3331
Development 122, 3331-3342 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV3517
Kinase-negative mutant epidermal growth factor receptor (EGFR) expression
during embryonal stem cell differentiation favours EGFR-independent
lineages
Jie-Xin Wu and Eileen D. Adamson*
The Burnham Institute, La Jolla Cancer Research Center, 10901, N. Torrey Pines Road, La Jolla CA 92037, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
EGF receptors are expressed on most fetal and adult cells
but their precise roles are not well known. We previously
reported that, in P19 embryonal carcinoma cells, the
expression of kinase-negative EGFR inhibits retinoic acid
(RA)-induced differentiation to nervous tissue, suggesting
that EGFR plays a role in differentiation (J.-X. Wu and E.
D. Adamson (1993) Dev. Biol. 159, 208-222). Embryo stem
(ES) cells differentiate into a wide range of tissue types
after the removal of the cytokine LIF from the culture
medium. We demonstrate here that the induction of some
early markers of differentiation, tissue-type plasminogen
activator (tPA), AFP and keratins 8 and 19 is inhibited,
whilst brachyury and myosin are increased, in clones containing kinase-negative mutant EGFR. After an extended
period of differentiation, the cell types present in mutant
and control cultures differed. Mutant clones produced
frequent cardiac and skeletal muscle as the predominant
differentiated cell types in vitro; other cells types were
sparse or absent. Teratocarcinomas formed by EGFR∆kinase-expressing ES cells contained frequent skeletal
and cardiac muscle as well as apoptotic nuclei, while
normal ES cells produced no detectable muscle and less
apoptoses. Since mutant differentiated cultures had slower
growth rates and increased levels of cell death, we
concluded that: (1) inactive EGFR does not allow some cell
types to survive and/or proliferate; (2) tissues that do not
require EGFR for their survival, development or function
predominate in long-term mutant cultures; (3) EGFR
activity is not necessary for cardiac and skeletal muscle or
endoderm formation and (4) Impaired survival of EGFdependent lineages leads to preferential selection of muscle
in differentiating ES cells.
INTRODUCTION
of the EGFR during development, we have used embryonal
carcinoma (EC) cells as models. P19 EC cells can differentiate into three main directions depending on the retinoic acid
(RA) concentration. After aggregate culture in the presence of
0.5 µM RA and subsequent outgrowth on adhesive plastic, P19
cells differentiate into neurons and glial-type cells marked by
the intermediate filament markers, neurofilament protein NF-1
and glial fibrillary acidic protein (GFAP), respectively. We
have demonstrated that the expression of EGFR mRNA and
protein are induced at least 10- to 20-fold during RA-induced
differentiation from very low levels found in the undifferentiated stem cells (Joh et al., 1992). In fact, we have been unable
to detect EGF binding in P19 or any EC line tested except F9
cells (Adamson and Hogan, 1984). The low level of receptor
protein present in undifferentiated cells appears to be intracellular while differentiated cells express cell surface EGFR
(Weller et al., 1987).
Previously, we introduced an expression vector into P19
cells that encodes the extracellular and transmembrane
domains of EGFR but is deficient in all but 43 amino acids of
the intracellular component of the polypeptide. We have shown
that the truncated kinase-deficient form of the protein disrupts
The proto-oncogene epidermal growth factor receptor (EGFR,
ErbB1, HER-1) is expressed by most cell types of the body
with some exceptions including parietal endoderm (Adamson
and Meek, 1984) and mature skeletal muscle (Lim and
Hauschka, 1984a,b). The function of the receptor varies
according to the cell type and its degree of differentiation.
Expression of the gene starts as early as the 4- to 8-cell preimplantation embryo and increases rapidly (Wiley et al., 1992)
on trophectoderm cells where receptors change gradually from
an apical to a basal position in the completed epithelium
(Dardik et al., 1992). Its presence on the trophectoderm apical
surface suggests, together with data from other laboratories,
that this receptor may play a role in the implantation of the
embryo. Clearly, a main role for the ligands that bind the
receptor to create a signal, is the stimulation of DNA synthesis,
cell growth and mitosis. However, a large number of
pleiotropic effects have been observed after ligand stimulation
of the EGFR, indicating the complexity of the signal pathways
and hence the diversity of the effects and roles of the receptor.
In order to simplify the analysis of the expression and roles
Key words: cell death, apoptosis, tPA, AFP, Brachyury, Endo A,
myosin, muscle, epidermal growth factor receptor, growth factor,
stem cell
3332 J.-X. Wu and E. D. Adamson
the activity of the endogenous EGFR by heterodimerization to
produce an inactive form. The expression of the truncated
EGFR, driven by the CMV promoter and enhancer, was
activated after the differentiation of the EC cells. At the same
time as endogenous EGFR was produced, the truncated version
was also synthesized and we demonstrated that it inhibited
differentiation and the production of nerve cells (Wu and
Adamson, 1993). This was partially accomplished by the
increased rate of cell death (2- to 6-fold) in mutant cultures
compared to controls during the 6 days of differentiation (our
unpublished results). We concluded that EGFR plays a role in
the differentiation of wild-type P19 cells to nervous tissues
and, by extrapolation, in neural development in vivo.
To determine other roles and activities of the EGFR, we are
using totipotent ES cells that can give rise to chimeric mice
after their introduction into a host blastocyst and transfer into
a recipient female. ES cells are able to respond to all the signals
that occur in vivo during development, differentiation and
organogenesis. These cells are also able to take part in gonadal
development and incorporate into the germ line of chimaeras.
