57BL/6 females. The blot was probed
with the SacI/HindIII genomic probe indicated
above. As expected, heterozygous agouti offspring show a 5.5- and a
7.5-kb band indicating that the targeted RI allele has been
transmitted through the germ line of RI male chimeras.
C, PCR analysis using the yolk sac DNA of embryos
obtained from interbreeding RI heterozygous male and female
offspring. The oligos used for PCR analysis in the top panel
correspond to the 5' and 3' end of RI exon 3 and should yield a
171-bp product if the wild type allele is present. RI homozygous
mutant embryos lack this 171-bp product consistent with the disruption
of exon 3 by insertion of the neomycin cassette into both alleles of
the RI gene. In a separate reaction shown in the bottom
panel, 5'- and 3'-oligonucleotides internal to the neomycin
resistance cassette were used which yield a 550-bp product if the
neomycin cassette is present and no product in wild type animals.
D, 10 µg of total protein from wild type and mutant
homogenates used in the kinase assays was run on an SDS-PAGE gel and
transferred to nitrocellulose. The blot was subsequently probed with an
affinity-purified polyclonal antibody to RI. nt,
nucleotide.
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Mice carrying a heterozygous mutation in RI were viable, fertile,
and morphologically indistinguishable from wild type littermates; however, interbreeding of RI heterozygotes to generate RI
homozygotes resulted only in wild type and heterozygous offspring in a
1:2 ratio. To determine the point at which RI homozygous embryos were no longer viable, embryos from heterozygous intercross matings were analyzed at successive stages of development. Only resorption sites were found upon initial inspection of embryos at E11.5; however,
RI mutant embryos were found in Mendelian ratios from E7.5 up to
E10.5 (Table I). PCR genotyping of yolk
sac DNA using 5'- and 3'-oligonucleotides to RI exon 3 confirmed
that the mutant embryos do indeed carry the disruption in exon 3 (Fig.
2C). To confirm the absence of RI protein in homozygous
mutant embryos, mutant and wild type or heterozygote embryos were
homogenized directly in sample buffer and run on an SDS-PAGE gel
followed by probing with an affinity-purified polyclonal antibody to
RI (Fig. 2D). Western blot analysis confirms that RI
protein is indeed absent in the phenotypically mutant embryos.
To determine whether loss of the RI regulatory subunit results in
increased or decreased PKA activity, we performed kinase assays on
pooled E8.5 mutant and wild type or heterozygote embryos (Fig.
3E). Although the total PKA
activity is decreased by ~40%, basal PKA activity is increased
substantially in RI mutant embryos. The reduction in total PKA
activity in the absence of the RI subunit is consistent with
previous research in cell culture that has demonstrated that the PKA
catalytic subunit is unstable when not complexed to regulatory subunits
(35), suggesting that there is an increase in free C subunit in RI
mutant embryos that is also more rapidly degraded. Approximately 52%
of the kinase activity in RI mutant embryos is still cAMP-regulated,
and Western blots confirm the presence of RII in RI mutant
embryos (Fig. 3E, inset). Interestingly,
up-regulation of RII is not observed in RI mutants, which is
consistent with observations made in cell culture where PKA catalytic
subunit was overexpressed (35). In fact, a decrease in RII levels is
seen in the RI knockout embryos that is also observed in primary
embryonic fibroblasts isolated from these embryos (Fig.
7B, inset).
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Fig. 3.
RI mutant embryos
are severely growth-retarded and have increased basal PKA
activity. A, morphology of E7.5 wild type and
RI mutant embryos. Mutant embryos are smaller than wild type
littermates but have a well formed A-P axis, including head-folds,
node, and allantois. B, morphology of E8.5 wild type
and RI mutant embryos. Mutant embryos show severe growth retardation
and developmental delay resembling presomite head-fold stage embryos.
The cardiogenic plate is present but no heart tube has formed.
