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RESEARCH ARTICLE 3711
DEVELOPMENT AND STEM CELLS
Development 139, 3711-3721 (2012) doi:10.1242/dev.085597
© 2012. Published by The Company of Biologists Ltd
Differential requirements for -catenin during mouse
development
Stefan Rudloff* and Rolf Kemler
SUMMARY
Embryogenesis relies on the precise interplay of signaling cascades to activate tissue-specific differentiation programs. An important
player in these morphogenetic processes is -catenin, which is a central component of adherens junctions and canonical Wnt
signaling. Lack of -catenin is lethal before gastrulation, but mice heterozygous for -catenin (Ctnnb1) develop as wild type. Here,
we confine -catenin amounts below the heterozygous expression level to study the functional consequences for development. We
generate embryonic stem (ES) cells and embryos expressing -catenin only from the ubiquitously active ROSA26 promoter and
thereby limit -catenin expression to ~12.5% (ROSA26/+) or ~25% (ROSA26/) of wild-type levels. ROSA26/+ is sufficient to maintain
ES cell morphology and pluripotent characteristics, but is insufficient to activate canonical target genes upon Wnt stimulation. This
Wnt signaling deficiency is incompletely restored in ROSA26/ ES cells. We conclude that even very low -catenin levels are able to
sustain cell adhesion, but not Wnt signaling. During development, ROSA26/ as well as ROSA26/+ partially rescues the knockout
phenotype, yet proper gastrulation is absent. These embryos differentiate according to the neural default hypothesis, indicating
that gastrulation depends on high -catenin levels. Strikingly, if ROSA26/+ or ROSA26/ is first activated after gastrulation,
subsequent development correlates with the dosage of -catenin. Moreover, molecular evidence indicates that the amount of catenin controls the induction of specific Wnt target genes. In conclusion, by restricting its expression we determine the level of catenin required for adhesion or pluripotency and during different morphogenetic events.
INTRODUCTION
The development of multicellular organisms is governed by
complex signaling cascades, which lead to defined gene expression
patterns to balance the proliferation, migration and differentiation
of embryonic cells. The highly conserved canonical Wnt signaling
pathway and cadherin-mediated cell adhesion at adherens
junctions provide two examples of major importance throughout
embryogenesis (Nelson and Nusse, 2004). Vital to both of these
processes is -catenin, encoded by Ctnnb1. In cells expressing
classical cadherins (i.e. E-cadherin or N-cadherin), -catenin binds
to the intracellular part of these transmembrane proteins and
interacts with -catenin, forming a chain of proteins connecting the
cell membrane with the actin filament network of the cell (Kemler,
1993). Extracellularly, cadherins of the same isotype connect
neighboring cells, and these homophilic interactions are the basis
for the cell sorting processes that take place in embryogenesis
(Takeichi, 1988). Interrupting adherens junctions at any point in
this chain of proteins severely weakens the adhesive bond among
cells, leading to incorrect cell sorting and migration, compromising
development.
Canonical Wnt signaling is important for patterning the early
embryo, organogenesis and maintenance of tissue-specific stem
cells (Reya and Clevers, 2005). The molecular mechanisms that
lead to the activation of the canonical Wnt signaling pathway are
complex, involving the interplay of a multitude of inhibitors,
activators and their co-factors at the cell membrane, in the
Max-Planck Institute of Immunobiology and Epigenetics, Department of Molecular
Embryology, 79108 Freiburg, Germany.
*Author for correspondence ([email protected])
Accepted 16 July 2012
cytoplasm and in the nucleus. Briefly, in the absence of canonical
Wnt signals, the so-called destruction complex sequentially
phosphorylates serine/threonine residues at the N-terminus of catenin, priming the protein for subsequent polyubiquitylation and
proteasomal degradation (Aberle et al., 1997). Upon binding of a
canonical Wnt ligand to receptors of the Frizzled and LRP family,
the destruction complex is sequestered to the cell membrane and
disintegrated (Zeng et al., 2008). Consequently, -catenin
accumulates in the cytoplasm, translocates to the nucleus and, in
cooperation with LEF/TCF transcription factors, controls the
transcription of canonical Wnt target genes (Daniels and Weis,
2005). Embryos lacking -catenin (Haegel et al., 1995; Huelsken
et al., 2000) or expressing a canonical Wnt signaling-incompetent
protein (Valenta et al., 2011) have gastrulation defects and are
lethal in early postimplantation development. An arrest in
development is also seen upon expression of a dominant-active
form of -catenin that is missing the phosphorylation sites
important for degradation. This results in accumulating amounts of
-catenin protein, which leads to precocious activation of Wnt
target genes and the uncoordinated premature generation of
mesodermal cells (Kemler et al., 2004). Hence, tight control of the
expression level as well as the activity of -catenin is essential for
gastrulation.
Interestingly, -cateninflox/– mice that express -catenin only
from one allele develop indistinguishably from wild-type mice. In
this regard it would be interesting to determine the limits of the
physiological range of -catenin expression within which
development progresses normally. Here, we generated a conditional
Ctnnb1 allele making use of the ubiquitously active ROSA26
promoter (Zambrowicz et al., 1997), and analyzed the
consequences of predetermined levels of -catenin in vitro and in
vivo. Using different Cre mouse lines, differential amounts of catenin in various morphogenetic processes before and after
DEVELOPMENT
KEY WORDS: -catenin gene dosage, Pluripotency, Gastrulation, Mouse morphogenesis
gastrulation are examined. We found that even low levels of catenin expression are sufficient to maintain cadherin-dependent
adhesion. By contrast, the expression of developmental stagespecific Wnt target genes requires different amounts of -catenin,
providing a molecular link between -catenin dosage and the extent
of morphogenesis.
