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INTRODUCTION |
Phosphorylated phosphoinositides generated by phosphoinositide
(PI)1 3-kinases play crucial
roles in signaling through receptors at the cell surface as well as in
membrane trafficking mechanisms in Golgi and endosomal compartments,
cytoskeletal organization, and regulation of apoptosis (1-9). The
genes for many PI 3-kinases have been isolated from a wide range of
tissues and organisms. They are divided into three major classes based
on their amino acid sequence, the homology among their lipid-kinase
domains, and substrate specificities (6, 9). Class I PI 3-kinases phosphorylate the 3' sites of PtdIns, PtdIns 4-phosphate, and PtdIns 4,5-diphosphate to form PtdIns 3-phosphate, PtdIns
3,4-diphosphate, and PtdIns 3,4,5-trisphosphate. Class I PI 3-kinases
can be further divided into two subclasses. The class IA PI
3-kinases, which include at least three isozymes,
,
, and
,
are heterodimers of a catalytic 110-kDa subunit (p110) and a regulatory
subunit of 85 or 55 kDa (p85/p55) (10-14). The interaction of p85/p55
and p110 via the inter-Src homology 2 domain on p85/p55 and the
amino-terminal 123 amino acids of p110 is critical for achieving
maximal activity of this class of PI 3-kinases in mammalian cells at
37 °C (11, 15, 16).
Despite a wealth of knowledge of the biochemistry and cell biological
effects of phosphoinositides, very little information is available on
the particular physiological roles played by these different enzymes
and isozymes during mammalian development. Inhibitors of PI 3-kinases
such as wortmannin (17) and LY294002 (18) are very useful for probing
the functions of these enzymes but as with all inhibitor studies suffer
from questions of specificity as to which enzymes and isozymes are
being inhibited at the dosages used in a particular study. In contrast,
specific mutations in genes encoding these enzymes would provide
greater specificity. We have therefore begun a systematic program of
generating mice carrying deletion alleles of the PI 3-kinases. Here we
report the successful targeting of the
isoform of p110 to create a deletion allele that, in the homozygous state, leads to embryonic lethality at E9.5 due to a severe defect in the proliferative capacity
of the embryo.
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MATERIALS AND METHODS |
Cloning of Pik3ca Genomic Sequence--
7.5 × 105 plaques from a 129SV mouse genomic library in the
FIX II vector (Stratagene) were screened (19) with probes derived from Pik3ca cDNA. Of three positive phage clones, one
(F1) was selected for further study and subcloning, and an 8-kb
SalI fragment was isolated and found to contain the first
three coding exons of Pik3ca (see Fig. 1).
Genetic Mapping of the Pik3ca Gene--
A tetranucleotide repeat
(CCTT) within an intron of Pik3ca was used for chromosome
mapping using the Jackson Lab C57BL/6J and Mus spretus
backcross DNA panel map service. Two PCR primers flanking the
tetranucleotide repeat (forward, GTA GGG ATG AAG GTG GAG AGG; backward,
AGC TGA CTC AAT GGA AGT AGG G) were used for the amplification. PCR
reaction was performed on 50 ng of DNA in a 20-µl volume containing
400 nM each primer, 200 nM of dNTP, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2,
and 0.5 unit of Taq polymerase. Reactions were denatured at
94 °C for 5 min and then subjected to 30 step cycles consisting of
94 °C for 20 s, 57 °C for 60 s, and 72 °C for
30 s. PCR products were loaded on 3.5% MetaPhore agarose gel and
separated at 70 V for 3 h.
Vector Construction and Pik3ca Gene Targeting in 129SV ES
Cells--
The targeting construct was made in the pPNT vector (20)
using an 8-kb SalI fragment from phage F1 and resulting in
replacement of the first coding exon with a neomycin resistance gene
transcribed in reverse orientation from a mouse PGK promoter (see Fig.
