of 31
Article
Basal delamination during mouse gastrulation
primes pluripotent cells for differentiation
Graphical abstract
Highlights
d
An
in vitro
3D model captures the main features of the
gastrulating mouse epiblast
d
Proliferation-mediated crowding triggers basal delamination
of aPKC high cells
d
Delamination sensitizes cells to Wnt activation, leading to
Brachyury expression
Authors
Nanami Sato, Viviane S. Rosa,
Aly Makhlouf, ..., Alexander Meissner,
Magdalena Zernicka-Goetz,
Marta N. Shahbazi
Correspondence
mshahbazi@mrc-lmb.cam.ac.uk
In brief
Sato and Rosa et al. develop a 3D
embryonic stem cell model and show
that, during gastrulation, proliferation-
induced crowding triggers basal cell
delamination. In turn, delamination
safeguards differentiation, as it sensitizes
cells to Wnt pathway activation.
Proliferation
& crowding
Brachyury
expression
Wnt
aPKC levels
Brachyury
Basal
delamination
Sato et al., 2024, Developmental Cell
59
, 1252–1268
May 20, 2024
ª
2024 MRC Laboratory of Molecular Biology.
Published by Elsevier Inc.
https://doi.org/10.1016/j.devcel.2024.03.008
ll
Article
Basal delamination during mouse gastrulation
primes pluripotent cells for differentiation
Nanami Sato,
1
,
9
Viviane S. Rosa,
1
,
9
Aly Makhlouf,
1
,
10
Helene Kretzmer,
2
,
10
Abhishek Sampath Kumar,
2
Stefanie Grosswendt,
2
,
3
,
4
Alexandra L. Mattei,
2
Olivia Courbot,
5
,
6
Steffen Wolf,
1
Jerome Boulanger,
1
Frederic Langevin,
1
Michal Wiacek,
1
Daniel Karpinski,
1
Alberto Elosegui-Artola,
5
,
6
Alexander Meissner,
2
Magdalena Zernicka-Goetz,
7
,
8
and Marta N. Shahbazi
1
,
11
,
*
1
MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK
2
Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany
3
Max Delbruck Center for Molecular Medicine, 13125 Berlin, Germany
4
Berlin Institute of Health (BIH) at Charite
́
—Universit
atsmedizin, Berlin, Germany
5
Cell and Tissue Mechanobiology Laboratory, The Francis Crick Institute, London NW1 1AT, UK
6
Department of Physics, King’s College London, London WC2R 2LS, UK
7
University of Cambridge, Cambridge CB2 3EL, UK
8
California Institute of Technology, Pasadena, CA 91125, USA
9
These authors contributed equally
10
These authors contributed equally
11
Lead contact
*Correspondence:
mshahbazi@mrc-lmb.cam.ac.uk
https://doi.org/10.1016/j.devcel.2024.03.008
SUMMARY
The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed.
This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak,
and their priming toward primitive streak fates. How these different events are coordinated remains un-
known. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that cap-
tures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this
system in combination with microfabrication and
in vivo
experiments, we found that proliferation-induced
crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon
delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak
markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that
the right cell types become specified at the right place during embryonic development.
INTRODUCTION
The emergence of the mammalian body plan takes place during
gastrulation, a critical stage that entails a complex choreography
of morphogenetic movements, cell fate specification events, and
a burst of proliferation.
1
All these events need to be finely coor-
dinated to ensure the correct cell types are specified at the right
place and to prevent congenital malformations or even embry-
onic lethality.
2
However, dissecting the mechanisms underlying
this coordination remains challenging due to the difficulty of
breaking down the contribution of individual components in the
in vivo
developing embryo.
Inmouseembryosatembryonicday6.5(E6.5),theextra-embry-
onic tissues generate a gradient of Wnt, Bmp, and Nodal signaling
that triggers the onset of gastrulation and primitive streak (PS) for-
mation in posterior embryonic epiblast cells.
3
Gastrulation starts
with the breakdown of the basement membrane,
4
which is fol-
lowed by the ingression of cells in the PS. At the cellular level,
ingressingcellsundergoapicalconstriction,followedbydelamina-
tion on the basal side. At the molecular level, ingression has been
proposed to be regulated by a complementary pattern of localiza-
tion of actomyosin and apical polarity proteins.
5
,
6
Basal mitotic
rounding also contributes to the process of ingression.