However, in culture, developmental cues are quite limited or
absent and ES cell differentiation is a random and incomplete
process leading to a mixture of cell types in a process that
cannot yet be directed to one or another particular cell type (but
see Bain et al., 1995). The tissues produced frequently are
visceral endoderm in the early stages, parietal endoderm which
persists, cardiac, skeletal and smooth muscle, fibroblasts and
various epithelial cell types in the later stages. The most
obvious cell type is the cardiac myocyte since groups of these
beat rhythmically in the dish. ES cells give rise to teratocarcinomas after subcutaneous injection into syngeneic adult hosts.
Keratin pearls, epidermal epithelia, secretory and ciliated
epithelia, pseudostratified epithelia and occasionally a little
muscle can be observed in tumours.
Using an improved dominant negative (kinase-minus)
EGFR expression vector, we have now transfected the E14 ES
cell line and derived several mutant clones resistant to G418
by the cotransfection of the bacterial neomycin-resistance
plasmid, pMCneo. Control clones were transfected with the
empty cassette and were also selected with G418. We
measured several parameters of differentiation and came to the
conclusion that ES cell differentiation is modified by the
expression of the mutant EGFR since the cell types remaining
after selective elimination appeared to be those that did not
require the activity of the EGFR for their production and/or
activity. Muscle was the principal differentiated tissue
produced in mutant clones. The selective death and slower
growth rates of EGFR-dependent cells could
account for abnormal proportions of each
cell type during differentiation.
27 µM 2-mercaptoethanol, 1000 U/ml LIF, 0.5 u/ml penicillin and 0.5
mg/ml streptomycin. For differentiation, cells were trypsinized and
seeded at one million/10 ml in 10 mm Petri dishes for 5 days (or unless
otherwise indicated) before reseeding in a tissue-culture dish for the
indicated number of days. The medium used to stimulate differentiation was the same ES medium without LIF. In some cases, all-trans
retinoic acid (0.6 µM RA) was used as an additional inducer of differentiation.
Tumour analyses
Teratocarcinoma formation was compared between the two cell types
by subcutaneous injection of 107 cells into each of two sites in nu/nu
mice. The tumours that developed (in two weeks) were weighed and
were processed for histological analyses by standard procedures by
staining with haematoxylin and eosin. Formaldehyde-fixed tissue was
examined for apoptosis (see below). Frozen sections were also
analyzed by immunocytochemical procedures (see below).
Expression vector construction and electroporation of ES
cells
The expression vector for truncated EGF-receptor contains an EcoRI
fragment (2.3 kb) from the cDNA (Joh et al., 1992) inserted into the
CXN2 vector (Niwa et al., 1991). The promoter is β-actin (1.7 kb)
with an enhancer of 200 bp derived from CMV and with β-globin
poly(A) sequences (0.9 kb) downstream. This 4.9 kb fragment was
cloned into the unique BamHI site of vector NNTG1 (Fig. 1).
NNTG1 is derived from the human keratin gene and encodes 2.3 kb
5′ sequences, which were inserted upstream of the EGFR vector, and
3.5 kb 3′ sequences downstream (Neznanov et al., 1993). The role
of the flanking K18 sequences is to insulate the vector sequences
from inactivation after insertion into DNA and to endow dose dependency to concatamer inserts (Neznanov et al., 1993). A control
vector lacking the EGFR insert was prepared similarly. For electroporation, the completed plasmids, K18-CXN-mEGFR (25 µg), K18CXN (25 µg), and pMCNeo (5 µg), were linearized with NotI, NotI
and BglI, respectively. DNA (20 µg vector plus 2 µg pMCNeo) was
added to cells (8×106) in single-cell suspensions in 0.7 ml culture
medium in the cuvette. The mixture was electroporated with 275 V
for 5 mS and allowed to stand on ice for 10 minutes. Each reaction
mix was plated into two 100 mm gelatinized plates. The selective
drug G418 (400 µg/ml) was added after 24 hours. After 9 days most
cells had died and clones were visible. About 20 clones were isolated
from each dish after 13 to 15 days. Mutant clones are designated
Km, control clones are Kc. The inhibitory activity of our truncated
EGF receptor construct was demonstrated earlier (Wu and Adamson,
1993). The construct was used successfully, to inhibit EGF receptor
function in two cell types by Moshier et al. (1995) and by Huang et
al. (1996).
Antibodies
We have previously described the rabbit polyclonal antibody to
purified EGF receptor from mouse liver (Weller et al., 1987; Wu and
2.3 kb -EGF receptor
K18
K18
MATERIALS AND METHODS
Cells and their culture
E14 embryo stem cells (Handyside et al., 1989)
were cultured on gelatin-coated plastic dishes
without feeder cells in Dulbecco’s modified
Eagles Medium (DMEM) with high glucose and
sodium pyruvate (10 mM) and glutamine (2 mM)
in the presence of 15% bovine fetal serum (BFS),
CMV-enh
-actin-Pro
-glob-pA
Fig. 1. The plasmid construct used to introduce the truncated EGFR gene. K18, cassette
derived from the human keratin 18 gene that insulates the internal sequences from
inactivation and renders site-independent and dose-dependent insertion of vectors in
genomic DNA (Neznanov et al., 1993; Thorey et al., 1993). TM, transmembrane domain
of the mouse EGFR cDNA in a total of 2.3 kb. The expression of the kinase-negative
EGFR is driven by the β-actin promoter and the CMV enhancer (Niwa et al., 1991). B,
BamI; E, EcoRI; X, XhoI.