MATERIALS AND METHODS
Generation, culture and teratoma formation of ROSA26::-catenin
embryonic stem cell lines
Full-length Ctnnb1 was PCR amplified from a pYX-Asc plasmid (IMAGE
Consortium) and cloned into the unique XbaI site of the pROSA26-1
targeting vector (Soriano, 1999). The targeting vector also included a splice
acceptor and a floxed lacZ-neomycin (-geo) selection cassette upstream
of Ctnnb1. The splice acceptor mediates the expression of -geo or Ctnnb1,
respectively, joined to the endogenous, otherwise non-coding ROSA26
transcript.
W4/S129S6 wild-type and -catflox/– embryonic stem (ES) cells cultured
under standard conditions (DMEM with 15% FCS, LIF, feeder cells) were
electroporated with the linearized targeting vector. Cells were selected with
400 g/ml G418. Surviving clones were PCR screened for homologous
recombination and confirmed by Southern blot. Selected ES cells were
transiently transfected with a Cre-IRES-GFP plasmid, sorted, and checked
for Cre-mediated recombination by PCR (for primers see supplementary
material Table S1) and Southern blot.
For stimulation of canonical Wnt signaling, ES cells were treated with
100 ng/ml recombinant mouse Wnt3a (R&D Systems) for 4 hours and
directly lysed in Trizol reagent (Invitrogen). For embryoid body (EB)
differentiation, 500 cells were allowed to form aggregates in hanging drops
(30 l) in ES cell culture medium without LIF for 2 days. EBs were
cultured in suspension for 3 days and then plated in gelatin-coated wells.
The medium was changed every 2 days. After 2 weeks, the EBs were
assessed for contractile cells or fixed for immunofluorescent staining. For
teratoma formation, 2⫻106 cells suspended in 200 l PBS were injected
into the flanks of nude mice. Teratomas were collected after 4 weeks and
analyzed.
Generation of ROSA26::-catenin mice and isolation of embryos
Targeted wild-type ES cells were injected into C57BL/6 blastocysts.
Chimeras were backcrossed to -catflox/flox mice (Brault et al., 2001) on a
C57BL/6 background. Compound -catflox/flox;ROSA26::-catenin mice
were then crossed with either Zp3::Cre (de Vries et al., 2000), Sox2::Cre
(Hayashi et al., 2002), Cdx1::Cre (Hierholzer and Kemler, 2009),
Foxn1::Cre (Soza-Ried et al., 2008) or Wnt1::Cre (Danielian et al., 1998)
mice. Detection of the vaginal plug was considered as embryonic day (E)
0.5. Embryos were isolated at various stages from E7.0 to E18.5 and
treated according to the follow-up experiments.
For in vitro culture, E7.0 embryos were dissected in M2 medium
(Sigma) and cultured in embryo culture medium (DMEM without Phenol
Red, 8 mM L-glutamine, 2% penicillin/streptomycin, 2 mM sodium
pyruvate, 0.002% -mercaptoethanol, 2⫻ non-essential amino acids, 20
g/ml ascorbic acid) containing 50% heat-inactivated filtered rat serum
(Millipore) for 12 hours with or without 200 ng/ml Wnt3a. Mice and
embryos were genotyped with the primers listed in supplementary material
Table S1.
Immunostaining, skeletal staining and lacZ staining
For immunostaining, ES cells and 7 m cryosections of embryos were
fixed in 4% paraformaldehyde for 15 minutes on ice, and immunodetection
was performed as described (Messerschmidt and Kemler, 2010). The
following primary antibodies were used: Nanog (Messerschmidt and
Kemler, 2010) at 1:50; Oct3/4 (monoclonal mouse, Santa Cruz
Biotechnology) at 1:200; Sox2 (monoclonal mouse, Chemicon) at 1:500;
GP84 (Vestweber and Kemler, 1984) at 1:200; vimentin (monoclonal
mouse, Exbio) at 1:200; N-cadherin (monoclonal mouse, BD Biosciences)
at 1:100; -tubulin 3 (mouse, Sigma) at 1:500; NF160 (monoclonal mouse,
Abcam) at 1:200; Pax6 (polyclonal rabbit, Covance) at 1:300; -catenin
(rabbit polyclonal, Cell Signaling) at 1:200; Gata4 (polyclonal rabbit, Santa
Development 139 (20)
Cruz Biotechnology) at 1:100; and Troma-1 (Kemler et al., 1981) undiluted
supernatant. Tagged secondary goat antibodies against mouse, rabbit and
rat were: Alexa Fluor 488 or 594 (Molecular Probes) at 1:500. Nuclei were
stained with 10 M DAPI (Molecular Probes) added to the embedding
medium.
For histological analysis, fixed embryos or teratomas were dehydrated,
embedded in paraffin and sectioned at 7 m. The sections were rehydrated
and stained with Hematoxylin and Eosin. Immunohistochemical detection
was performed on rehydrated sections with primary antibodies against Ecadherin, N-cadherin and nestin (mouse monoclonal, Chemicon) at 1:30.
The signals were detected with the EnVision Plus System (Dako) using the
DAB peroxidase substrate (Sigma). For analysis of skeletal phenotypes,
E18.5 embryos were stained with Alcian Blue/Alizarin Red as described
previously (Mallo and Brändlin, 1997). For lacZ staining, the embryos
were treated as described (Stemmler et al., 2005).
Western blotting and cell surface co-immunoprecipitation
ES cells were homogenized in lysis buffer and cell lysates were probed for
-catenin at 1:1000, E-cadherin (monoclonal mouse, BD Biosciences) at
1:3000 and Gapdh (monoclonal mouse, Calbiochem) at 1:25,000 as
described (Kemler et al., 2004). Nuclear and cytoplasmic extracts were
prepared with the ProteoJET Kit (Fermentas) according to the
manufacturer’s protocol. Upon lysis of nuclei, this fraction was further
treated with benzonase nuclease (Sigma) to break up the chromatin.