7
,
8
Upon
ingression, mesoderm progenitor cells undergo an epithelial-to-
mesenchymal transition (EMT), which leads to the downregulation
of the cell-cell adhesion protein E-cadherin, the dismantling of
the epithelial phenotype, and the acquisition of a mesenchymal
morphology.
9
In terms of cell fate, posterior epiblast cells become
regionalized in response to varying levels of Bmp, Wnt, and Nodal
signaling.ProximalepiblastcellsupregulateBrachyuryexpression
anduponingressiongiverisetomesodermderivatives,whiledistal
epiblast cells generate definitive endoderm and axial mesoderm.
3
Theabovementionedchangesincellfateandtissueshapehappen
concomitantly with a burst of proliferation. Prior to PS formation,
epiblast cells have a cell-cycle length of approximately 9 h, while
in the PS this is decreased to 5–6 h.
7
,
10
,
11
Epiblast stem cells (EpiSCs) represent a tractable system to
dissect the mechanisms that regulate lineage specification at
1252
Developmental Cell
59
, 1252–1268, May 20, 2024
ª
2024 MRC Laboratory of Molecular Biology. Published by Elsevier Inc.
This is an open access article under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
).
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Developmental Cell
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, 1252–1268, May 20, 2024
1253
gastrulation.
12
,
13
When cultured in the presence of fibroblast
growth factor 2 (bFgf2) and Activin-A, they show a transcrip-
tional profile that is comparable with the anterior PS of the gas-
trulating mouse embryo.
14
They display a primed pluripotent
state of high transcriptional heterogeneity and lineage
biases.
15
,
16
However, because they are cultured in 2D, they
fail to recapitulate the shape changes that take place at gastru-
lation. Recently, several stem cell models of the embryo that
mimic certain aspects of gastrulation have been developed.
17
Gastruloids robustly undergo symmetry breaking and tissue
patterning but lack proper tissue organization,
18
while models
formed by aggregation of embryonic stem cells (ESCs) and ex-
tra-embryonic cells recapitulate tissue organization and cell
identities, but with limited efficiency.
19–22
Here,wehaveestab-
lished a self-renewing three-dimensional (3D) culture of EpiSCs
that recapitulates the main transcriptional and architectural fea-
tures of the gastrulating epiblast. Using this system in combina-
tion with microfabrication and
in vivo
experiments, we demon-
strate that cell delamination safeguards differentiation at the PS.
RESULTS
Identification of conditions to preserve epithelial
architecture and pluripotency in 3D
As a first attempt to develop a model of the gastrulating epiblast,
we embedded mouse ESCs in 3D Matrigel in the presence of
EpiSC medium (FA). Although this maintained epithelial integrity
and pluripotent gene expression for up to 5 days, by day 7 cells
lost expression of the pluripotency markerSox2 and failed to pro-
liferate upon passaging (
Figure 1
A). We therefore systematically
tested different combinations of growth factors and inhibitors
(
Table S1
) and used tissue shape as a readout. From all the com-
binations tested, only Activin-A, together with the Wnt inhibitor
XAV939, preserved epithelial integrity (
Table S1
), but long-term
growth was compromised. Adding bFgf2 allowed the expansion
of epithelial 3D structures that expressed pluripotency genes and
early post-implantation factors (
Figures 1
A, 1B,
S1
A, and S1B), in
agreement withpreviousfindings.
23
However, wealsodetecteda
high expressionof differentiation markers (
Figure1
C). Theculture
of ESCs in 3D gels has been shown to activate the Bmp pathway,
leading to differentiation.
24
,
25
Using a Bmp reporter ESC line,
26
we observed that cells cultured in bFgf2-Activin-A-XAV939
(FAX) displayed active Bmp signaling (
Figure 1
D and 1E). The
addition of the Bmp inhibitor Noggin decreased the activity of
the Bmp pathway and the levels of the PS marker Brachyury
(
Figures 1
D–1F), without affecting the expression of pluripotency
genesorearlypost-implantation factors(
FiguresS1
AandS1B).A
time course experiment revealed that Brachyury and the endo-
derm marker Sox17 appeared only after 2 weeks of 3D culture,
while the neuroectoderm marker Sox1 could not be detected
(
Figure 1
G). Epithelial integrity was maintained, as demonstrated
by the basal localization of Integrin
b
1 and the apical localization
of the Par complex component aPKC, the tight junction protein
ZO1, and the lumenal protein Podocalyxin (
Figures 1
H,
S1
C,
and S1D). Recently, new culture formulations that preserve an in-
termediate formative pluripotent state in 2D have been devel-
oped.