Mutant EGFR affects ES cell differentiation 3333
Adamson, 1993). TROMA-1 and TROMA-3 are rat monoclonal antibodies to keratins 8 and 19 (Brûlet et al., 1980; Kemler et al., 1981a,b)
kindly provided by Dr R. Kemler. Hybridoma supernatant to chicken
(cardiac and skeletal) myosin heavy chain (MF 20) was obtained from
the Developmental Studies Hybridoma Bank maintained by the
Department of Pharmacology and Molecular Sciences, Johns Hopkins
University School of Medicine, Baltimore, MD and the Dept of Biological Sciences, University of Iowa, Iowa City, IA under contract
from NICHD.
Immunofluorescent staining
ES cells were seeded onto gelatinized glass coverslips and induced to
differentiate for up to 6 days in medium containing RA. The cells
were fixed in methanol at −20°C for 5 minutes and stored desiccated
and frozen until used. The cells were stained with undiluted TROMA3 and TROMA-1 monoclonal antibody supernatants for 1 hour,
washed and then reacted with fluorescein-labeled goat anti-rat IgG.
The coverslips were mounted in 90% glycerol in PBS and observed
using a Nikon epifluorescence Biophot microscope fitted with
automatic exposure meter and camera. Frozen sections of teratocarcinomas were similarly processed for indirect immunofluorescence
analysis for myosin H-chain using M20 MCAb.
Immunoblotting (Western)
Cells were lysed in Laemmli sample buffer and the optical density at
280 nm measured to estimate the concentration of protein. Equal
amounts of protein in each sample were analyzed on 6% polyacrylamide gels containing SDS, electrotransferred on to PVDF
membranes (Immobilon, Millipore Corporation, Bedford, MA) and
myosin was visualized using monoclonal antibodies (MF 20) followed
by peroxidase-labeled anti-mouse IgG antibodies and the ECL kit as
described by the manufacturer (Amersham Corporation, UK).
RESULTS
Evidence for the expression of the truncated EGFR
in transfected clones
ES14 cells were electroporated in the presence of the linearized
plasmid expression vector for truncated mouse EGF receptor
In situ apoptosis by terminal transferase dUTP nick endlabelling (TUNEL)
The procedure described by the manufacturers (ApoTagTMPlus,
Oncor, Gaithersburg, MD) was used to detect the presence of
apoptotic nuclei in paraffin wax embedded tumor tissues. Briefly, this
consisted of labelling new 3′-OH ends generated by DNA fragmentation. Digoxigenin-labelled dUTP was incorporated into the fragmented DNA by terminal deoxynucleotide transferase, and detected
by peroxidase-labelled anti-digoxigenin and substrate for peroxidase.
The sections were counterstained with haematoxylin. A brown
coloured nuclear stain indicates the cells undergoing apoptosis; the
nucleus becomes contracted during this process. Sections were photographed at 400× on a Leitz Dialux 22 microscope.
Immunoprecipitation
ES clones in 35 mm dishes containing 10 5 cells were metabolically
labelled with [35S]methionine and cysteine (TranSlabel, ICN, Irvine,
CA) for 2 hours. Cells were lysed in RIPA buffer containing protease
inhibitors and aliquots were taken for total radioactive protein measurement as previously described (Wu and Adamson, 1993). Samples
containing equal amounts of radioactive protein were analyzed on
6% polyacrylamide gels containing SDS. After fixing, staining and
drying the gels, fluorographic records were made on X-Omat film
(Kodak).
Northern blotting
Cells in two 100 mm dishes were harvested at each of the indicated
days of differentiation and total RNA was extracted using guanidium
thiocyanate as described (Chomczynski and Sacchi, 1987). 30 µg
RNA from each sample was electrophoresed and blotted using
standard procedures (Sambrook et al., 1989). A [32P]dCTP-labeled
cDNA encoding tissue-type plasminogen activator (tPA) (Strickland
et al., 1980) was used as a probe (a kind gift from S. Strickland). Other
probes were for α-fetoprotein (AFP), a marker of visceral endoderm
(PstI fragment of pBR322.AFP2, obtained from Dr S. Tilghman);
Brachyury, a marker for pre-mesodermal tissues (EcoRI fragment of
pSK75 kindly supplied by Dr B. G. Herrmann, Wikinson et al., 1990),
ErbB2 (fragment from pSV2neuN kindly supplied by Dr R. A.
Weinberg, Bargmann et al., 1986); and ErbB3 (EcoRI-HindIII
fragment from pTZ19U/ErbB3, kindly supplied by Dr G. Plowman).
A probe that detects L32 mRNA, a ribosomal protein gene (Dudov
and Perry, 1984), or mouse β-actin (cDNA fragment from pHβA-1
obtained from Dr L. Kedes, Ponte et al., 1984) were used as controls
for equal loading of RNA in gel slots.
Fig. 2. Biosynthesis of EGF receptor polypeptides in ES clones.
Cells were metabolically labeled with [35S]methionine as described
in the Methods section. Immunoprecipitation with rabbit EGFreceptor antisera (A,C) or with non-immune sera (B,D) were
analyzed on 6% PAGE-SDS gels, which were then processed for
autoradiography. (A,B) The results for three mutant clones analyzed
on the day after the start of differentiation indicated above.