1). Electroporation into TC1 ES cells (21) and selection for homologous recombination were by published procedures. Recombinant ES cell lines
were screened by PstI genomic Southern blot using probe B,
located outside the targeted region of homologous recombination, and
positive cell lines were confirmed by SacI genomic Southern blot using probe E (see Fig. 1). All studies described in this report
were performed on mice carrying the mutant alleles on a 129/SV inbred
background. Animal experiments were performed under protocols approved
by the National Human Genome Research Institute's Animal Care and Use
Committee under National Institutes of Health guidelines, "Using
Animals in Intramural Research."
Prenatal and Postnatal Genotyping of Wild Type and Deletion
Alleles of Pik3ca--
DNA was extracted from yolk sac (22) and tails
(19) by published methods. Two PCR reactions were performed for
genotyping. One pair of primers derived from the exon deleted in the
deletion allele (P110N, forward, CTC CCC AAT GGA ATG ATA GTG, and
P110N, reverse, TCT TTT CTT CAC GGT TGC CT) amplifies a 260-bp fragment only from the wild type. The second pair of primers derived from the
neomycin resistance gene (Neo1, 5'-AGA GGC TAT TCG GCT ATG ACT G, and
Neo2, 3'-TTC GTC CAG ATC ATC CTG ATC) amplifies a 430-bp fragment only
from the disrupted Pik3ca gene. PCR amplification was
performed per the manufacturer's protocols (Life Technologies, Inc.).
RNA extracted from 9.5-day-old embryos (22) was used for reverse
transcriptase (RT)-PCR per the manufacturer's protocols (Life
Technologies, Inc.). Four pairs of primers were used for PCR
amplification: P110N, forward, CTC CCC AAT GGA ATG ATA GTG, and
reverse, TCT TTT CTT CAC GGT TGC CT, 260 bp; Neo1, 5'-AGA GGC TAT TCG
GCT ATG ACT G, and Neo2, 3'-TTC GTC CAG ATC ATC CTG ATC, 431 bp;
p110
, RT-1, sense primer, CTG TAT AAT GCT GGG GAG GAT GC, and RT-2,
antisense primer, ATG CGG TAC AGG CCA GAG ATT C, 505 bp, amplify
825-1329 of p110
cDNA; p110
, RT-3, sense primer, TCT GTC CCC
TCT GAA TCC TGC TC, and RT-4, antisense primer, TAT ATC TCG CCC TTG TTC
TTG TCC, 416 bp, amplify 2253-2669 of p110
cDNA. To control for
spurious amplification due to DNA contamination, primers were designed
to cross intron-exon boundaries, and reactions without RT were included
in every assay.
Western Blot Analysis--
Cells obtained from
Pik3ca+/+, Pik3ca+/del,
and Pik3cadel/del E9.5 embryos were collected by
trypsinization on the third day of culturing. Cultured cells were
washed three times with 1× phosphate-buffered saline, and the cell
pellets were frozen on dry ice immediately. Cell pellets were lysed in
25 µl of deionized H2O on ice, the lysates were separated
on 6% SDS/polyacrylamide electrophoresis gels, and the proteins were
transferred to Immobilon-P transfer membrane according to the
manufacturer's protocols (Millipore). Filters were used for Western
blotting (23) with either rabbit anti-human p85
serum (Upstate
Biotechnology Inc.) or rabbit anti-human p110
(Santa Cruz
Biotechnology sc-7174) in 0.1% Tween-20/1× phosphate-buffered saline
for 1 h at RT, and the signal was detected using
peroxidase-conjugated donkey anti-rabbit antibody and chemiluminescence
according to the manufacturer's protocols (ECL, Amersham Pharmacia
Biotech). Filters were exposed to x-ray films (Kodak) for 25 min at
room temperature.
Proliferation Assay--
Pregnant mice from a
Pik3ca+/del × Pik3ca+/del cross were injected
intraperitoneally at day E9.5 with BrdUrd (Sigma, 100 µg/g body
weight). E9.5 embryos were separated from their yolk sacs, which were
used for determining Pik3ca+/+ and
Pik3cadel/del genotypes. Embryos were fixed in
4% paraformaldehyde at 4 °C overnight and then embedded in
paraffin. Sagittal sections were mounted onto silanized slides without
staining, dewaxed, hydrated in decreasing ethanol solutions, treated
with 3% H2O2, and washed in phosphate-buffered
saline. Sections were exposed to anti-BrdUrd antibody (Becton
Dickinson) and developed in goat anti-mouse IgG (Sigma) using
peroxidase-conjugated Elite ABC (Vector Lab) and Sigma Fast DAB as
described (24).