27
,
28
After 2 weeks of 3D culture in formative media, more
than 60% of the spheroids lost epithelial integrity and upregu-
lated expression of differentiation markers (
Figures S1
E–S1G),
indicating the 3D environment changes the signaling require-
ments of pluripotent cells. Therefore, we refer to the combination
of bFgf2, Activin-A, XAV939, and Noggin (FAXN) as the 3D EpiSC
medium.
Establishment of self-renewing 3D epiblasts
We next tested whether the identified conditions could capture
pluripotent cells directly from the embryo. To this end, we iso-
lated the epiblasts of E5.5 mouse embryos and embedded
them in Matrigel. As a control, we plated epiblasts in 2D Matrigel
with FA or FAXN medium (
Figure S2
A). Epiblasts cultured in 3D
FAXN gave rise to epithelial spheroids surrounding a central
lumen (
Figures 1
I and 1J), with an efficiency comparable with
ESC-derived spheroids (
Figure 1
H). Pluripotency genes were ex-
pressed at similar levels in all conditions, while the early post-im-
plantation factor
Fgf5
was decreased in 2D FAXN (
Figures S2
B
and S2C). Cells cultured in 2D FA expressed higher levels of
PS and endoderm markers (
Figures 1
I,
S2
D, and S2E), whereas
cells cultured in 2D FAXN upregulated Sox1 (
Figure S2
F). These
findings indicate that while Bmp inhibition promotes neuroecto-
derm differentiation in 2D, it blocks PS fates in 3D.
We next assessed the genomic integrity of self-renewing 3D
epiblasts. Proliferation rates were similar in 2D and 3D (
Fig-
ure S2
G), and the percentage of aneuploid cells was not affected
by the culture conditions (
Figure S2
H), in contrast to adult epithe-
lial stem cells.
29
Lastly, we could use conventional 2D EpiSCs as
a starting point to generate 3D EpiSCs (
Figures S2
I and S2J).
Figure 1. Inhibition of Wnt and Bmp signaling supports long-term self-renewal of 3D EpiSCs
(A) Immunostaining of mouse ESCs cultured in 3D Matrigel using different conditions. Scale bars, 25
m
m.
(B) Morphological characterization of spheroids from (A). Data are shown as a contingency bar graph, and the number of spheroids per category is indic
ated. 2
independent experiments. X
2
test. ****p < 0.0001; nd, not detected; ns, non-significant.
(C) Relative expression levels of
T
,
Gata4
, and
Cdh2
in cells cultured under different conditions. Data are shown as mean ± SEM. n = 6 samples, 3 independent
experiments. Kruskal-Wallis test. **p = 0.0099 (
Gata4
), **p < 0.0027 (
Cdh2
), ***p = 0.0005 (
T
).
(D) Immunostaining of mouse ESCs cultured in 3D Matrigel. Scale bars, 20
m
m.
(E and F) Ratio of Bmp+ (E) and Brachyury+ (F) cells in spheroids from (D). Data are shown as mean ± SEM. Each dot represents an individual spheroid. In (E)
,n=
99 (FAX) and 75 (FAXN) spheroids. In (F), n = 38 (FAX) and 47 (FAXN) spheroids. 3 independent experiments. Mann-Whitney U test. *p = 0.0153, ****p < 0.000
1.
(G) Ratio of differentiated cells in 3D EpiSCs. Data are shown as mean ± SEM. Each dot represents an individual spheroid. For Brachyury, n = 42, 40, and 53
, and
for Sox17/Sox1 n = 46, 40, and 48 spheroids. 2 independent experiments. Kruskal-Wallis test, **p = 0.0060, ****p < 0,0001; ns, non-significant.
(H) Morphological characterization of 3D EpiSCs. Data are shown as a contingency bar graph, and the number of spheroids per category is indicated. 2 in
de-
pendent experiments. X
2
test, ****p < 0.0001.
(I) Immunostaining of epiblast-derived cells cultured at early passage (P6–P8) in different conditions. Scale bars, 30
m
m.
(J) Morphological characterization of spheroids from (I) at early (P6–P8) and late (P18–P19) passages. Data are shown as a contingency bar graph, and
the number
of spheroids per category is indicated. 5 independent experiments. X
2
test; ns, non-significant.
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Developmental Cell
59
, 1252–1268, May 20, 2024