Differentiation was induced by the addition of retinoic acid (RA) to
aggregates in Petri dishes followed by culture for 5 days or
differentiation occurred spontaneously (sp) by the removal of LIF
from the medium. (C,D) The results for control clones. Only the
mutant clones in A express the 120×103 Mr exogenous EGF-receptor
polypeptide while very low levels of the endogenous normal receptor
protein was detected. In contrast, the control clones synthesized high
levels of full length EGF-receptor in late differentiation shown in C.
3334 J.-X. Wu and E. D. Adamson
(pK18-CXN-mEGFR) together with a neomycin-resistance
plasmid. At least ten mutant and ten control clones were
selected in medium containing 400 µg/ml G418 and these were
expanded and frozen as stocks. Three of each were chosen for
the analysis of expression of EGFR protein. The mutant clones
were selected on the basis of their positive staining in immunofluorescence tests using rabbit anti-EGFR antibody as
described in the Methods section (data not shown). To confirm
that the clones selected expressed full-length and truncated
EGFR proteins, we metabolically labeled the cultures and
immunprecipitated EGFR protein from lysates using the same
antibody. Wild-type EGFR protein (170×103 Mr) is not
detectable in undifferentiated ES cells but appears during
differentiation. Fig. 2A shows that the truncated 120×103 Mr
EGFR was expressed in mutant ES clones and it increased 1.4fold during RA-stimulated or spontaneous differentiation indicating the increased efficiency of the β-actin promoter in differentiated cells. Mutant EGF receptor protein was expressed
from 5-fold to 20-fold more than endogenous wild-type
receptor protein in mutant clones.
During differentiation a clear and notable difference was
observed between the control and mutant cultures. The level
of the wild-type EGFR was low (Fig. 2A, lanes 6-16) or absent
(see clone Km25, Fig. 2A, lanes 1-5) in the mutant cultures
while it was clearly increasing starting at 3 days after the start
of RA-induced differentiation and reaching readily detectable
levels that were maximal at about 9 days during control cell
differentiation (Fig. 2C, lanes 1-18). The low production of
EGFR protein in mutant clones was surprising since dominant
negative mutated receptor molecules are not known to inhibit
the synthesis of the wild-type receptor protein. Two possible
explanations are: (1) that more rapid degradation of the wildtype form occurs after the formation of inactive dimers with
Table 1. The tumorigenicity of the ES clones in athymic
mice
Cell type
Km25
Kc101 and Kc107
Av. tumour growth rate
±S.D. (No of tumours).
(mg/day in 15 days)
Range
21.0±13.1 (9)
26.5±19.8 (8)
7.9–34.0
6.6–46.3
There was no significant difference in the rate of tumour growth between
the controls and mutant clones. Ten million cells were injected into several
subcutaneous sites in each mouse.
the truncated polypeptide; (2) that cell death occurs in cells that
express high levels of mutant receptor. The results described
below support the second possibility.
Morphological appearance and growth of mutant
cell lines
There was no detectable difference in the morphology of
mutant ES cells (Km clones) compared to control cells (Kc
clones). They grew at the same rates and were tightly clustered
small cells when undifferentiated. This was expected because
wild-type ES cells, similar to most EC cells, do not express
surface EGFR and do not respond to EGF (Adamson and
Hogan, 1984) and therefore they are not expected to behave
differently in the presence of mutant receptor protein. After the
removal of LIF, when endogenous EGFR synthesis is induced,
the ES cell aggregates differentiated to give similar outer layers
of endoderm in all clones. In other words, embryoid body
formation appeared normal.
Tumour formation by ES cell clones
ES cells are tumourigenic when placed in subcutaneous
Fig. 3. Tumour sections
stained with Hematoxylin and
Eosin. (A) Km25 and (B) Km
207; sections from mutant
(mut) tumours display large
areas of fibrous-like tissue,
heavily infiltrated with blood
cells. (C,D) Kc101; control
tumours (con) have large
areas of carlilage (c) and
epithelia (e) some were
columnar and resembled
neuroepithelium (ne). Bar,
50 µm.
Mutant EGFR affects ES cell differentiation 3335
locations in athymic or syngeneic animals. The
tumours produced (teratocarcinomas) usually contain a
wider range of recognizable differentiated cell types
than during differentiation in vitro. Control and mutant
cells (ten million) were injected into nu/nu mice to
determine if the growth rate or the differentiated tissues
produced in the tumours differed. Tumours grew at
similar rates in both cell types to achieve a large mass
after 2 weeks. Tumours produced from mutant clones
were slightly smaller on average but this was not
statistically significant because of the wide range in
sizes (Table 1). Histological analyses (Fig. 3) revealed
that there were differences in the predominant tissues
appearing in the control and mutant tumours. The
control cells produced tumours with more frequent and
larger areas of highly differentiated tissues including
cartilage (Fig. 3C). Epithelioid organs such as ciliated
secretory epithelia (bronchial-like), cysts and glands
lined by epithelial cells (Fig. 3D) were present. These
tissues occurred in tumours from only one (Km 207)
out of three mutant ES clones tested and was much less
frequent, in smaller amounts and less-well-differentiated (Fig. 3A,B).
Further observations using higher magnification of
H&E-stained sections revealed a striking difference in
the composition of mutant tumours. Striated muscle
was evident in many areas of the Km tumours: both
skeletal muscle (Fig. 4B) and cardiac muscle (Fig. 4C)
were found frequently while control tumours (Kc) had
very little. Control tumours contained a predominance
of epithelia with little if any muscle (Fig. 4C). Frozen
sections of similar teratocarcinoma tumours examined
by indirect immunofluorescence with an antibody to
myosin heavy chain, confirmed this observation (data
not shown).