C, morphology of E9.5 wild type and RI mutant
embryos. E9.5 mutant embryos are severely growth-retarded, have failed
to form a contractile heart tube, and have not initiated the turning
process. D, comparison of an E9.5 RI mutant embryo
with wild type embryos from several stages of development. Mutant
embryos at E9.5 resemble E8.0 wild type embryos. Although RI mutants
are approximately the same size as E8.0 wild type embryos, mutants
differ in that they have more somites, an allantois that is thickened
at the base, a more radical entry of the foregut, and bilateral
cardiocytes that have failed to fuse ventrally to form the heart. Scale
bars in A-D = 500 µM. The abbreviations
used are as follows: al, allantois; cp,
cardiogenic plate; fg, foregut; hp, head process;
ht, heart; nd, node; tr, trunk;
ys, yolk sac. E, basal and total kinase
activity in E8.5 wild type (WT) and RI mutant embryos.
KO, knockout. Mutant (n = 2) and wild
type (n = 4) embryos were pooled after removal of yolk
sacs for PCR genotyping and homogenized in kinase assay buffer. Kinase
assays were performed in triplicate in the presence and absence of cAMP
using Kemptide as the substrate. Homogenates were also assayed in the
presence of protein kinase inhibitor to demonstrate that the measured
kinase activity was PKA-specific. Inset, Western
analysis using polyclonal antibodies to RI or RII from
homogenates used for kinase assays.
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Phenotype of RI Mutant Embryos--
One of the most striking
features of RI mutant embryos when first observed was severe growth
retardation combined with developmental delay. Although not
morphologically evident in mid- and late-streak stage embryos, the
phenotype became clearly apparent from the head-fold stage forward
(Fig. 3B). Examination of over 30 RI mutant embryos at
E9.5 revealed a very consistent mutant phenotype. The
anterior-posterior axis of RI mutant embryos is clearly apparent, with a prominent head structure followed by a greatly reduced trunk
structure compared with wild type E9.5 littermates (Fig. 3C
and Fig. 4, A and B). The E9.5 RI mutant
embryo is equivalent in size to an E8.0 wild type embryo (Fig.
3D). The most striking feature of the RI E9.5 mutant
embryo when compared with an equivalently sized E8.0 wild type embryo
is the absence of a definitive heart tube (Fig. 3, B and
D). The cardiogenic plate is present (Fig. 3B),
but rather than being displaced more caudally by the formation of the
heart tube, it is tightly juxtaposed to the proencephalon. The absence
of a heart tube also results in a more radical angle of entry of the
foregut (Fig. 3D).
At E8.5, RI mutant embryos can be easily recognized by their small
size, flattened morphology (Fig. 3B), and ruffled yolk sac
membrane (not shown). Mutant embryos at this stage resemble presomite
head-fold stage wild type embryos and are situated appropriately within
the yolk sac and amniotic membranes. At E7.5 the developmental delay is
less apparent, although E7.5 RI mutant embryos are smaller than wild
type littermates and have the characteristic flattened appearance
and ruffling of the yolk sac membrane.
Abnormal Cardiac Morphogenesis in RI Mutant Embryos--
As
mentioned in the previous section, although the cardiogenic plate is
present in E8.5 mutant embryos, no heart tube is formed. Morphological
and histological examination of the cardiogenic plate reveals the
presence of bilateral cardiocytes located within the putative
intraembryonic coelomic cavities that have failed to fuse and thus form
the pericardial cavity (Fig. 4,
C and D). Examination of one E11.5 litter
containing three RI mutant embryos revealed the presence of
bilaterally located cardiocytes within enlarged coelomic cavities that
contracted rhythmically at room temperature in M2 media at 36 beats/min
for several hours (not shown). Histological examination of these
embryos revealed the presence of healthy bilateral aggregations of
cardiocytes, whereas the embryos themselves were completely necrosed.
