Nature Structural & Molecular Biology
nature structural & molecular biology
https://doi.org/10.1038/s41594-024-01311-9
Article
Tead4
and
Tfap2c
generate bipotency and
a bistable switch in totipotent embryos to
promote robust lineage diversification
Meng Zhu
1,2,7
, Maciej Meglicki
1
, Adiyant Lamba
1
, Peizhe Wang
3
,
Christophe Royer
4
, Karen Turner
5
, Muhammad Abdullah Jauhar
2
,
Celine Jones
6
, Tim Child
6
, Kevin Coward
6
, Jie Na
3
&
Magdalena Zernicka-Goetz
1,2
The mouse and human embryo gradually loses totipotency before
diversifying into the inner cell mass (ICM, future organism) and
trophectoderm (TE, future placenta). The transcription factors TFAP2C
and TEAD4 with activated RHOA accelerate embryo polarization. Here we
show that these factors also accelerate the loss of totipotency. TFAP2C and
TEAD4 paradoxically promote and inhibit Hippo signaling before lineage
diversification: they drive expression of multiple Hippo regulators while
also promoting apical domain formation, which inactivates Hippo. Each
factor activates TE specifiers in bipotent cells, while TFAP2C also activates
specifiers of the ICM fate. Asymmetric segregation of the apical domain
reconciles the opposing regulation of Hippo signaling into Hippo OFF and
the TE fate, or Hippo ON and the ICM fate. We propose that the bistable
switch established by TFAP2C and TEAD4 is exploited to trigger robust
lineage diversification in the developing embryo.
In mammals, the highly differentiated sperm and egg fuse to generate
a totipotent zygote that gives rise to all the cells in the body and to the
extraembryonic tissues. Totipotency gradually decreases during the
first few cell divisions (Fig.
1a
). At the eight-cell stage, each cell (blas
-
tomere) becomes polarized along the outside–inside axis, forming a
cap-shape structure on the outside domain called the apical domain
1
–
5
.
Asymmetric segregation of apical domains produces a 16-cell embryo
with polar outside cells that will become the trophectoderm (TE, future
placenta) and apolar inside cells that will become the inner cell mass
(ICM, future epiblast and yolk sac)
6
,
7
. ICM- and TE-specific transcrip
-
tion factors were found to be co-expressed in blastomeres before
lineage diversification
4
,
8
–
12
. However, what mechanisms lead to the
co-expression of opposite lineage markers and to reconciliation of
this bipotency into one of the two fates remain unclear.
Asymmetric segregation of the apical domain results in asym
-
metric regulation of Hippo signaling (Fig.
1b
). In outer cells, the api
-
cal domain sequesters and inhibits positive regulators of the Hippo
pathway, such as ANGIOMOTIN (AMOT) and ANGIOMOTIN-LIKE 2
(AMOTL2) (ref.
13
), resulting in a Hippo OFF state and translocation of
YAP or TAZ to the nucleus. Nuclear YAP interacts with the transcription
factor TEAD4 to induce high expression of TE fate specifiers, including
Cdx2
and
Gata3
and repress pluripotency transcription factors such as
Sox2
(refs.
8
,
14
–
18
). In contrast, the Hippo ON state in ICM cells, which
lack an apical domain, leads to YAP phosphorylation, its cytoplasmic
Received: 6 February 2024
Accepted: 9 April 2024
Published online: xx xx xxxx
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1
Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
3
Centre for Stem Cell Biology and Regenerative
Medicine, School of Medicine, Tsinghua University, Beijing, China.
4
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
5
Oxford Fertility, Institute of Reproductive Sciences, Oxford, UK.
6
Nuffield Department of Women’s and Reproductive Health, Level 3, Women’s Centre,
John Radcliffe Hospital, University of Oxford, Oxford, UK.
7
Present address: Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston,
MA, USA.
e-mail:
magdaz@caltech.edu
Nature Structural & Molecular Biology
Article
https://doi.org/10.1038/s41594-024-01311-9
Tead4
and
Tfap2c
are known to be essential for the expression
of
Cdx2
after embryo polarization and therefore for formation of the
TE lineage at the blastocyst stage
22
–
25
.