In situ apoptosis tests (TUNEL assays) revealed
differences in the frequency of apoptotic figures in
tumours, with the highest level in the Km-derived cell
tumours (Fig. 5, Mut) compared to control cell
tumours (Kc 107, Fig. 5, Con). Km 207 tumours had
the highest density of apoptosis of the Km tumours
and this correlated with the presence of some differentiated tissues. Cell death in Kc tumours was present
but occurred at a lower frequency. This suggested that
rates of cell death may differ between the two cell
types during differentiation and this could account for
the observations described above.
Cell death in vitro
ES cells in aggregate cultures were analyzed for cell
death and growth rates during differentiation induced
Fig. 4. High-power magnification of teratocarcinoma
sections stained with H & E. (A) Control tumours (Kc)
contained largely epithelial (arrows), fibroblasts and
smooth muscle-like cells. (B) Mutant ES cell tumours were
rich in striated myotubes (thick arrows) in which the thin
and thick banding pattern typical of skeletal muscle was
seen (small arrows). (C) Cardiac muscle (thick arrows) was
also observed more frequently in Km tumours. Bar in A
indicates 50 µm in all panels.
3336 J.-X. Wu and E. D. Adamson
Fig. 5. In situ TUNEL assays for apoptotic
cells. (A) Little apoptosis is seen (arrows) in
Kc102 tumours (con). (B) Section of a
Km207 (mut) teratocarcinoma showing
numerous brown-stained apoptotic nuclei,
singly and in groups largely in epithelial
cells. Clusters of undifferentiated ES cells
are seen in the upper half. Bar, 50 µm.
40
A
Growth Rate
Km25
30
Km27
Kc107
Kc106
20
10
0
0
2
4
6
8
Days of Differentiation
Dead cells x 10-4 in 5d / 106 initial cells
100
B
80
60
40
20
0
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAAAAA
AAA
Kc106
Kc107
Km25
Km207
Fig. 6. Growth and death in ES cell cultures after the removal of LIF.
(A) The number of cells, both adherent and suspended, that were
Trypan blue negative was recorded over 7 days of differentiation. Cells
were counted on the day indicated and normalized to the exact number
(10,000) of cells seeded Triplicate samples, repeated once were used to
generate averages and standard deviatons. See inset for cell types.
(B) The accumulated dead cells after 5 days of culture (± s.d.).
Fig. 7. Northern blot analysis of tissue-type plasminogen activator
(tPA) expression in differentiating ES clones. Top panel shows that
only control clone Kc18 started to express tPA on the 3rd day (lane
8) while mutant clones were not expressing detectable tPA at this
time. To indicate that the gels were loaded with similar amounts of
total (30 µg) RNA, the same blot was stripped and analyzed for the
expression of β-actin.
Mutant EGFR affects ES cell differentiation 3337
Fig. 8. Immunofluorescent analysis for
the expression of keratin 19 (TROMA 3)
in parietal endoderm cells produced from
ES differentiation. Cultures were fixed
on the 6th day of differentiation and
stained with rat monoclonal antibody to
TROMA 3. (A) Mutant clones (mut)
were negative in this assay while the
edges of colonies of B,C, control clones
(con), were positive. Bar, 20 µm.
by the withdrawal of LIF. Each experiment was performed at
least twice with similar results and two clones of each type
were examined. We analyzed growth rates (Fig. 6A) (cells not
stained by Trypan blue) in cultures during 7 days of differentiation. This type of analysis in ES cells is subject to underestimates of the number of live cells because the disaggregation
of cell clumps is incomplete and cells can be injured by
extended enzyme treatments. Assuming that a similar error is
applicable to all cell populations, the growth rates were about
three-fold lower in mutant clones. The numbers of dead cells
that accumulated in the medium in 5 days consistently showed
that cell death was 2-fold more frequent in mutant clones
compared with Kc clones (Fig. 6B).
Early markers of differentiation
Early stages of the differentiation of EC cells are often marked
by the appearance of the secreted protease, tissue-type plasminogen activator (tPA), produced by parietal endoderm and
other cells and by the presence of intermediate filament
proteins, TROMA 1, TROMA 2 and TROMA 3 (K8, K18 and
K19), also characteristic early markers of differentiation. We
tested for these markers in differentiating ES clones in total
RNA. In all three mutant clones examined, there was no
evidence for tPA gene activation which is expected to occur
on the 3rd day in control and wild-type clones (Fig. 7, lane 8).
The approximately equal loading of the gel was ascertained by
stripping the blot and reprobing for β-actin (Fig. 7, lower
panel). A positive signal for tPA mRNA in RNA extracted
from control cells at 3 days of differentiation was a consistent
finding and occurred in other control clones in addition to those
shown in Fig. 7.
Similarly, control clones produced outgrowths that
expressed TROMA-1 and TROMA-3 cytoskeletal proteins one
day earlier than mutant clones, appearing at 6 days in controls
but not in mutant clones (Fig. 8). These markers are typical of
parietal endoderm cells and all clones eventually produced
similar staining levels, showing no difference in TROMA
expression at later stages of differentiation. We concluded that
the formation or activity of the primitive endoderm cells that
give rise to visceral and parietal endoderm was delayed by the
presence of mutant receptors. This conclusion was confirmed
by northern blots probed with a cDNA to AFP, a visceral
endoderm-specific marker. Fig. 9 shows that the control clone,
Kc107, started to produce AFP after 5 days of aggregate
culture in the absence of LIF (Fig. 9, lane 5). By 5 + 5 days in
culture, a large amount of AFP mRNA was detected (Fig. 9,
lane 6) whereas it took 10 days culture of mutant cells (Km27)
before a low level of AFP was detected (Fig. 9, lane 3).