For cell surface co-immunoprecipitation, ES cells were washed with
PBS and incubated on ice in 50% culture medium in PBS supplemented
with 10 l/ml rabbit serum containing the GP84 antibody for 1 hour. After
three washes with ice-cold PBS, the cells were lysed in 0.5% NP40 lysis
buffer for 20 minutes on ice. Then, 500 ng cell lysate was incubated with
20 l Dynabeads Protein G (Invitrogen) overnight at 4°C. The beads were
washed three times in PBST (PBS containing 0.1% Tween 20) and bound
proteins eluted by boiling in SDS loading buffer for 5 minutes.
Whole-mount in situ hybridization and qRT-PCR
Whole-mount in situ hybridization using digoxigenin-labeled riboprobes
was performed as described (Correia and Conlon, 2001). For quantitative
(q) RT-PCR, total RNA was isolated with Trizol (Invitrogen) following the
manufacturer’s instructions. Then, 500 ng RNA was reverse transcribed
using oligo(dT) primers (Roche), diluted 1:25 and amplified in qPCR using
Absolute qPCR ROX Mix (Thermo Scientific) in combination with the
Mouse Universal Probe Library (Roche). Results were obtained from at
least three experiments performed in triplicate using the CT method.
Primers and probes are listed in supplementary material Table S2.
RESULTS
Expressing -catenin from the ROSA26 locus
Wild-type (wt) and -cateninflox/– (-catflox/–) ES cells were targeted
to generate mice and to characterize the ROSA26::-catenin allele
in vitro. In targeted -catflox/– ES cells, transient expression of Cre
recombinase results in the simultaneous deletion of the
endogenous, floxed Ctnnb1 allele and the removal of the selection
cassette from the ROSA26 locus, activating the ectopic expression
of -catenin (ROSA26/+ ES cells) (supplementary material Fig.
S1). ROSA26/ ES cells were generated by a second round of gene
targeting. ES cells expressing -catenin only from the ROSA26
promoter maintained their characteristic growth morphology in
culture (supplementary material Fig. S2A-D).
-catenin mRNA levels in wt, -catflox/–, ROSA26/ and
ROSA26/+ ES cell lines were determined by qRT-PCR (Fig. 1A).
As expected, -catflox/– ES cells had ~50% -catenin mRNA
compared with wt ES cells. The mRNA levels in ROSA26/ or
ROSA26/+ ES cells were further reduced to a half or a quarter,
respectively, of that of -catflox/– ES cells. From this it can be
concluded that the ROSA26 promoter generates ~25% -catenin
transcripts compared with the endogenous Ctnnb1 promoter. To
establish whether the differences in mRNA levels led to different
DEVELOPMENT
3712 RESEARCH ARTICLE
-catenin dosage in development
RESEARCH ARTICLE 3713
Fig. 1. -catenin expression from the
ROSA26 locus is markedly reduced
compared with wild type but is
functionally intact. (A,B)-catenin transcript
(A) and protein (B) are found in gradually
decreasing amounts in wild-type (wt), catflox/–, ROSA26/ and ROSA26/+ mouse ES
cells. Gapdh provided a loading control.
(C)Cell surface immunoprecipitation (csIP)
using an antibody that recognizes the
extracellular domain of E-cadherin (GP84)
followed by western blotting for -catenin or
E-cadherin shows an interaction in wt and
ROSA26/+ ES cells. A non-specific antibody
was used as a control (ctrl). (D) Stimulation
with Wnt3a for 4 hours results in significant
upregulation of brachyury (T) in all ES cell
types except ROSA26/+. Treated wt and catflox/– ES cells show comparable levels of
activation, whereas stimulated ROSA26/ ES
cells express T at significantly reduced levels.
At least three samples for each genotype/
condition were analyzed in triplicate. *P<0.05,
**P<0.01, ***P<0.001, #P>0.05 (not
significant). Error bars indicate s.e.m.
Taken together, we see that -catenin produced from the
ROSA26 locus is functional in cadherin-mediated cell adhesion as
well as in canonical Wnt signaling. However, owing to the reduced
transcriptional activity of the ROSA26 promoter, the signaling
function of -catenin is almost absent in ROSA26/+ ES cells and
is compromised in ROSA26/ ES cells.
In vitro differentiation potential of ROSA26::catenin ES cells
To characterize ROSA26/+ and ROSA26/ ES cells in more detail,
qRT-PCR analysis was performed for the pluripotency transcription
factors Pou5f1 (Oct3/4), Nanog and Sox2, as well as for other
stemness markers such as Klf4 and Tert, and comparable expression
levels to -catflox/– or wt ES cells were found (Fig. 2A-C;
supplementary material Fig. S3D,E). In ROSA26/+ ES cells,
nuclear localization of Oct3/4, Nanog and Sox2 was observed,
similar to the other three ES cell types (Fig. 2D-F). Thus, despite
the different amounts of -catenin protein among the four ES cell
types, the expression of pluripotency genes appears unaffected.
From these results, we conclude that low levels of -catenin are
sufficient to maintain the ES cell phenotype.
In vitro differentiation potential was investigated using embryoid
body formation in hanging drops and replating on gelatin-coated
dishes, followed by immunofluorescent staining for differentiation
markers (Fig. 2G-I). In general, all four ES cell lines underwent
epithelial-mesenchymal transition with downregulation of Ecadherin and expression of the mesenchymal markers vimentin and
N-cadherin (cadherin 2) (Fig. 2G,H). However, ROSA26/+ ES cells
exhibited a notable bias toward differentiation into neurons, as
shown by the abundance of cell aggregates positive for -tubulin 3
DEVELOPMENT
amounts of -catenin protein, western blots on serial dilutions of
total cell lysates from all four ES cell types were performed (Fig.