Apoptosis Assay of E9.5 Embryos
Sections--
Pik3ca+/+ and
Pik3cadel/del E9.5 embryos were separated
from their yolk sacs, which were used for genotyping. Embryos were
fixed in 4% paraformaldehyde at 4 °C overnight then transferred
into 70% EtOH. Sagittal and parasagittal sections from the midline of
embryos were stained by standard hematoxylin and eosin staining and the number of apoptotic cells assayed by TUNEL assay (25).
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RESULTS |
Pik3ca Gene Maps to Mouse Chromosome 3--
A novel
tetranucleotide repeat (CCTT)n within the fourth intron
of Pik3ca was identified when this fragment was sequenced to
determine the exon/intron boundaries. The repeat was found to be
polymorphic between strains of C57BL/6J and M. spretus. A
backcross between these two strains provided by the Jackson Laboratories Backcross DNA Panel Map Service was used to map the locus.
Pik3ca maps cleanly to chromosome 3 at 12.9 centimorgans between Il7 and D3Mit21, with no crossovers with markers
D3Bir4 and #Sno. This location is syntenic with the region
between D3S3682 and D3S1564 on the long arm of human chromosome 3 where
human PIK3CA maps (SHGC-12912).
Construction of Targeting Vector and Gene Targeting--
A
clone (F1) isolated from a 129SV mouse genomic library (Stratagene)
contained the first four coding exons of Pik3ca (Fig. 1). The first coding exon contains 76 bp
of 5'-untranslated sequence and the first 352 bp of coding sequence
including the translation initiation codon (numbering according to
GenBankTM accession number U03279).

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Fig. 1.
Vector construction and Pik3ca
gene targeting in 129SV ES cells. In the wild type allele,
restriction sites (SI, SalI; P,
PstI; X, XbaI; EV,
EcoRV; H, HindIII; Sc,
SacI; EI, EcoRI) and four exons are
shown as white rectangles. The targeting construct was made
from the SalI 8-kb fragment by replacing the first coding
exon (black rectangle) with neomycin resistance (PGK-Neo).
Counter-selection used herpes simplex virus thymidine kinase
(hsv-tk). A correct homologous recombination (recombinant
allele) is detected using probe B from outside the region of homologous
recombination and probe E from inside for Southern blots of
PstI and SacI digested DNA. Cells containing only
wild type alleles (labeled W) are shown in lanes
1, 2, and 4; a cell with a recombinant
allele (labeled R) is shown in lane 3.
Confirmation of correct targeting is shown in lanes 6-8 in
which three ES clones found positive using probe B had the recombinant
allele with SacI digestion and probe E. Lane 5 is
normal control.
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The targeting construct (Fig. 1) contains 4.8 kb of sequence 3' and 3 kb 5' to the first coding exon. Homologous recombination of the
targeting construct shown in Fig. 1 results in deletion of this exon.
The transcript of the deleted allele lacks the translation initiation
codon as well as the domain that mediates the binding to the inter-Src
homology 2 domain of a regulatory subunit that is required for
catalytic activity of p110 in mammalian cells (11). The next
downstream, in-frame ATG occurs at base 15 of the second coding exon
(ATTGGCATGCCAGT); this ATG would allow the rest of the
transcript to be translated in frame, although it sits within an
incomplete Kozak consensus context (26) that lacks a G in position
+4.
Eight out of seventy-two (~11%) ES colonies showed the expected
recombinant pattern by genomic Southern blot analysis (Fig. 1). The
recombinant ES cells were injected into blastocysts (C57BL6) to
generate chimeric males. The chimeras transmitted the disrupted Pik3ca gene to the next (F1) generation.