Another early marker of pre-mesoderm tissues in mouse
embryogenesis, Brachyury (Wilkinson et al., 1990), gave the
opposite result. Brachyury (By) mRNA was strongly expressed
in mutant differentiating ES Km27 cells by day 5 (Fig. 9,
middle panel, lane 3) whilst control clones expressed barely
detectable levels at all three stages examined. This result
appears to indicate that muscle or other mesodermal tissues
might be more predominant in mutant ES cell cultures.
Morphological appearance of differentiated cultures
The spontaneous differentiation of E14 ES cells after the
removal of LIF is a slow process occurring over at least 14
days when beating cardiac muscle is observed as a dominant
feature. Later cultures become enriched in matrix-containing
Fig. 9. Northern blot analysis of 20 µg total RNA extracted from
mutant (Km) and control (Kc) ES cell clones. Cells were stimulated
to differentiate in aggregate cultures by the removal of LIF from the
medium for 5 days or for 5 days followed by 5d in tissue culture
dishes. Labeled probes for marker gene products, α-fetoprotein
(AFP), brachyury (By) and ribosomal protein (L32) were used. AFP
production is delayed in mutant clones, while brachyury is elevated.
3338 J.-X. Wu and E. D. Adamson
areas of parietal endoderm
cells and by frequent areas of
myotubes. Dishes containing
mutant cell lines (Fig. 10A)
always contained fewer cells
compared to control or wildtype cells (Fig. 10C). The other
distinct difference was the
earlier and increased appearance of actively beating
cardiac-type muscle cells in the
mutant cultures compared to
control cultures after prolonged
spontaneous differentiation (in
the absence of LIF and RA).
Fig. 10 illustrates some
features of the differences
observed in the phase-contrast
microscope in mutant and
control
cultures.
Skeletal
muscle was observed in both
types of cultures whether spontaneously or RA induced but
there was always more in the
mutant cultures in late differentiation stages. The most
prominent feature of the
mutant cell cultures were the
frequent large colonies of small
densely packed cells that
resembled ES cells that had
failed to differentiate (Fig.
10A,E,F).
Fig. 10. Phase-contrast
micrographs to compare the
appearance of mutant (m) and
control (c) clones in culture after
various days of differentiation.
(A,C) RA-induced differentiation
for 4+9 days, endoderm and
fibroblast-like cells predominate.
(B,D) After 4+23 days of
differentiation (RA-induced),
mutant cultures are sparse (B)
while control cultures (D) contain
more fibroblast-like cells.
(E,F) Mutant clones on day 5+59
of spontaneous differentiation,
showing large areas of ES-like
(es) cells and fusing myoblasts
(mu). (G,H) Control cells on day
5+59 of spontaneous
differentiation have areas with
thyroid-like cells (th) and
cartilage-like cells (ca).
(I,J) Control cultures on day 5+53
of RA-induced differentiation
have numerous areas of
complexly organized structures
that are absent from mutant
cultures. Bar, 50 µm.
m
m
c
c
m
m
c
c
c
c
Mutant EGFR affects ES cell differentiation 3339
Fig. 11. Immunoblot to
demonstrate the presence of the
myosin heavy chain in ES cell
differentiated cultures after
various days of differentiation.
Lanes 1-6, control clones (Kc
106 and Kc107) after 0 and 15
days of RA-induced
differentiation of which 6 days
were in serum-free medium
(SFM) and 26 days, the last
17days being SFM. Mutant
clones in lanes 7-12 (Km 25,
Km 207, Km 27) were
harvested at the same times as
control cultures as indicated
above the panel. Lanes 13-16,
various mutant clone cultures
were harvested after
spontaneous differentiation. Equal amounts of protein were analyzed by 6% SDS-PAGE, blotted and detected with a monoclonal antibody to
chicken muscle myosin heavy chain. The myosin polypeptide gives a signal at about 200×103 Mr which is stronger in mutant clones than in
control or wild-type cultures (wt). Marker proteins (×10−3 Mr) migrated as shown on the left.
Biochemical evidence for the altered course of late
differentiation in mutant clones
We described above that the appearance and levels of mRNA
for tPA in mutant clones was delayed for this early marker of
differentiation. In contrast, the appearance of the brachyury
marker that precedes mesodermal differentiation was markedly
enhanced. As a later marker of mesodermal-type differentiation, the induction of the striated muscle myosin heavychain gene indicates the appearance of muscle cells in cultures.
When RA was used to induce differentiation, the process was
faster. In the experiment shown in Fig. 11, RA-stimulated
differentiation is shown on the left and spontaneous differentiation on the right. In this experiment, we used serum-free
medium during the later stages of culture to remove cells that
were serum-dependent. The mutant cultures at harvesting were
rich in beating cardiac cells. In support of this observation, we
found that only the mutant cells and not the control or wildtype cells expressed high levels of the myosin gene after
prolonged culture. Clone Km27 gave the strongest RA-stimulated myosin gene expression (Fig. 11, lane 11) while Km25
and Km207 produced highest levels of cardiac muscle during
spontaneous differentiation (Fig. 11, lanes 14 and 16, respectively). We concluded that the presence of the truncated EGF
receptor altered the outcome of the differentiation of ES cells
in that muscle formation was frequent and this occurred at the
expense of epithelium formation.