1B). Levels of -catenin protein closely mirrored the mRNA levels,
as -catflox/–, ROSA26/ and ROSA26/+ ES cells expressed ~50%,
~25% and ~12.5% relative to wt ES cells. Furthermore, subcellular
fractionation revealed that -catenin is present in the cytoplasm and
in the nuclei of all four ES cell types (supplementary material Fig.
S3A).
These ES cells with defined levels of -catenin were studied
for the dual function of -catenin in cell adhesion and Wnt
signaling. In immunofluorescence for -catenin, ROSA26/+ ES
cells showed clear membrane staining, indicating that the low
amount of protein produced can complex with E-cadherin
(cadherin 1) (supplementary material Fig. S2I-L). Cell surface
immunoprecipitation experiments using an antibody specific for
the extracellular part of E-cadherin [GP84 (Vestweber and
Kemler, 1984)] revealed a similar association with -catenin in
wt and ROSA26/+ ES cells, demonstrating the correct assembly
of the cadherin-catenin complex even in ES cells with the lowest
amount of -catenin (Fig. 1C). The Wnt signaling function was
examined by stimulating the ES cells with Wnt3a and studying
the activation of the canonical Wnt target genes Axin2, Cdx1 and
brachyury (T) (Fig. 1D; supplementary material Fig. S3B,C).
Without Wnt3a treatment, T is expressed at very low levels in all
four ES cell lines. However, upon stimulation, clear differences
became apparent. In wt and -catflox/– ES cells, T expression was
induced at least 11-fold and reached comparable levels in both
ES cell types. By contrast, ROSA26/ ES cells only showed a 5fold activation, whereas in ROSA26/+ ES cells no increase was
detected.
3714 RESEARCH ARTICLE
Development 139 (20)
Fig. 2. ROSA26::-catenin ES cells maintain
the expression of pluripotency markers but
have limited differentiation potential in
vitro. (A-C)The core pluripotency transcription
factors Nanog, Oct3/4 and Sox2 are similarly
expressed in all four ES cell types. (D-F)Nanog,
Oct3/4 and Sox2 are detected in
immunofluorescent stainings of ROSA26/+ ES
cells. (G-I)Some differentiated ROSA26/+ cells
are positive for the mesenchymal markers
vimentin and N-cadherin and have
downregulated E-cadherin, whereas the
majority of cells preferentially differentiate into
neurons marked by -tubulin 3 (-T3) and
neurofilament 160 (NF160). At least three
samples for each genotype were analyzed in
triplicate. #P>0.05 (not significant). Error bars
indicate s.e.m. Scale bars: 100m.
expression was greatly reduced (arrows, Fig. 3D) and a marked
increase of N-cadherin and nestin was observed (Fig. 3E,F).
In summary, keeping ES cells in an undifferentiated state does not
require high levels of -catenin. Furthermore, low -catenin
expression levels are sufficient to induce differentiation in vitro and
in teratomas; however, the developmental fate of ES cells expressing
-catenin only from the ROSA26 locus is biased toward neural fates.
ROSA26::-catenin partially rescues -catenin
knockout embryos
Next, we induced the switch in -catenin expression from the
endogenous to the ROSA26 locus during embryonic development,
Fig. 3. ROSA26::-catenin ES cells
preferentially differentiate into
neuroectoderm. (A-C) Hematoxylin and
Eosin-stained teratoma sections derived from
-catflox/– (A) and ROSA26/+ (B,C) ES cells.
-catflox/– teratomas are composed of
derivatives of all three germ layers. The
diagonal line separates two images taken
from different regions of the same teratoma,
demonstrating the diverse differentiation
pattern. ROSA26/+ ES cells preferentially
form neuroectodermal cell aggregates,
neural rosettes and neural tube-like
structures. (D-F)Immunohistochemical
analysis of ROSA26/+ teratomas reveals only
a few E-cadherin-positive cells (arrows), and
the upregulation of the proneural markers Ncadherin and nestin. Scale bars: 100m.
DEVELOPMENT
(-T3) and Nefm [neurofilament 160 (NF160)] (Fig. 2I). Contractile
cardiomyocyte precursors were never detected in ES cells
expressing -catenin only from the ROSA26 locus, in contrast to
controls, in which these structures were rather frequent. Thus, the
in vitro differentiation of ES cells expressing low amounts of catenin suggested a shift toward neural differentiation and low
mesodermal cell fate specification. Further support for this came
from the analysis of teratomas. Whereas -catflox/– ES cells
differentiated into derivatives of all three germ layers (Fig. 3A),
teratomas derived from ROSA26/+ ES cells were largely composed
of neuroectodermal cell conglomerates (Fig. 3B,C). In
immunohistochemistry of ROSA26/+ teratomas, E-cadherin
-catenin dosage in development
RESEARCH ARTICLE 3715
using several Cre transgenic mouse lines. In a first set of
experiments, Cre recombinase expression was driven by the zona
pellucida glycoprotein 3 promoter (Zp3::Cre) or by the promoter
of the Sox2 transcription factor (Sox2::Cre), resulting in the
deletion of endogenous Ctnnb1 before gastrulation (Fig. 4).
Zp3::Cre recombination is restricted to the maternal genome, but
recombined alleles are present in embryonic and extra-embryonic
tissues (de Vries et al., 2000). Sox2::Cre, if paternally inherited, is
only active in the epiblast starting at E5.5, leaving extra-embryonic
tissues unaffected (Hayashi et al., 2002).