These observations support the conclusion that at least some mesodermal
cells arising from the posterior epiblast of RI mutants have
ingressed through the primitive streak and migrated in the appropriate
lateral and anterior direction together with the definitive endoderm to
arrive at the heart field (36). Interestingly, the RI mutants never initiate the turning process that would normally occur at the 7-8
somite transition (37). The absence of turning is also evident in the
BMP 2 and GATA 4 knockouts that display aberrant cardiac morphogenesis
(38, 39). In the case of BMP 2, a failure in the closure of the
proamniotic canal results in the formation of a single heart tube
outside the amniotic membrane (39). In the GATA 4 knockout, a failure
of lateral to ventral folding results in the formation of fully
developed bilateral heart tubes located dorsal to the aberrant foregut
(38). In the RI mutants, we hypothesize that the failure of the
bilateral cardiocytes to fuse ventrally arises from a deficiency in the
total number of mesodermal cells fated to become cardiocytes.
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Fig. 4.
RI mutant
embryos have deficiencies in cranial and trunk mesenchyme.
A, scanning electron microscopy of the head of an E9.5
wild type embryo. Note the well developed forebrain, midbrain, and
hindbrain resulting from complete closure of the neural tube. The first
and second branchial arches are present and lie adjacent to the
pericardium. The otic vesicle has delaminated from the surface
ectoderm, and the optic vesicle is visible. B, scanning
electron microscopy of the head of an E9.5 mutant embryo. Note the open
neural tube in the forebrain and midbrain region. Branchial arches are
absent, and cranial mesenchyme is severely deficient. The otic vesicle
is present and has delaminated from the surface ectoderm.
C, E8.5 wild type embryo showing ventral heart tube
formation, including both endocardial and myocardial elements suspended
within the pericardium and attached via the dorsal mesocardium.
D, equivalent section through an E9.5 mutant embryo
showing the absence of a ventral linear heart tube and pericardium.
Precardiac mesoderm is present on either side of the midline of the
pharynx. Note the persistence of the intraembryonic coelomic cavities
that have failed to fuse and form the pericardium. E,
whole mount in situ staining of an E9.5 RI mutant and an
E9.5 wild type embryo using a Pax 3 antisense riboprobe. Note the
staining in the 10-12 tiny somites that lie adjacent to the neural
tube. Somites in the RI mutant are segmented but are greatly reduced
in size and number when compared with the E9.5 wild type littermate.
F, ventral view of E9.5 RI mutant compared with an
E9.5 RI wild type littermate. Note the absence of fusion of somites
across the midline, indicating the presence of notochord tissue.
Scale bars in A-D = 100 µm;
E and F = 500 µm. The abbreviations used
are as follows: am, amnion; cm,
cardiomyocytes; co, intraembryonic coelomic cavity;
da, dorsal aorta; en, endocardium; fg,
foregut; hp, head process; mc, myocardium;
ng, neural groove; pcm, precardiac mesoderm;
ph, pharynx; ysm, yolk sac membrane.
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Axial and Paraxial Mesoderm Is Formed and Expresses Appropriate
Markers but Is Greatly Reduced in Size--
As mentioned previously,
the anterior-posterior axis of RI mutant embryos is well defined,
including axial and paraxial mesodermal derivatives. A morphological
node is apparent in E7.5 mutant embryos (Fig. 3A), and the
notochord is clearly present in rostral sections of E9.5 mutant embryos
(not shown). Although paraxial mesoderm is formed and is segmented in
RI mutants, the somites are very small and irregularly shaped (Fig.
4, E and F). The absence of fusion of somites
along the ventral midline suggests that notochord material must be
present along the rostro-caudal axis of RI mutants (Fig.
4F), as the complete absence of notochord normally results in ventral fusion of somites (40-42). Although the somites are small
and irregularly shaped, they do express the dermomyotomal marker Pax 3, suggesting that dorsolateral differentiation of the somites is
progressing normally (Fig. 4E).