DISCUSSION
In this study of the effect of the over-expression of a truncated
EGFR, we used a construct driven by the β-actin promoter
together with the CMV enhancer (Niwa et al., 1991). It was
predicted that this construct (Fig. 1) would be active in undifferentiated ES cells in contrast to the CMV promoter used in
our previous study (Wu and Adamson, 1993) and this proved
to be the case. The 120×103 Mr truncated protein expressed,
however, was the same from both constructs and was detected
in immunofluorescence assays as exogenous receptor protein
on the surface of the mutant clones derived by G418 selection
(data not shown). There was no apparent effect of the
expression of the mutant receptor in ES cells before differentiation. We conclude that the growth, morphology, survival and
phenotype of the mouse ES cell does not depend on the EGFR.
This is not surprising since the low level of receptor protein
detected in ES and EC cells appears to be unreceptive since
the application of EGF does not elicit a response (Weller et al.,
1987).
The observed differences between mutant EGFR and control
clones indicate that the expression of the kinase-negative
mutant receptor affects the differentiation of ES cells. Differentiation is retarded as early as the 3rd day. The early marker,
tPA, does not appear on the third day of differentiation, AFP
does not appear on the 5th day of differentiation and TROMA
1 and TROMA 3 (keratins 8 and 18) do not appear on the 6th
day of differentiation in mutant clones as they do in control
clones. EGF is known to stimulate the production of tPA and
urokinase in squamous cells (Niedbala et al., 1990) and this
could explain the slow production of tPA in differentiating
mutant ES clones.
Several effects of the lack of active EGFR in mutant cells
likely contribute to the retardation of differentiation and to the
reduced expression of endogenous EGFR. Normally, as ES
cells differentiate in the absence of LIF, EGF (and other)
receptors appear and cells are able to produce a proliferative
signal to the nucleus in response to growth factors in the serumrich medium. Cells that lack this response are less able to
survive and also grow at slower rates (Fig. 6A). Cell death
assays support this hypothesis, because mutant cells produce
2-fold more dead cells in the medium than control cultures
during the first 5 days of differentiation (Fig. 6B). Apoptotic
nuclei seen in EC and ES cell aggregates during cystic
embryoid body formation, is part of embryonic programmed
cell death (Coucouvanis and Martin, 1995). At later stages of
differentiation, we were able to show that apoptosis is more
frequent in mutant cells, using the TUNEL procedure in teratocarcinomas (Fig. 5). The prevention of apoptosis by EGF has
been previously described for ovarian granulosa cells (Tilly et
3340 J.-X. Wu and E. D. Adamson
al., 1992) and in kidney development (Koseki et al., 1992;
Coles et al., 1993). Death of cells with inactive EGF-receptors
is one possible mechanism for the low expression of endogenous EGF-receptors in differentiated mutant ES cultures.
Slower proliferation rates will compound this effect so that the
population of mutant cells have lower levels of normal EGFR.
The net result of negative selection of certain cell types during
ES cell differentiation is the skewing of differentiation
pathways caused by the limited capacity of the surviving EGFreceptor-poor cells.
The normal response to EGFR signalling is the gradual accumulation of transcription factors that (a) ensure survival, (b)
maintain cell cycling and (c) commit the cells towards differentiation. In the absence of the signals provided by EGFR, the
pathways of differentiation are altered. Fig. 12 summarizes the
results obtained and the conclusions that we made. We have
shown that very low levels of 170×103 Mr receptor protein are
produced by mutant clones (Fig. 2) compared to control clones
especially during RA-induced differentiation. This results in
the increased survival of EGFR-independent cells such as those
that express brachyury (Fig. 9) followed later by the production of higher levels of muscle myosin in mutant cultures (Fig.
11). EGFR-dependent cell types such as cartilage and epithelial tissues are scarce in mutant cultures and tumours (Figs 3,4)
compared to controls. We conclude that the predominant
surviving cells are those that need little or no EGFR to differentiate and function. Cardiac and skeletal muscle are such
tissues. In the case of skeletal muscle, it has been documented
that, although EGFR protein is present and functional on
myoblast cells during the proliferation stages, the receptors are
not essential to myoblast cell proliferation and they become
inactive after fusion of the cells into myotubes (Olwin and
Hauschka, 1988; Lim and Hauschka, 1994). Our results
indicate that cardiac muscle can be added to the list of tissues
that do not require EGF-receptor expression to proliferate, to
differentiate or to function in contractility.
EGF-receptor protein expression is lost by the ES cell
adapted to in vitro culture but is present in the inner cell mass
(ICM) in the blastocyst in vivo (Wiley et al., 1992; Dardik et
al., 1992). Indeed, the function of the receptor has been shown
to be essential to the implantation of the blastocyst and the
survival of the ICM in vivo in some strains of the EGFR-null
mouse (Threadgill et al., 1995). Microinjection of the mutant
EGFR construct used here into >200 zygotes (C57Bl/6×SJL F2
hybrids) and subsequent transfer of embryos into the uteri of
recipient animals yielded only 21 pups and none were positive
for the transgene (data not shown). Therefore, in vivo, the
mutant construct in preimplantation embryos appears to
produce a lethal behaviour, much like the CF-1 EGFR-null
embryos of Threadgill and colleagues.