Sole expression of ROSA26/+ or ROSA26/ was able to
ameliorate the -catenin knockout phenotype (Fig. 4A). However,
compared with -catflox/– embryos (Fig. 4B), the formation of
proper embryonic structures was incomplete, regardless of whether
such embryos were generated with Zp3::Cre or Sox2::Cre (Fig.
4C,D). Morphologically, E8.5 mutant embryos had a sac-like
morphology, exhibiting invaginations (Fig. 4C-H,K,L). The entire
embryonic structure was composed of two layers, of which the
outer layer that faces the yolk sac cavity was reminiscent of the
visceral endoderm, whereas the inner layer had characteristics of a
pseudostratified epithelium. Further marker analysis demonstrated
that the inner layer expressed transcription factors of early neural
progenitors, such as Sox2 and Pax6 (Fig. 4E,F), and showed
enhanced expression of N-cadherin (Fig. 4G), whereas E-cadherin
expression was very low (Fig. 4H).
To assess whether gastrulation took place in mutant embryos,
expression of the early mesodermal marker T was analyzed at E7.5
(Fig. 4I,J). In contrast to controls, no T message was detected in
ROSA26/+ or ROSA26/ embryos (Fig. 4J). From these results we
conclude that the low amount of -catenin in pregastrulation
ROSA26/+ or ROSA26/ embryos is insufficient to establish the
posterior-anterior Wnt signaling gradient, a prerequisite for
gastrulation. This was further supported by the introduction of the
Wnt reporter BATGal (Maretto et al., 2003) into ROSA26/ mutant
embryos, which failed to become activated. To determine whether
the Wnt reporter in ROSA26/ embryos could be force-activated,
E7.0 embryos were cultured in vitro with or without recombinant
Wnt3a for 12 hours (Fig. 4K,L). Without stimulation the BATGal
DEVELOPMENT
Fig. 4. ROSA26::-catenin
partially rescues the catenin knockout. (A,B)At
E7.5, no embryonic structures
are found in -catenin
knockouts (A), whereas catflox/– embryos are of wildtype appearance (B).
(C,D)Morphologically,
ROSA26/+ or ROSA26/
embryos have a sac-like,
bilayered epithelial structure,
regardless of whether extraembryonic tissues were
recombined or not. The inner
layer is reminiscent of
embryonic ectoderm and the
outer has characteristics of
visceral endoderm. (E-H)The
early neural markers Sox2 (E)
and Pax6 (F) are expressed in
the inner epithelial layer,
whereas E-cadherin
expression is almost absent
(H) and N-cadherin strongly
upregulated (G). (I,J)In situ
hybridization for the early
mesodermal marker T shows
the typical posterior
expression pattern in catflox/– embryos (I), whereas
no signal is detected in
mutants (J). (K,L)Additional
stimulation of ROSA26/
embryos in vitro with Wnt3a
for 12 hours is sufficient to
activate the canonical Wnt
reporter BATGal (L). Reporter
activity is not detected in
mock treated embryos (K).
Scale bars: 100m.
3716 RESEARCH ARTICLE
Development 139 (20)
reporter remained inactive (Fig. 4K). However, Wnt3a addition
resulted in the activation of the reporter in a group of cells (Fig.
4L). The positioning of these BATGal-positive cells within the
mutant embryos appeared to be random (inset in Fig. 4L), which
was most likely due to the exposure of the entire embryo to the
exogenous Wnt3a signal. Nevertheless, these experiments
demonstrate that -catenin originating from the ROSA26 locus is
able to activate the Wnt reporter gene, and that the amount of catenin is critical. These results provide convincing evidence that
at gastrulation, high expression levels of -catenin are required for
proper development.
-catenin dosage effects on development after
gastrulation
In order to circumvent the defects during gastrulation, we made use
of Cdx1::Cre, which at E7.5 initiates recombination in all three
germ layers in the posterior half of the embryo caudal to the heart
anlage (Hierholzer and Kemler, 2009). Interestingly, ROSA26/
and ROSA26/+ embryos generated with Cdx1::Cre exhibited
remarkable differences in the formation of embryonic structures
(Fig. 5A-F). At E9.5, ROSA26/+ embryos showed a truncated tail
bud region (Fig. 5A) and caudal development was severely
impaired, such that at E14.5 these embryos consist of a head
attached to internal organs including lung, liver and intestine (Fig.
5D). The urogenital system and mesoderm-derived tissues making
up the body wall (muscles, ribs and limbs) were highly
underdeveloped or absent. By contrast, E9.5 ROSA26/ embryos
had tail buds of normal length (Fig. 5B). However, most distally
they showed an improperly folded neural tube that remained open
as development progressed (arrowheads, Fig. 5B,E). At E14.5,
ROSA26/ embryos had developed small kidneys (asterisk, Fig.
5E), adrenal glands and gonads, but lacked a tail, hind limbs and
showed malformations including a persistently open cloaca (arrow,
Fig. 5E). Skeletal preparations of E18.5 ROSA26/ embryos
showed deformed shortened fused ribs, vertebrae and digits, and
rarely also rudimentary bones of the upper hind limb
(supplementary material Fig. S4). Although ROSA26/ embryos
developed until birth, they did not survive. These results clearly
indicate that the degree of caudal development is -catenin dosage
dependent. This was also seen using the BATGal reporter (Fig. 5AC). Activation of this Wnt reporter was severely impaired in the
caudal half of ROSA26/+ embryos (Fig. 5A). By contrast,
ROSA26/ embryos showed a BATGal activity pattern comparable
to that of controls (Fig. 5B,C). It is noteworthy that the BATGal
reporter, which could not be activated before gastrulation (Fig. 4K),
showed normal activity thereafter.