Phenotype of Mice Carrying the Pik3ca Deletion--
Genotyping of
98 liveborn F2 mice from breeding of F1
Pik3ca+/del heterozygotes showed 32 were
Pik3ca+/+ and 66 were
Pik3ca+/del with no
Pik3cadel/del mice identified (Table
I). The absence of
Pik3cadel/del offspring was highly significant
(p = 5 × 10
13, exact binomial
test); the ratio of Pik3ca+/+ to
Pik3ca+/del mice (32:66) fit the expected 1:2,
consistent with a purely recessive lethality in the homozygote.
Pik3ca+/del were fertile and showed no
abnormalities in growth or development.
To identify the stage of embryogenesis at which lethality is occurring
in Pik3cadel/del embryos, embryos at different
stages of embryonic development were examined and genotyped (Table I).
At 10.5 days of embryonic development, no viable
Pik3cadel/del homozygote embryos were observed
among 19 genotyped. Genotyping of 60 E9.5 embryos showed 17 Pik3ca+/+, 31 Pik3ca+/del, and 12 Pik3cadel/del as well as 9 apparent resorptions
that could not be genotyped. Although there appeared to be a deficiency
of Pik3cadel/del embryos, a deficiency that
could be accounted for by the nine resorptions, the 17:31:12 ratio was
not significantly different from the expected ratios of 15:30:15
(chi-square with Yates' correction). At E8.5, 17 embryos showed normal
Mendelian ratios (Table I). From these data, we conclude that
homozygote embryos survive to E8.5, begin to die at E9.5, and do not
survive to E10.5.
Most Pik3ca del/del homozygote embryos at E9.5
appeared to be viable, with obvious cardiac function, and histological
analysis of E9.5 embryo sections stained with hematoxylin and eosin
revealed no defect in organogenesis. They were, however, smaller than
normal embryos, with fewer somites (average 22) compared with the
normal embryos (average 25) from the same litter. The head region of E9.5 Pik3cadel/del homozygote embryos was
smaller as compared with normal, and hemorrhage was observed at
forehead, snout, and other parts of the body of the
Pik3cadel/del embryos (Fig.
2). Heterozygous embryos were normal in
appearance.

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Fig. 2.
Phenotype of three
Pik3cadel/del embryos. Hemorrhage is
observed at the forehead, snout, and other parts of the body.
A and B represent the most typical examples of
the distribution of hemorrhage. C was a less typical example
in which a hemorrhagic bubble was observed on the dorsum of the
embryo.
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Pik3Ca Transcripts--
Northern blot analysis of RNA from wild
type embryos and adult mouse tissues revealed three transcripts (7.5, 5.4, and 4.4 kb) detected by a cDNA probe containing the coding
region of Pik3ca cDNA (data not shown). The different
transcripts result from alternative splicing in untranslated portions
of the gene and are not the result of alternative splicing of coding
exons (data not shown). All three transcripts appeared to be expressed
at similar levels from E7 to E17 of embryonic development and in
different tissues of adult mice, although there was variability in the
expression levels of the different sized transcripts among various
tissues. Whole mount in situ hybridization at E9.5 showed
that the Pik3ca transcript was expressed uniformly
throughout the whole embryo (data not shown).
RT-PCR analysis of RNA obtained from Pik3ca+/+
and Pik3cadel/del embryos revealed that the
deletion allele was transcribed. As shown in Fig.
3, Pik3ca transcripts were
present in both Pik3ca+/+ and
Pik3cadel/del embryos, when assayed by RT-PCR of
two segments downstream of the deletion (primer pair RT-1 and RT-2 and
primer pair RT-3 and RT-4), whereas the portion of the transcript
deleted by the targeted event was missing from
Pik3cadel/del RNA (primer pair p110-F and
p110-R). As control, the neomycin resistance gene transcript was
amplified from Pik3cadel/del embryos.

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Fig. 3.