Tead4
knockout (KO) embryos
do not form a blastocyst and express the pluripotency factors, OCT4
and NANOG, in all the blastomeres, even the outer ones
24
. Combined
depletion of maternal and zygotic TFAP2C also blocks blastocyst for-
mation
22
. Additionally, TFAP2C and the TE specifier GATA3 have been
shown to directly couple TE-specific gene induction with suppression
of pluripotency
26
. Moreover, embryonic stem cells overexpressing
retention and subsequent degradation
8
,
14
,
16
. High Hippo signaling in
the ICM, and the lack of nuclear YAP, TAZ and TEAD4, promote the
expression of the pluripotency transcription factors, such as
Sox2
and
Nanog
17
,
19
. Polarized outer cells with moderate YAP or TAZ activity
that express both the ICM gene
Sox2
and the TE gene
Cdx2
have been
observed
17
,
20
. Hippo signaling in these outer cells disrupts polarization,
leading to their repositioning to the ICM
21
. How these ‘conflicted’ outer
cells arise and how Hippo signaling itself is established before cell fate
specification remain long-standing questions.
2-cell stage
16-cell stage
1.
Ezrin
–RFP mRNA
0.5
0.4
0.3
0.2
0.1
0
Ezrin
only
TTRhoA
ICM ratio (NANOG + SOX17)
**
c
d
e
Ezrin
only
TTRhoA
CDX2 NANOG
SOX17
Bright field
Single plane
Three-dimensional
a
ICM
Epiblast
Primitive endoderm
TE
b
Apical domain
TEAD4
YAP
LATS
YAP
p
AMOT
AMOTL2
AMOT
AMOTL2
NANOG
SOX2
Hippo OFF
Hippo ON
Sort polarized cells
Blastocyst reconstruction
Apical domain
ZGA
Zygote
2-cell
4-cell
Early 8-cell
Mid 8-cell
stage
Late
blastocyst
Mid
blastocyst
Lineage specification and segregation
Morula
Totipotency
2. With
Tfap2c
+
Tead4
+
RhoA-Q63L
mRNA
Fig. 1 | Premature expression of TFAP2C, TEAD4 and activated Rho GTPase
are sufficient to advance the first cell fate decision.
a
, A schematic of
preimplantation development. ZGA, zygotic genome activation.
b
, A schematic
of differential Hippo signalling in TE (top) and ICM (bottom) lineages in the
morula stage mouse embryo.
c
, A schematic of blastocyst reconstruction
assay. Two-cell stage embryos injected with
Ezrin
–RFP (
Ezrin
only, control) or
Tfap2c
+
Tead4
+
RhoA
mRNA (TTRhoA) were cultured until the early 16-cell
stage. Sixteen polarized cells from each genotype were sorted, re-aggregated,
cultured until the mid-blastocyst stage and the proportion of ICM examined.
d
, Representative images of the reconstructed blastocysts from
Ezrin
-only or
TTRhoA embryos. Embryos were immunostained to reveal CDX2 (TE), NANOG
(epiblast) and SOX17 (primitive endoderm).
e
, Quantification of the ratio of ICM
from reconstructed blastocysts from
Ezrin
-only or TTRhoA embryos. The ICM
ratio is calculated as the number of cells positive for NANOG or SOX17 divided
by the total number of cells (positive for CDX2, NANOG or SOX17). Each dot
indicates the data point obtained from one embryo. Data shown as mean ± s.e.m.
N
= 19 embryos for EZRIN-only group and
N
= 16 embryos for TTRhoA group.
N
= 2
experiments. **
P
= 0.0266, two-sided Student’s
t
-test. Scale bars, 15 μm.
Nature Structural & Molecular Biology
Article
https://doi.org/10.1038/s41594-024-01311-9
TFAP2C or TEAD4 alone upregulate TE specification genes
14
,
27
. Thus,
TEAD4 and TFAP2C have been recognized as TE fate specifiers in late
embryos at the blastocyst stage.