Reduced Primitive Streak and Abnormal Migration of Mesoderm in
RI Mutants--
Due to the deficiencies observed in mesodermal
derivatives, we chose to look at earlier stages of mesoderm formation
in RI mutant embryos. Histological examination of E6.5 RI mutant
embryos compared with E6.5 wild type littermates revealed aberrant
migration of mesodermal cells away from the primitive streak. Although
wild type late-streak stage embryos always exhibit very tightly ordered columns of mesodermal cells that extend smoothly in a lateral and
anterior direction, the mutants display highly disorganized movement
away from the primitive streak (Fig. 5,
A and B). Quantitation of mesodermal cells that
have exited the streak in the embryonic portion of late-streak stage
RI mutant embryos indicates that mutants have approximately
one-third the number of mesodermal cells that have exited the streak
and moved laterally and anteriorly (Fig. 5C). Reduction in
primitive streak mesoderm is also clearly visible 2 days later in the
E8.5 RI mutant embryo, where a significant reduction in
brachyury mRNA is observed (Fig. 5D).
An accumulation of presumptive mesoderm beneath the primitive streak
that has failed to migrate out laterally and anteriorly is observed in the E9.5 embryo (not shown).
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Fig. 5.
Deficiencies in mesoderm formation begin at
gastrulation. A, representative
eosin-hematoxylin-stained 8-µm section through a wild type E6.5 late
streak stage embryo. Note the thick and highly ordered columns of
mesodermal cells that have exited the primitive streak and moved
laterally and anteriorly. Inset, outline of the embryo
showing the approximate location of the section. B,
representative eosin-hematoxylin-stained 8-µm section through an
RI mutant late streak stage embryo. Note the reduced number of
mesodermal cells that have exited the streak and the highly
disorganized manner in which the cells have exited the streak to move
anteriorly. Inset, outline of the embryo showing the
approximate location of the section. C, quantitation of
the number of mesodermal cells that have exited the streak in the
embryonic portion of wild type (n = 2) and RI mutant
(n = 2) late-streak stage embryos. Data are the total
number of mesodermal cells ± S.D. that have exited the streak
from four representative 8-µm sections for each embryo. Sections were
at the same level for each embryo. D, whole mount
in situ staining of an E8.5 wild type and RI mutant
embryo using a brachyury antisense riboprobe. Note
the greatly reduced staining in the primitive streak region as compared
with the wild type embryo. Scale bars in A and
B = 50 µm; D = 500 µm. The
abbreviations used are as follows: ant, anterior;
ect, ectoderm; end, endoderm;
mes, mesoderm; pos, posterior;
ps, primitive streak.
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Loss of RI Alters Cell Morphology, Migration, and Localization
of C Subunit in Primary Embryonic Fibroblasts--
To address the
hypothesis that increased PKA activity inhibits growth factor signaling
involved in the proliferation and migration of mesoderm from the
primitive streak stage forward, we isolated mesodermal primary
embryonic fibroblasts from E8.5 RI mutant and wild type embryos.
Fibroblasts isolated from these embryos were extremely unusual with
respect to overall morphology. Rather than showing the typical spindled
morphology of wild type fibroblasts, RI mutant fibroblasts are
flattened and show a box-shaped or rounded morphology. Examination of
the actin cytoskeleton revealed that the RI mutant fibroblasts have
multiple actin organizing centers that extend in all directions and an
extensive subcortical actin cytoskeleton, whereas wild type fibroblasts
display sparse parallel actin fibers running from the leading to the
lagging edge of the cell (Fig. 6,
A and B). To assess localization of C subunit,
confocal imaging was used on wild type and RI knockout E8.5 primary
embryonic fibroblasts. C subunit is uniformly distributed throughout
the cytoplasm of the wild type fibroblasts (Fig. 6C); however, in RI knockout fibroblasts, C subunit was preferentially localized to the perinuclear space (Fig. 6D). Examination of
RI localization revealed a uniform cytoplasmic distribution in wild type fibroblasts (Fig. 6E), similar to that observed for
C, whereas no staining was observed in RI knockout fibroblasts
(Fig. 6F). Cell cycle analysis of wild type and RI mutant
fibroblasts showed no difference in the percentage of cells in
G0/G1, S, and G2/M (Fig.
7A). cAMP activation curves
revealed high basal PKA activity in RI mutant fibroblasts consistent
with the unregulated PKA activity observed in the RI knockout
embryos (Fig. 7B). A small decrease in total PKA activity
was observed compared with wild type fibroblasts combined with a
decrease in RII levels in RI knockout fibroblasts (Fig.