Pik3ca mRNA transcription in
Pik3ca+/+ and
Pik3cadel/del embryos as determined
by RT-PCR using total RNA isolated from E9.5 embryos. The
genotypes of the embryos were first determined by PCR using yolk sacs
separated from the embryos. Four pairs of primers were used for RT-PCR:
pair p110F, p110R amplifies the first coding exon; primer pair neoF,
neoR amplifies the neomycin resistance gene; primer RT-1 and RT-2 and
primer RT-3 and RT-4 are located at the positions shown and generate
fragments of 504 and 416 bp, respectively. Results demonstrate that RNA
from the Pik3cadel allele is transcribed but
lacks the portion of the transcript encoding the p85 binding domain.
Size standard is HaeIII digest of x174. KO,
knock-out; N, normal.
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Proliferation and TUNEL Assay in Embryos--
The PI 3-kinases
have been implicated both in signaling through growth factors (8, 27,
28) and in blocking apoptosis through signaling through the Akt Ser/Thr
kinase (29, 30). The delayed embryonic development and lethality of
Pik3cadel/del embryos and the inability of cells
derived from these embryos to expand in culture could be due either to
failure to proliferate or to excessive apoptosis or both. We tested
E9.5 Pik3cadel/del embryos for their ability to
proliferate using BrdUrd incorporation in vivo (24) as well
as examining them for excess apoptosis by TUNEL assay (25). In wild
type mice, the cells in three rapidly dividing regions of an E9.5
embryo revealed heavy BrdUrd labeling, whereas the same regions of
Pik3cadel/del littermate embryos incorporated
very little BrdUrd (Fig. 4). In contrast
to the severe proliferative defect, no abnormality in apoptosis was
seen (Fig. 5). The number of cells
undergoing apoptosis was counted and averaged in six sagittal and
parasagittal sections; wild type embryos contained an average of 58.8 apoptotic cells per section, whereas
Pik3cadel/del contained an average of 48.0 apoptotic cells per section (p > 0.05, Student's
t test). The apoptotic cells were mostly located in
forebrain and hindbrain with a similar distribution in both genotypes
(Fig. 5).

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Fig. 4.
Proliferation assay of wild type
(panels A-C) and
Pik3cadel/del (panels
D-F) embryos. Sagittal sections of E9.5 embryos
harvested 1 h after intraperitoneal injection of the pregnant
mouse with BrdUrd. A and D are from midbrain
region, B and E are from region of rhombomere,
and C and F are from neural tube. Heavy brown
staining of most cells in A-C from wild type embryos
indicates that proliferation (as evidenced by BrdUrd incorporation) was
taking place. The nearly complete absence of similar staining in the
Pik3cadel/del embryos shows that despite
generally normal development up to E9.5, embryonic cells had ceased to
proliferate. KO, knock-out; N, normal.
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Fig. 5.
TUNEL assay of wild type and
Pik3cadel/del embryos at E9.5.
A-C are from wild type, with B and C
showing higher magnification; D-F are from the
Pik3cadel/del embryo, with E and
F showing higher magnification of similar locations as shown
in B and C. No significant difference in BrdUrd
incorporation into nuclei by terminal transferase was seen in
Pik3cadel/del embryos as compared with wild type
embryos, indicating that there is no significant difference in
apoptosis between wild type and Pik3cadel/del
embryos.
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Mouse Embryo Fibroblast Culture--
Cells from wild type,
heterozygote, and homozygote embryos were obtained by explanting E9.5
embryos. Mouse embryonic fibroblasts grew normally from explants of
wild type and Pik3ca+/del heterozygous embryos.
In contrast, when explants were obtained from E9.5
Pik3cadel/del embryos, cells would adhere to
tissue culture plates but could not be induced to expand in culture in
Dulbecco's modified Eagle's medium supplemented with 20% fetal calf
serum and a variety of growth factors (bombesin, phorbol 12-myristate
13-acetate, and lysophosphatidic acid), used either singly or in
combination. These growth factors were chosen because they were
reported to bypass a block in mitogenic signaling in cultured cells
caused by antibodies to Pik3ca (28).
Western Analysis of Regulatory p85/p55 in Homozygous Deletion
Embryos--
The deletion mutation in Pik3ca eliminates the
p85/p55 binding site, but the transcript containing the deletion could
theoretically still be translated into a protein lacking the first 122 residues of the full-length p110
. Antibody directed against residues
189-390 of p110
was used for Western blot analysis of fibroblasts
from wild type and Pik3cadel/del embryos.