For further analysis, RNA was isolated from the tail bud region
of E9.5 embryos and the expression levels of -catenin determined
by qRT-PCR; these were similar to those found in ES cells
(supplementary material Fig. S5). To shed some light on how the
different -catenin levels influence caudal development, the
expression profiles of Wnt target genes and genes important for the
formation and maintenance of the tail bud and presomitic
mesoderm were assessed. Generally, the expression levels of
known and putative Wnt targets, such as Cdx1, Fgf8, T and Wnt3a,
as well as Tcf1 and Lef1, were significantly reduced in ROSA26/+
mutant embryos (Fig. 6A-F). In ROSA26/ mutants, the expression
of most of these genes reached at least 60% of that of the control
group of embryos. Interestingly, intermediate or even elevated catenin expression did not further increase the transcription of
target genes. Especially notable are the expression profiles of
Wnt3a (Fig. 6D), which was expressed in ROSA26/ embryos at
comparable levels to controls, and of Cdx1 (Fig. 6A), which
remained at very low levels in both mutants. In contrast to this and
to the expression profiles of Tcf1 and Lef1 (Fig. 6E,F), an increase
in the expression of Tcf3 and Tcf4 was only observed for
ROSA26/+ embryos (Fig. 6G,H). Based on these observations, it
can be concluded that -catenin-responsive genes are detected at
very low levels in ROSA26/+ embryos and that doubling the catenin dosage is sufficient to induce a significant increase.
DEVELOPMENT
Fig. 5. -catenin dosage correlates with
developmental progress in postgastrulation
embryos. (A-C)BATGal reporter activity in E9.5
mouse embryos. (A)ROSA26/+ embryos have a
severely truncated tail bud and reduced Wnt
reporter activity caudal to the forelimbs.
(B,C)ROSA26/ embryos (B) have a BATGal
reporter activity that is indistinguishable from
that of -catflox/– embryos (C). However,
morphologically, they display a neural tube
closure defect (arrowheads). (D-F)Midsagittal
sections of E14.5 embryos. (D)ROSA26/+
embryos consist of a head attached to a bundle
of inner organs. Brain structures posterior to the
thalamic region are severely underdeveloped or
missing. An enclosing body wall and the
urogenital system are absent; the heart is not
shown in this section. (E,F)In ROSA26/ embryos
(E), brain development is complete. Parts of the
body wall, as well as the urogenital system
develop; however, the kidneys (asterisk) are
smaller than in controls (F) and malformations
are apparent (open cloaca, arrow). Hind limbs
and a proper tail are missing. The caudal neural
tube closure defect persists (arrowhead). Lu,
lung; Li, liver; In, intestine; #, toes. Scale bars:
500m.
-catenin dosage in development
RESEARCH ARTICLE 3717
Another important finding is that each gene seems to require a
specific level of -catenin in order to be expressed at the wild-type
level.
Tissue-specific expression of ROSA26::-catenin
To address whether the expression of ROSA26/+ or ROSA26/ can
support the development and maintenance of specific organs or
tissues at later stages of development, we made use of Wnt1::Cre
and Foxn1::Cre transgenic mice. Wnt1::Cre-specific deletion of
Ctnnb1 results in dramatic brain malformations and defective
craniofacial development (Brault et al., 2001). Exclusive
expression of ROSA26::-catenin in these areas rescues brain
development and the formation of neural crest-derived facial
structures in a dosage-dependent manner (Fig. 7A-C). ROSA26/
mutant embryos were born, breathed, but died within the first
hours, probably because of their inability to feed (Fig. 7D). In
contrast to all other Cre mouse lines used in this study, expressing
ROSA26::-catenin with the help of Foxn1::Cre in the thymic
anlage, epidermis and hair follicles (Soza-Ried et al., 2008),
resulted in viable offspring (Fig. 7F,G). The knockout of -catenin
in the Foxn1 expression domain is neonatal lethal due to skin
lesions (J. Swann, personal communication). Strikingly, even one
ROSA26::-catenin allele was sufficient to restore skin epithelial
integrity. Although mutant ROSA26/+ and ROSA26/ pups are of
reduced size at postnatal day (P) 14 compared with control
littermates they recover with age. Again, a -catenin dosagedependent phenotype is detected for these mice. ROSA26/+ mice
exhibit an irregular fur pattern, in which broad stripes of hair loss
DEVELOPMENT
Fig. 6. Expression profiles of known and putative Wnt target genes in the tail bud. Different Wnt target genes expressed in isolated E9.5
mouse tail buds have differential expression profiles. (A)Cdx1 expression is reduced to ~30% relative to wt in ROSA26/ and ROSA26/+ embryos,
reaches a plateau level and shows a second upsurge for the highest amount of -catenin. (B)Fgf8 expression is less than 20% in ROSA26/+ tail
buds, and shows an intermediate, but still significantly reduced, level in ROSA26/ embryos (~60%). (C)T expression levels are similar for all but
ROSA26/+ embryos, in which it is ~30%. (D)Wnt3a behaves similar to T, but shows a secondary increase in expression like Cdx1. (E,F)Tcf1 and
Lef1 expression profiles are analogous to that of Fgf8. (G,H)In contrast to all other genes analyzed, the expression of Tcf3 and Tcf4 remains
constant for all embryos, but increases significantly in ROSA26/+ tail buds. n, number of embryos. *P<0.05, **P<0.01, ***P<0.001, #P>0.05 (not
significant). Error bars indicate s.e.m.
3718 RESEARCH ARTICLE
Development 139 (20)
are followed by intervals of regrowth (Fig. 7F). ROSA26/ animals
have continuous fur cover that is thinner than that of wt mice (Fig.
7G).