7B, inset). HPLC analysis of wild type E8.5
primary embryonic fibroblasts reveals significant quantities of type II
holoenzyme along with type I holoenzyme (Fig. 7C). Quantitation of the type II and type I holoenzyme peaks indicates a
type II to type I ratio of ~3.5 to 1 in the wild type cells and as
expected no type I kinase in the mutants (Fig. 7C).
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Fig. 6.
Confocal imaging of the actin-based
cytoskeleton, C, and RI
localization in E8.5 RI knockout and wild type primary
embryonic fibroblasts. Confocal image of Texas Red
phalloidin-stained wild type E8.5 primary embryonic fibroblasts
(A) and RI mutant E8.5 primary embryonic fibroblasts
(B). Fibroblasts were seeded onto glass coverslips in
12-well dishes, fixed, permeabilized, incubated with Texas Red
phalloidin, and subsequently washed and coverslipped. The actin
cytoskeleton (red) was visualized by confocal microscopy
with appropriate filters. Nuclei were counterstained with
m-phenylenediamine (green). C staining of wild
type E8.5 primary embryonic fibroblasts (C) and RI
knockout E8.5 primary embryonic fibroblasts (D) are shown.
Cells were seeded onto glass coverslips in 12-well dishes, fixed,
permeabilized, blocked, and incubated with a rabbit polyclonal antibody
to C followed by biotin anti-goat antibody and avidin-fluorescein.
C localization (green) and nuclei counterstained with
propidium iodide (red) were visualized as above. RI
staining in wild type E8.5 primary embryonic fibroblasts (E)
and RI knockout E8.5 primary embryonic fibroblasts (F)
are shown. Cells were treated exactly as in the C staining, but a
rabbit polyclonal antibody to RI was used in lieu of the C
antibody. Scale bars = 100 µm.
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Fig. 7.
Analysis of cell cycle parameters, kinase
activity, holoenzyme assembly, and cell migration in
RI knockout (KO) and wild
type (WT) E8.5 primary embryonic fibroblasts.
A, cell cycle analysis of RI mutant and wild type
E8.5 primary embryonic fibroblasts. Fibroblasts grown in 60-mm dishes
in 10% FBS/DMEM in 5% CO2 at 37 °C were washed,
trypsinized, washed, and resuspended in 200 µl of DAPI buffer.
Fibroblasts were subsequently run through a Coulter counter to measure
the DAPI fluorescence over 20,000 cells. DNA histograms were
subsequently analyzed for cell cycle parameters using the
"multicycle" software package. B, cAMP activation
curves with RI wild type E8.5 primary embryonic fibroblasts and
RI knockout E8.5 primary embryonic fibroblasts. Cells in 60-mm
dishes were washed and lysed in homogenization buffer, sonicated, and
stored at 80 °C until the day of the assay. Samples were thawed
and homogenates assayed in triplicate with various concentrations of
cAMP using the standard kinase assay described under "Experimental
Procedures." cAMP activation curves were performed three times with
similar results. Inset, Western analysis from
homogenates of wild type and RI knockout primary embryonic
fibroblasts using polyclonal antibodies to RI or RII.
C, kinase assays on fractions obtained by HPLC DEAE
ion-exchange fractionation of homogenates from E8.5 wild type and RI
knockout primary embryonic fibroblasts. Cells were grown in 60-mm
dishes, washed, lysed, sonicated, and stored at 80 °C until the
day of the assay. On the day of the assay, 1-2 mg of protein
homogenate was loaded on a 2-ml DEAE ion-exchange column and separated
by HPLC into 1-ml fractions over a 0-250 mM salt gradient.
Fractions were assayed the same day by kinase assay as described under
"Experimental Procedures." D, in vitro
wound assay monitoring the migration of RI mutant and wild type
fibroblasts. Fibroblasts were seeded onto glass coverslips in 12-well
dishes and allowed to grow to confluency. An incision was made across
the center of the coverslip using a p200 pipette tip; the media were
replaced, and migration of fibroblasts back into the artificial wound
was monitored using an Leica inverted scope interfaced with a Kodak
DC290 digital camera and accompanying software. Magnification = ×100.