Although this region of p110
was not deleted in the knock-out
construct, no p110
protein was seen (Fig.
6A). Furthermore, by comparing
two different amounts of protein (18 and 36 µg) from wild type and
heterozygous embryos, the amount of p110
was clearly reduced in the
heterozygotes, consistent with loss of expression of p110
resulting
from the targeting event. Another difference between
Pik3ca+/+ and
Pik3cadel/del embryos was also apparent when
rabbit anti-human p85 polyclonal antibody (Upstate Biotechnology Inc.)
was used for Western blotting of cells derived from
Pik3ca+/+ and
Pik3cadel/del embryonic tissue. An 85-kDa
fragment recognized by rabbit anti-human p85 (Upstate Biotechnology
Inc.) polyclonal antibody in the wild type appeared to be substantially
increased in Western blot of Pik3cadel/del
embryo tissues (Fig. 6B). Other proteins detected by the
antibody were either unchanged or decreased in intensity in lysate from Pik3cadel/del embryos.

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Fig. 6.
Expression of p110
and p85/p55 by Western analysis of
Pik3ca+/+ (lanes labeled
N), Pik3ca+/del (lanes
labeled C) and Pik3cadel/del
(lanes labeled KO) cells. A,
anti-p110 antibody with either 36 or 16 µg of protein from cells
derived from the three different genotypes N, C,
and KO. The arrow indicates the absence of the
fragment corresponding to p110 in the
Pik3cadel/del (lane KO) as compared
with the normal Pik3ca+/+ (lane N)
and Pik3ca+/del (lane C). The
fragment in the lane from Pik3ca+/del embryos is
diminished in intensity, consistent with there being half-normal levels
of p110 protein in heterozygous embryos carrying one targeted
allele. B, anti-p85 antibody using either 40 or 25 µg of
protein from cells derived from E9.5 embryos with the three different
genotypes N, C, and KO. The
arrow indicates the markedly increased intensity of the
fragment corresponding to p85 in the
Pik3cadel/del (lane KO) as compared
with the normal Pik3ca+/+ (lane N)
and Pik3ca+/del (lane C), despite
loading less protein in the KO lane.
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DISCUSSION |
PtdIns 3,4,5-trisphosphate has been implicated in the response to
growth factor signaling through tyrosine kinase receptors for
platelet-derived growth factor, insulin, insulin-like growth factor 1, colony-stimulating factor 1, nerve growth factor, hepatocyte growth
factor, and stem cell growth factor (8). Phosphoinositides phosphorylated at the 3 position of the inositol ring, including PtdIns
3,4,5-trisphosphate, are generated by the type IA PI
3-kinases, which also interact with Ras proteins in a
GTP-dependent manner (31). In addition, Akt Ser/Thr protein
kinases, p70 ribosomal S6 kinase and protein kinase C are considered to
be downstream targets of PI 3-kinases by receptor-stimulated signaling
(6). Other PI 3-kinases have also been found to participate in protein trafficking (7), cytoskeletal organization (32), and regulation of
apoptosis (29, 30). Given that there are so many PI 3-kinases involved
in so many cellular processes, it is important to ask to what extent
each of the isozymes of PI 3-kinase is either necessary or sufficient
for carrying out these many functions of PI 3-kinases. In particular,
is the
isozyme of PI 3-kinase essential for normal embryonic
development or is it redundant because a deficiency could be
compensated for by other activities, such as the
isozyme?
We created mice homozygous for a deletion within Pik3ca by
gene targeting and demonstrated loss of expression of p110
by Western analysis using an antibody directed against a region of the
protein not involved in the deletion event.
Pik3cadel/del homozygotes have delayed
development and undergo intrauterine death between E9.5 and E10.5 of
embryonic development. The developmental delay and growth failure were
due to an inability of Pik3cadel/del embryos to
proliferate in response to mitogenic stimulation. Consistent with this
severe proliferation defect was our finding that all attempts to grow
cells obtained from explants of E9.5 Pik3cadel/del embryos from 129/SV inbred matings
failed. Cells from Pik3cadel/del embryos failed
to expand in culture and appeared resistant to the mitogenic effects of
fetal calf serum as well as defined growth factors such as bombesin,
lysophosphatidic acid, and phorbol 12-myristate 13-acetate. These
results were consistent with the findings of Nave et al.