In summary, tissue-specific expression of ROSA26::-catenin
produces dosage-dependent phenotypes at embryonic and postnatal
stages. Interestingly, for Foxn1::Cre, the expression of a single
ROSA26::-catenin allele is sufficient to substitute for endogenous
Ctnnb1 in terms of skin integrity and viability of the animals; no
immunological defects were found.
DISCUSSION
In the work presented here, we take advantage of the ROSA26 locus
as a stable source for -catenin transcription and were able to limit
the amount of -catenin to ~25% or ~12.5% relative to wild-type
levels upon ablation of endogenous Ctnnb1. The ROSA26 locus was
identified in a random retroviral gene-trapping approach
(Zambrowicz et al., 1997), and is characterized by its ubiquitous and
moderated expression throughout embryonic development and in
adult tissues (Kisseberth et al., 1999). Based on the expression of the
-geo fusion gene used for selection of targeted ES cells, we detected
similar expression levels in various tissues of E14.5 embryos by
qRT-PCR (supplementary material Fig. S6A), as well as in X-Galstained sections (supplementary material Fig. S6B).
Monoallelic expression of ROSA26::-catenin seems unable to
cope with the dual demand for -catenin that is necessary to supply
adherens junction complexes and to initiate Wnt target gene
transcription. In ROSA26/+ ES cells, -catenin is predominantly
found at the cell membrane in a complex with E-cadherin, as in wt
ES cells. The strong association between these proteins even at low
-catenin concentrations can be explained by the nature of the
assembly process of cadherin-catenin complexes (Ozawa et al.,
1989). -catenin co-translationally binds to the cytoplasmic domain
of E-cadherin already in the endoplasmic reticulum, protecting the
cadherin molecule from degradation and aiding in its transport to
the cell membrane (Chen et al., 1999; Curtis et al., 2008).
Furthermore, it was shown using recombinant proteins that Ecadherin is able to outcompete -catenin from associations with
Apc or Lef1 (Hülsken et al., 1994; Orsulic et al., 1999). Therefore,
the inability to activate the transcription of canonical Wnt target
genes in ROSA26/+ ES cells could be explained by insufficient
amounts of free -catenin due to the constant sequestration of
newly synthesized -catenin protein to E-cadherin.
Despite its high affinity for E-cadherin, we could still detect
small amounts of -catenin in nuclear extracts of ROSA26/+ ES
cells. Nevertheless, canonical Wnt target genes were not induced
in these ES cells upon stimulation of the Wnt signaling pathway.
An explanation for this observation can be found in Weber’s law,
which states that a stimulus is always perceived with respect to
its background level. Goentoro and Kirschner proposed that
Weber’s law applies to canonical Wnt signaling in developing
DEVELOPMENT
Fig. 7. Tissue-specific expression of ROSA26::-catenin shows dosage-related phenotypes. (A-E)Wnt1::Cre recombination results in
increased morphogenesis of craniofacial structures in a -catenin dosage-dependent manner. Lateral (A-C) and frontal (A⬘-C⬘) views of ROSA26/+,
ROSA26/ and control embryos at E16.5. Neonatal ROSA26/ pups (D) have open eyes and a misshapen snout compared with controls (E).
(F,G)Mice expressing ROSA26::-catenin in the Foxn1 domain. ROSA26/+ mice (F) have a disturbed fur pattern, with broad stripes of fur-covered
and naked skin. ROSA26/ mice (G) have thicker fur than heterozygous mutants; however, it is still less dense than that of control mice.
Xenopus embryos (Goentoro and Kirschner, 2009). According to
their hypothesis, the embryos respond to the fold-change value
in -catenin levels upon Wnt signaling and not to the absolute
amount of -catenin after Wnt induction. Therefore, within a
certain range, the exact levels of -catenin present in individual
cells before the signaling event do not determine the signaling
outcome, as long as the fold-changes are maintained. Thus, the
-catenin expression level of ROSA26/+ ES cells might generate
insufficient fold-changes upon Wnt stimulation and thus Wnt
target genes are not turned on. By contrast, in ROSA26/ ES
cells the fold-change in -catenin levels upon Wnt stimulation is
sufficient to activate the transcription of Wnt target genes.
However, their absolute expression level is markedly reduced
compared with control ES cells, which indicates a more complex
regulation of target gene expression than just the fold-change in
-catenin levels.
Absent or reduced signaling cues promote the differentiation
of ROSA26/+ and ROSA26/ ES cells toward neural fates,
which is in accordance with a hypothesis known as the default
model of differentiation (Glinka et al., 1997; Hemmati-Brivanlou
and Melton, 1997; Kamiya et al., 2011). Under culture
conditions, ROSA26/+ ES cells exhibit normal ES cell
morphology and express pluripotency-associated transcription
factors similar to wt ES cells. These findings raise the question
of how -catenin contributes to maintain pluripotency and add
new aspects to the controversial role of Wnt signaling in this
process (Wray and Hartmann, 2012). Independent reports on the
derivation of -cat–/– ES cell lines with normal pluripotency
marker expression profiles (Lyashenko et al., 2011; Wray et al.,
2011) support the idea that ES cells can be maintained without
-catenin. In this regard, it was shown that plakoglobin is able
to substitute for -catenin in adherens junctions, highlighting
the importance of E-cadherin-mediated cell adhesion for
pluripotency. E-cadherin was proposed to be at the core of a
distinct signaling network that, parallel to Wnt signaling,
promotes the ES cell state (Xu et al., 2010). Furthermore, during
reprogramming, the forced expression of E-cadherin
significantly enhances the yield of induced pluripotent stem cell
colonies (Redmer et al., 2011). It is generally believed that catenin prevents the differentiation of ES cells through the
inhibition of Tcf3-mediated repression of pluripotencyassociated genes (Sokol, 2011). Alternatively, TCF-independent
mechanisms have been proposed in which direct interactions of
-catenin with either Oct3/4 or Satb1 were shown to promote the
expression of pluripotency markers (Savarese et al., 2009; Kelly
et al., 2011). Therefore, the small amount of -catenin produced
from the ROSA26 locus suffices to sustain pluripotency either by
supporting cadherin-mediated cell adhesion or through Wnt
signaling-independent mechanisms.