However, TEAD4 and TFAP2C were recently shown to play unique
roles also in the early embryo. Their expression is initiated already at
the two-cell stage and its gradual increase is required for embryo polari
-
sation at the eight-cell stage
28
. Indeed, we found that co-expressing
TFAP2C and TEAD4 with activated RHOA, a GTPase required for apical
domain formation
3
, induces precocious embryo polarization at the
four-cell stage rather than at the typical eight-cell stage
28
. Moreover,
TFAP2C was recently shown to promote the expression of both ICM and
TE genes in a bipotency program before lineage diversification
11
,
12
,
29
.
How the bipotent state is reconciled into two robust lineages, the TE
and ICM, remains unclear.
In this Article, we show that TFAP2C and TEAD4 initiate expression
of opposite lineage markers to create bipotency and an intermedi
-
ate level of Hippo signaling in all bipotent blastomeres before cell
fate specification. Our results suggest that YAP negative feedback
via Hippo signaling
30
is exploited from the two- to eight-cell stages
to diminish totipotency and generate a bistable switch. This switch is
ultimately controlled by the presence or absence of the apical domain.
Inheritance of the apical domain inhibits intermediate Hippo signaling
(Hippo OFF), sustains YAP activity and establishes TE. In contrast, in
cells lacking the apical domain, intermediate Hippo signaling becomes
Hippo ON, extinguishes YAP activity and establishes ICM. In this way,
opposing regulation of Hippo signaling in bipotent blastomeres creates
a bistable state that can robustly diversify into the two first lineages.
Results
TFAP2C, TEAD4 and RHOA are sufficient to advance cell fate
commitment
We previously showed that ectopic expression of TFAP2C, TEAD4 and
RHOA (named TTRhoA hereafter) accelerates embryo polarization.
Mouse embryos typically polarize at the eight-cell stage; however,
embryos expressing TTRhoA polarize already at the four-cell stage
28
.
Given that the potential of polarized outer cells to regenerate ICM
is progressively lost as blastomeres become committed to the TE
fate
20
, we wished to determine whether these three factors are also
sufficient to accelerate TE commitment. To test this, we performed
a blastocyst reconstruction assay (Fig.
1c
). We microinjected both
blastomeres of two-cell embryos with EZRIN–red fluorescent protein
(RFP) messenger RNA (mRNA) (to mark the apical domain), with or
without TTRhoA, using a technique that does not impair development
and allows the embryo to develop beyond implantation
31
. Polarized
blastomeres were sorted from nonpolarized blastomeres at the mid-
16-cell stage, re-aggregated and allowed to develop to the blastocyst
stage. We determined the proportion and number of ICM cells relative
to total cells in reconstructed blastocysts (Fig.
1c–e
and Extended
Data Fig. 1a–f ). Compared with controls, blastocysts derived from
polarized blastomeres expressing TTRhoA had a lower number of
ICM cells and therefore a lower ICM ratio (Fig.
1e
and Extended Data
Fig. 1b). These results indicate that TTRhoA overexpression not only
accelerates the timing of embryo polarization
28
but also accelerates
commitment to the TE fate in blastomeres.
TFAP2C, TEAD4 and RHOA advance apical domain aging and
inhibit Hippo signaling
Hippo signaling pathway components at the apical domain in the TE are
inactive, leading to nuclear localization of unphosphorylated, active
YA P
7
,
14
. In contrast, active Hippo signaling in the ICM generally triggers
YAP phosphorylation (p-YAP), cytoplasmic retention and degradation
32
.
Thus, the Hippo pathway negatively regulates YAP activity.
To determine how the acceleration of polarization and TE commit
-
ment in TTRhoA blastomeres affects Hippo signaling, we expressed the
apical marker, EZRIN–RFP, with or without TTRhoA in one blastomere
at the late two-cell stage. The uninjected blastomere served as a nonin
-
jection control (Fig.
2a
). We found that none of the EZRIN–RFP blasto
-
meres in control early eight-cell embryos displayed an apical domain
3
,
5
,
whereas about 30% of the blastomeres in EZRIN–RFP + TTRhoA had
an apical domain already at the early eight-cell stage
28
(Fig.
2b,c
and
Extended Data Fig. 2).
To investigate the consequences of precocious apical domain for-
mation on Hippo signaling, we wished to determine YAP localization in
TTRhoA embryos versus controls. To this end, we upregulated expression
of TT at the two-cell stage, introduced activated RHOA at the four-cell
stage and examined embryos at the mid eight-cell stage (Fig.