(33), who reported PI 3-kinase is involved in signaling through phorbol
12-myristate 13-acetate, but contrasts with the findings of Roche
et al. (28), who reported that neither bombesin nor
lysophosphatidic acid uses signaling pathways involving PI 3-kinase to
stimulate growth.
Because of the observation that PI 3-kinases activate the AKT protein
kinase and inhibit apoptosis in the nervous system (29, 30), we were
particularly interested in determining whether there was increased
apoptosis in the Pik3ca
/
embryos,
particularly in the developing brain. In fact, embryos did not
demonstrate increased numbers of apoptotic cells, either in the region
of the developing brain or elsewhere. We conclude from these
experiments that the PI 3-kinase encoded by Pik3ca is
absolutely required for normal embryonic growth and development; the
phenotype seen in Pik3cadel/del mice results
from an inability to proliferate in response to growth factor signaling
rather than from unregulated apoptosis. However, we cannot
eliminate the possibility that abnormalities in other
phosphoinositides phosphorylated at the 3 position of inositol by
the class IA PI 3-kinase we disrupted in these mice may
play a role in the embryonic lethality through other unknown mechanisms.
Given the wide variety of cellular processes in which PI 3-kinases are
involved, a puzzling aspect of the phenotype of
Pik3cadel/del homozygotes is that the embryos
manage to survive to E9.5 of embryonic development. There may be an
alternative supply of PI 3-kinase that is either lost or becomes
inadequate by E9.5 in face of a sharp increase in demand for PtdIns
3,4,5-trisphosphate that occurs normally at this stage of development.
For example, a maternal contribution of p110
in the oocyte may only
be sufficient to support limited early development. Alternatively,
embryonic forms of PI 3-kinase with limited temporal expression might
compensate early in development but not after E9.5. Finally, other PI
3-kinase catalytic subunits, such as the p110
subunit, might be
sufficient to allow development to occur prior to E9.5.
The period between E9.5 and E10.5 is a time of markedly increased
proliferation and differentiation as organogenesis and placentation accelerate rapidly in the embryo (34). A four-chambered heart begins to
beat, and angiogenesis allows the vascular system to expand in order to
deliver nutrients and remove waste products. We might hypothesize that
as the E9-E9.5 embryo faces markedly increased demands for
proliferation, growth, and differentiation, intrauterine death of
Pik3cadel/del embryos occurs at this stage
because the increased demand exceeds generate residual capacity of PI
3-kinase pathways to phosphatidylinositols required for proliferation.
It is interesting to note that the Pik3cadel/del
embryos demonstrated areas of extravasated blood, suggestive of
defective angiogenesis. A role in angiogenesis for
phosphatidylinositols phosphorylated by PI 3-kinase at the 3 position
of inositol is supported by studies in which wortmannin inhibited
angiogenesis in a chick embryo chorioallantoic membrane system
(35).
One interesting finding in the Pik3cadel/del
homozygous embryos was an apparent increase in p85/p55 as
determined by Western blot analysis of embryo tissue obtained at E9.5.
These data raise the intriguing possibility that one component of the
deleterious effect of the Pik3cadel allele on
embryonic development may be mediated through excess p85/p55 rather
than simple loss of function of p110
. Overexpression of p85 can
interfere with the binding of PI 3-kinases to other proteins by
disrupting the binding between the Src homology 2 domains of the p85
subunits of the PI 3-kinases with their targets (36). There is very
little information on the regulation of transcription and turnover of
p85/p55 and an investigation into the mechanism of this elevation of
p85/p55 is greatly hampered by our inability to propagate
Pik3cadel/del cell lines. The increase in p85
protein seen in the Pik3cadel/del embryos points
to a previously unrecognized aspect of p85 regulation that deserves
further investigation.