During embryogenesis, the time point of ROSA26::-catenin
activation is critical for the developmental outcome of the embryos.
When expressed before gastrulation, ROSA26/+ or ROSA26/
mutant embryos possess an epithelial structure, which represents
an improvement compared with knockout embryos for which no
embryonic organization is observed (Haegel et al., 1995). However,
neither ROSA26/+ nor ROSA26/ is able to activate the BATGal
reporter or endogenous Wnt target genes such as T. Given the
crucial role of T in the induction of mesoderm (Arnold et al., 2000),
mutant embryos transfate into neuroectoderm, analogous to the
differentiation potential of ROSA26/+ or ROSA26/ ES cells.
Similar phenotypes are also found in mice, in which canonical Wnt
signaling is disrupted through the knockout of Wnt pathway
RESEARCH ARTICLE 3719
components upstream of -catenin (Liu et al., 1999; Hsieh et al.,
2003; Biechele et al., 2011). In these knockout mice, -catenin
cannot accumulate due to its uninterrupted degradation; however,
its adhesive function is unchanged. From this, we conclude that
gastrulation requires a high level of -catenin and that low catenin levels are able to sustain normal cell adhesion in vivo.
Interestingly, the necessity for such high -catenin levels seems not
to exist in postgastrulation development. Instead, we observed a
strong correlation between the -catenin gene dosage, the degree
of morphogenesis among ROSA26/+, ROSA26/ or control
embryos, and the induction of -catenin target genes.
From our gene expression data, it appears that Wnt3a is already
expressed at ~50% in ROSA26/+ and at wild-type levels in
ROSA26/ tail buds. Despite the normal expression level of Wnt3a
in ROSA26/ embryos, their phenotype is highly reminiscent of the
Wnt3a knockout (Takada et al., 1994), showing that Wnt3a alone
is not sufficient to control tail bud development. Other factors
known to be involved in the caudal extension of the embryo are
Fgf8 and Cdx1 (van den Akker et al., 2002; Naiche et al., 2011).
However, Fgf8 expression reaches only ~60% and Cdx1 expression
remains below 30% in ROSA26/ embryos relative to controls.
These findings highlight the need for proper expression of Fgf8 and
Cdx1 in tail bud morphogenesis. Moreover, we also show that
different Wnt target genes require different levels of -catenin to
be activated, adding another regulatory level to canonical Wnt
signaling. A likely reason for the diminished transcription of Wnt
target genes in ROSA26::-catenin mutant embryos is the reduced
expression levels of Tcf1 and Lef1. Since these transcription factors
are required for the activation of canonical Wnt target gene
transcription, their reduced expression levels might cause globally
decreased expression of Wnt target genes. In support of this notion,
a recent publication proposed an additional regulatory circuit of
Wnt signaling wherein Tcf3-mediated repression of Lef1
transcription is alleviated upon stimulation of the pathway (Wu et
al., 2012). In agreement with their hypothesis, we observe that, in
ROSA26/+embryos, the expression level of Tcf3 is increased and
those of Lef1 and Tcf1 are greatly reduced. Thus, at low expression
levels, -catenin seems unable to counteract Tcf3-mediated
repression of Lef1 and Tcf1. However, in ROSA26/ embryos, the
expression levels of Lef1 and Tcf1 are partially restored. Based on
these findings, we reason that canonical Wnt signaling activity
itself depends on the amount of -catenin. Once -catenin
expression is at least as high as that in -catflox/– mice, adequate
Wnt signaling activity is restored. The expression levels of all
LEF/TCFs and that of most target genes remain unchanged, even
in mice that have an elevated expression level of -catenin (catflox/+ plus ROSA26/ or ROSA26/+). Thus, we conclude that the
Wnt signaling machinery is able to cope with small increases in catenin expression.
Tissue-specific expression of ROSA26::-catenin also shows catenin dosage-dependent phenotypes. However, different tissues
seem to require different levels of -catenin for their
morphogenesis and functionality. For example, expression of
ROSA26/ using Wnt1::Cre supports the formation of midbrain
and craniofacial structures, although mutant pups die within a few
hours after birth. Strikingly, mice expressing ROSA26/+ in the
Foxn1 expression domain develop to term and thrive normally.
This shows that low -catenin levels that merely support its
adhesive function are sufficient to restore the integrity of the skin
epithelial barrier in vivo. Nevertheless, ROSA26/+ mice show an
abnormal hair cycle, which is less severe in ROSA26/ animals,
probably owing to improved Wnt signaling.
DEVELOPMENT
-catenin dosage in development
By expressing -catenin from the ROSA26 promoter we were
able to separate the adhesive from the signaling function of catenin without introducing any additional mutations into the
molecule. More importantly, we show that a given level of catenin controls particular morphogenetic events in development,
which is also transferable to the induction of specific -catenin
target genes.
Acknowledgements
We thank Riana Vogt for excellent technical support, preparing reagents and
cell culture; Benoît Kanzler for blastocyst injection; Caro Johner and Manfred
Mellert from the animal facility; and Andreas Hierholzer, Ignacio del Valle and
Daniel Messerschmidt for comments and discussions regarding the preparation
of the manuscript.
Funding
This work was funded by the Max-Planck Society.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.085597/-/DC1
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DEVELOPMENT
-catenin dosage in development