How does the exogenous mutant EGF-receptor behave in a
cell with normal endogenous receptor? Previous studies
(Boni-Schnetzler and Pilch, 1987; Cochet et al., 1988) have
shown that the activity of the wild-type EGF-receptor is
mediated by ligand-stimulated receptor dimerization and that
stimulation of the tyrosine kinase activity leads to the
pleiotropic responses of the cell. Several groups have
described the dominant-negative effect of a mutant receptor
dimerizing with and inactivating signal transduction from the
wild-type receptor. Recently, evidence was presented for
some types of signalling to proceed from normal receptor
ES CELLS
-LIF
DIFFERENTIATION
EGFR
DEPENDENT
EARLY
Visceral Endoderm
(AFP, Tr1)
EGFR
INDEPENDENT
Parietal Endoderm
(tPA, Tr3)
Fibroblasts
Premesoderm
(By)
LATE
Keratinocytes
Cartilage
Gut Endoderm
Bronchial Epithelium
Skeletal Muscle
+ Cardiac Muscle
(MHC)
Fig. 12. Summary of ES cell differentiation. LIF, Leukemia
Inhibitory Factor; AFP, α-fetoprotein; Tr1, TROMA-1; Tr3,
TROMA-3; tPA, tissue-type plasminogen activator; By, brachyury;
MHC, myosin heavy chain.
molecules even in the presence of kinase-negative mutant
molecules (Honegger et al., 1987; Kashles et al., 1991). It is
possible that a mutation is less dominant if the dimerizing
receptor molecules are from different species as in the cited
studies, or varies according to the mutation site and its extent
within the kinase domain. Another consideration must be
given to the possibility of the diversity of the fates of the
dimers and how this is affected by the ratio of the two partners.
In the ES cells described here, the expression of the truncated
receptor (amino acids 1-689) is always in excess of the
endogenous wild-type receptor molecule. We know that the
exogenous receptor is expressed on the cell surface and that
differentiated P19 cells expressing the mutant receptors are
incapable of forming tyrosine-phosphorylatable dimers in
response to EGF while control cells can do so (Wu and
Adamson, 1993). Therefore, it is likely that in ES cells the
excessive levels of truncated receptors will also inactivate the
endogenous receptors, although this could not be demonstrated because of the paucity of normal EGF-receptor protein
in these cells. We do not know the precise fate of the EGFreceptor molecules in mutant clones but we presume that heterodimers of wild-type and mutant proteins will form as well
as homodimers of mutant receptors.
The truncated receptor molecule contains the sequences 647
to 688 (including the transmembrane domain), shown to be
needed for internalization and turnover (Wiley et al., 1991 and
H. S. Wiley, personal communication) but it does not have the
more carboxy-terminal sequences needed for lysosome
targeting (French et al., 1994) and mitogenic signalling. We
believe that the truncated receptor will go to the default
pathway and be recycled to the surface to be effective again in
preventing the responses of the wild-type receptor. This type
of receptor lacks SH2 domains and is unlikely to be able to act
as an absorber of signalling components needed for other
Mutant EGFR affects ES cell differentiation 3341
growth factor pathways. There is a possibility, however, that
the truncated mutant EGF-receptor may form dimers with other
members of this receptor family, mErbB-2, mErbB-3 and
mErbB-4. It is possible that the protein products of c-ErbB-2
and c-ErbB-3 have functions that overlap with those of the
EGFR that may compensate for the loss of the EGFR protein.
However, we have been unable to detect any ErbB-2 or ErbB3 transcripts in differentiating ES cells by northern blotting
(data not shown) and so this seems unlikely. In any case,
truncated EGF receptor should also inactivate other ErbB
receptors by heterodimerization.
The results of this study indicate that the presence of mutant
EGF-receptor is injurious to the survival, growth and differentiation of ES cells. In the beginning, the expansion of selected
populations of committed cells might be compromised as in
inner cell mass cells in EGFR null mutant mice (Threadgill et
al., 1995) while later, the course of differentiation is altered.
The end result of the activities of the dominant-negative EGFreceptor in ES cells is differentiation to lineages that do not
require EGFR for their production. We have identified cardiac
muscle as one of these while skeletal muscle was already
known not to require EGF receptors (Olwin and Hauschka,
1988; Lim and Hauschka, 1994). We were unable to detect any
nerve cell differentiation in the E14 ES cell control clones and
so the formation of this tissue type in the presence of mutant
receptors could not be tested. In P19 cells, however, RAinduced differentiation to nerve and glial cells was inhibited
by the dominant-negative mutant receptor protein (Wu and
Adamson, 1993) and supports the idea that EGF-receptors are
required for differentiation to nervous tissues.
The results of this study also serve to underline some differences between ES cells in culture and ICM cells in the blastocyst and emphasize the need for in vivo studies to determine
how the maternal environment and the time and spatial
demands of the developmental process in vivo are regulated by
the activities of the EGF-receptor. The ES cell, however,
provides a simplified model system in which to test the cellular
and physiological effects of mutant genes in differentiating
embryonic cells in vitro.
We thank Dr S. Krajewsky for the TUNEL assays and Mr. M.
Hasham for photographic services. Excellent technical help was
provided by W. Matheny and D. Okamura. We thank Drs R-P Huang,
C. Niemeyer and D. Mercola for critical comments on the manuscript
and Dr Mercola for histological services and analyses. The β-actin
expression vector (pCXN2, Niwa et al., 1991) was kindly provided
by Dr K. Ozato. This work was supported by grants from the Public
Health Service, CA 28427 and P30 CA 30199.
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(Accepted 3 July 1996)