2d
). Both
EZRIN–RFP-only and TTRhoA mid eight-cell stage embryos had a mixture
of polarized and unpolarized cells, with more polarized cells in TTRhoA
embryos than in controls (Fig.
2e–h
). In control embryos, the polarized
and unpolarized blastomeres displayed similar nuclear-to-cytoplasmic
(N/C) ratios of YAP (Fig.
2e,f
and Extended Data Fig. 3a). In contrast, in
TTRhoA mid eight-cell embryos, the N/C ratios of YAP were higher in
polar versus apolar blastomeres (Fig.
2g,h
and Extended Data Fig. 3b).
Moreover, the levels of cytoplasmic p-YAP were lower in polar versus
apolar blastomeres of TTRhoA versus control embryos (Fig.
2h
). We often
detected nuclear p-YAP in blastomeres, which has been observed in other
contexts, particularly in sparsely plated cells
33
,
34
(Discussion). Overall,
these data suggest that Hippo signaling diminishes after polarization
of TTRhoA embryos, which indicates that TTRhoA not only advance
polarization timing but also enhance the function of the apical domain,
resulting in reduced Hippo signaling in blastomeres.
TFAP2c and TEAD4 activate TE and ICM genes to create a
bipotent state
We previously found that TTRhoA embryos had precocious expression
of the TE specifier
Cdx2
(ref.
28
). To determine whether this is related to
Fig. 2 |
Tfap2c
,
Tead4
and activated
RhoA
coordinate Hippo inactivation
with apical domain formation.
a
, A schematic showing the workflow for
experiments in
b
and
c
.
b
, Embryos injected with EZRIN–RFP only (as a
control) or TTRhoA mRNAs were analyzed at the early eight-cell stage to reveal
EZRIN–RFP and AMOT. The yellow squares indicate the magnified regions.
The arrows indicate magnified cells.
c
, Quantifications of apical membrane
enrichment of AMOT in cells expressing EZRIN–RFP or with TTRhoA. Data are
shown as individual data points with box and whisker plots (lower: 25%; upper:
75%; line: median; and whiskers: min to max). Each dot indicates an analyzed
cell.
N
= 12 cells for EZRIN–RFP and
N
= 8 cells for TTRhoA.
N
= 2 experiments.
***
P
= 0.0002, two-sided Mann–Whitney test.
d
, A schematic of TTRhoA
overexpression for experiments shown in
e
–
g
.
e
, Embryos overexpressing
EZRIN–RFP only (as a control) or TTRhoA, immunostained at mid eight-cell
stage for DNA (DAPI), YAP and EZRIN–RFP. The pink arrows indicate apolar
cells and yellow arrows indicate polar cells. Quantifications are shown in
f
.
f
, Quantification of the YAP N/C ratio in the polar or apolar cells of embryos
overexpressing EZRIN–RFP only or TTRhoA. Data shown as individual data
points with mean, cyan dots indicate polar cells and red dots indicate apolar
cells.
N
= 12 embryos for EZRIN–RFP only and
N
= 29 embryos for the TTRhoA
group,
N
= 4 experiments, ****
P
< 0.0001, two-way ANOVA test. YAP N/C ratios
between polar and apolar cells are statistically different in the TTRhoA group
but not in the EZRIN–RFP only group.
g
, Embryos overexpressing EZRIN–RFP
only (as a control) or TTRhoA analyzed at mid eight-cell stage for EZRIN–RFP
or p-YAP. The arrows indicate the apolar cells in TTRhoA overexpressing
embryos.
h
, Quantification of the cytoplasmic ratio of p-YAP between the polar
and apolar cells in embryos overexpressing of EZRIN–RFP only or TTRhoA.
Data shown as individual data points with mean indicated by the line. N = 7
embryos for EZRIN–RFP only and
N
= 11 embryos for the TTRhoA group,
N
= 4
experiments and *
P
< 0.05, Mann–Whitney test. The lower cytoplasmic level
of p-YAP in polar versus apolar cells in TTRhoA embryos versus controls.
For all quantifications, data are shown as individual data points with mean.
Scale bars, 15 μm.