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Migration assays using an in vitro wound model demonstrated
that RI mutant fibroblasts completed wound healing by ~18 h, whereas wild type fibroblasts were significantly slower at completing this process (Fig. 7D). This difference in migration and
"wound healing" was similar when fibronectin-coated dishes were
used in place of uncoated plastic. The significantly faster closure of
a scrape "wound" in cell culture by RI mutant fibroblasts is
superficially at odds with the observed deficiency in mesoderm formation and migration out of the primitive streak in RI knockout embryos. However, it is likely that the complex environment at the
primitive streak in vivo, involving
FGF-dependent receptor tyrosine kinase signaling, E
cadherin down-regulation, epithelial to mesenchymal transition, and
subsequent integrin-dependent migration of nascent mesoderm
over the endoderm is not adequately modeled by this in vitro assay.
The Deficits in Mesodermal Derivatives Can Be Rescued by Crossing
RI Mutants to C Mutants--
Based upon the hypothesis that the
defects observed in the mesoderm of RI mutants is a result of
increased basal PKA activity, we attempted to rescue the RI mutant
phenotype by crossing RI mutants to C mutant mice that carry a
null mutation in the C gene (24). As C homozygous mutant mice are
infertile and RI homozygous mutant mice are embryonic lethals, we
generated RI/C double heterozygotes
(RI+//C+/) by crossing RI
heterozygous females to C heterozygous males. The resulting
RI+//C+/ double heterozygous animals
were viable and were interbred to generate RI knockout animals on
the C heterozygous and C homozygous background. The first 32 offspring from a total of four litters yielded
RI//C+/ and
RI//C/ offspring at the expected
Mendelian ratios of 1:8 and 1:16.
RI//C+/ embryos were easily
identified by their morphology and display an intermediate phenotype
clearly indicative of partial rescue of the RI mutation (Fig.
8C). One of the most dramatic
features of RI//C+/ is the presence
of a definitive heart tube located within a fluid-filled pericardial
cavity (Fig. 8C). Morphological and histological examination of these embryos reveals a significant increase in all axial, paraxial,
and lateral plate mesodermal derivatives, including increased head
mesenchyme, increased somite size and number, and increased trunk
length. Unlike the RI mutants,
RI//C+/ embryos also seem to have
initiated the turning process (Fig. 8C).
RI//C/ embryos showed an even
greater degree of rescue compared with the RI mutant phenotype (Fig.
8, B and D).
RI//C/ shows dramatic increases
in trunk length, somite number and size, branchial arch and limb bud
development, and head mesenchyme. Unlike RI mutants,
RI//C/ also completes the turning
process and has a heart tube located within the pericardial cavity
(Fig. 8, B and D). Kinase activity measurements
from the various genotypes revealed a predictable decrease in total PKA
activity but also a graded decrease in basal PKA activity as C
alleles are deleted on the RI knockout background (Fig.
8E). Genotyping of newborn pups from several
RI+//C+/ interbreedings has failed to
yield viable RI knockout animals, suggesting that the lack of RI
protein on the C knockout background is still lethal. Examination of
RI//C/ embryos at midgestation
(E10.5) revealed an expanded fluid-filled pericardial cavity,
suggesting cardiovascular failure in the absence of RI at a later
stage in development.
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Fig. 8.
RI mutants can be
rescued on the C mutant background.
Morphology of an E9.5 RI+/+C+/+ wild type
littermate (A), E9.5
RI/C+/+ mutant (B), E9.5
RI/C+/ mutant (C), and
an E9.5 RI/C/ mutant
(D). Scale bars = 500 µm.
E, measurement of basal and total PKA activity from all
four genotypes. Animals were staged and embryos collected from two
litters (14 total embryos) to obtain rescued embryos with the genotypes
indicated. Embryos were homogenized, sonicated, and stored at
80 °C until the day of the assay.
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DISCUSSION |
We have utilized targeted disruption of PKA subunit genes to
determine the developmental and physiological roles of individual isoforms of the PKA family of genes in the mouse. Disruption of the
RII subunit that is expressed ubiquitously at all stages of
development did not lead to any developmental deficits, and the animals
were healthy and fertile (43). However, in this report we show that
disruption of the other ubiquitous R subunit, RI, results in a
dramatic developmental phenotype. This phenotype is characterized by
mesodermal insufficiency, and the early manifestations are caused by
inappropriately regulated C subunit activity because we can partially
rescue the RI mutant embryos by breeding them onto a C knockout
genotype that reduces the total level of C subunit in the mutants.
The observation that receptor-tyrosine kinases are critical for
mesoderm formation combined with the substantial literature on
PKA-dependent inhibition of receptor tyrosine kinase
signaling led to the hypothesis that increased PKA activity in the
RI mutants might be antagonizing growth factor-dependent
signaling in the primitive streak. This possibility is consistent with
the greatly reduced brachyury whole mount in
situ staining in the primitive streak of RI mutants and the
deficiencies in anterior mesoderm-derived structures. The accumulation
of nascent mesoderm at the base of the primitive streak also suggests
deficits in integrin-dependent cell migration over the
endoderm which also depends upon growth factor receptor-tyrosine
kinases and extracellular matrix signals (44). Targeted disruption in
mice of several key proteins involved in integrin-mediated signaling
and migration, including fibronectin and focal adhesion kinase (FAK),
result in the failure of embryos at gastrulation (45-47).
FAK-deficient and fibronectin-deficient embryos bear a striking
resemblance to each other and to RI mutant embryos, including growth
retardation, developmental delay, failure of cardiac morphogenesis, and
anterior mesoderm deficits. The similarities in the phenotypes of FAK,
fibronectin, and RI mutant embryos combined with the literature
describing PKA-dependent regulation of the actin
cytoskeleton support the hypothesis that growth factor/integrin
signaling may be affected (3, 4). Activation of PKA causes
dephosphorylation of paxillin via increasing a tyrosine phosphatase
activity (48). More recent evidence demonstrates that PKA can
phosphorylate and activate Shp2, a tyrosine phosphatase that localizes
to focal adhesion complexes and dephosphorylates both paxillin and FAK
(49, 50). Related studies reveal that the p21-activated kinases are
also critical for the integration of growth factor and
integrin-extracellular matrix signals with the actin cytoskeleton (51,
52). Significantly, PKA activity also negatively regulates
p21-activated kinase and thus interferes with its ability to mediate
anchorage-dependent growth factor responses (53). Isolation
of primary embryonic fibroblasts from E8.5 RI mutant embryos reveals
a profound disruption of the actin-based cytoskeleton. These
fibroblasts bear a striking resemblance to FAK-deficient and
Shp2-deficient primary embryonic fibroblasts, including a dramatic
increase in focal adhesions and condensed F-actin aggregation at the
cell periphery (47, 54). Expression of a dominant negative Shp2
tyrosine phosphatase prevents paxillin and FAK dephosphorylation and
results again in increased focal adhesions and impaired migration (49),
suggesting that successive cycles of phosphorylation/dephosphorylation
are required for directed cell migration and that PKA may also be
involved in this process.
We hypothesize that the defects observed in mesodermal derivatives
result from antagonism of growth factor-mediated production and
migration of mesoderm out of the primitive streak. The fact that we can
incrementally rescue all the mesodermal derivatives of the primitive
streak by reducing the total amount of C subunit supports the
hypothesis that excessive catalytic subunit activity is antagonizing
growth factor signaling in the primitive streak. These observations
support a model in which PKA plays a role as a general negative
regulator of growth factor signaling in the primitive streak, not
unlike its role as a general negative regulator of sonic
hedgehog signaling in vertebrates (19, 20). Whether the cAMP/PKA
pathway is normally involved in regulating receptor tyrosine kinase
signaling in the primitive streak via endogenous G-protein-coupled
receptors remains to be demonstrated.
Is there a specific role for RI in mouse embryo development? C
,
, and
TOP
ABSTRACT
INTRODUCTION
