540 | Nature | Vol 577 | 23 January 2020
Article
During embryonic development in mice, the anterior visceral
endoderm secretes LEFTY1 and CER1 to antagonize posteriorizing
morphogens, and OTX2 regulates the anterior visceral endoderm and
anterior–posterior axis^22. We observed some EPIs expressing T-box
transcription factor T (T, also known as brachyury or TBXT), an early
marker for primitive streak^26 , but repressing OCT4 at the EPIs near the
AME compartment boundary at 14 d.p.f. but not at 12 d.p.f. (Fig. 3c,
Extended Data Fig. 5a). Although we did not observe mutually exclusive
expression of SOX2 (anterior commitment) and NANOG (posterior
commitment)^27 in EPIs, we observed CER1, LEFTY1 and OTX2 expression
on one side of the 14-d.p.f. embryonic disc (Fig. 3d, e, Extended Data
Fig. 5b–e), which suggests formation of the anterior visceral endoderm.
In contrast to expression in mouse embryonic cells but not the extra-
embryonic compartment^22 , OTX2 was expressed in the PrEs of our 3D
embryos (Extended Data Figs. 3f, 5e). The location of T-expressing
EPIs was opposite to the anterior visceral endoderm side (Fig. 3d, e).
Next, we examined the expression of HESX1, as a marker of EPI^28 ,
early anterior differentiation or visceral endoderm^29. We observed that
HESX1 was uniformly expressed in 12-d.p.f. EPIs (Extended Data Fig. 5f ).
Some EPIs lost HESX1 but upregulated T expression at 14 d.p.f. (Fig. 3f).
Furthermore, T−HESX1+ and T+HESX1− EPIs localized on opposite sides of
the EPIs, representing anterior and posterior EPIs. scRNA-seq revealed
decreased expression of HESX1 in the PSA-EPIs (14 d.p.f.) (Extended Data
Fig. 5g). EPIs from 14-d.p.f. embryos branched to produce 2 populations:
T−HESX1+ and T+HESX1− cells (Extended Data Fig. 5h), which further
defines anterior and posterior cell fate. T+HESX1− EPIs enriched with
posterior and primitive streak genes, whereas T−HESX1+ EPIs showed
upregulated early anterior genes (Extended Data Fig. 5i, Supplemen-
tary Table 3).
Using serial sections of whole embryos, we observed that some
14-d.p.f. embryos formed a cell dissemination region^30 , in which some
T+ EPIs focally migrated from the embryonic disc, disrupted barriers
formed by PrE (with N-cadherin expression), invaded the space near
the visceral endoderm and co-expressed GATA6 (Fig. 3g, h, Extended
Data Figs. 6, 7). FLK1 (also known as KDR), a marker of extra-embryonic
mesoderm^31 , was highly expressed in the EPIs, but lost in T+ cells migrat-
ing from the embryonic disc (Extended Data Fig. 5j), which suggests
that the latter is not extra-embryonic mesoderm. Some FLK1+ cells local-
ized between PrEs and TrBs, indicating extra-embryonic mesenchyme
(Extended Data Fig. 5k). Compared with post-implantation EPIs, PSA-
EPIs significantly upregulated primitive streak genes (Extended Data
Fig. 5l). However, the absence of neural gene expression (Extended Data
Fig. 5m–o) suggests that the 14-d.p.f. embryos have not developed to
generate the initial nervous system yet. We conclude that our 14-d.p.f.
embryos were at the PSA stage, which meets the internationally recog-
nized ethical limit for human embryo culture. Together, we conclude
that 1 population of 14-d.p.f. EPIs underwent changes and has initiated
anterior–posterior polarity and primitive streak formation.
Continuous cell proliferation is key to evaluate embryonic develop-
mental status. Cell proliferation in 2D-cultured embryos occurs only
within 8–10 d.p.f., but not after 10 d.p.f.^2 , which implies that 2D embryos
do not survive much beyond 14 days. Our 3D embryos maintained con-
tinuous proliferation of EPIs, TrBs and PrEs at all stages (Extended Data
Fig. 5p–r). We predict that continuous cell proliferation may facilitate
human embryos to develop beyond 14 d.p.f. to initiate gastrulation.Development of the trophoblast lineage
The human placenta consists of three major TrB subpopulations: CTBs,
EVTs and STBs. In our 3D-cultured embryos, the TrBs surrounding EPIs
and PrEs presented a polarized localization of F-actin (Extended Data
Fig. 8a). CK7+ cells near the EPI–PrE bilayer had a single nucleus that
expressed TEAD4 and E-cadherin (Extended Data Fig. 8a–c), which
indicate a CTB identity. Multinucleated cells in the layer adjacent to
AME expressed hCG (Extended Data Fig. 8d, e), which suggests STB
identity. One outer-layer population highly expressed HLA-G, a marker
of EVTs, in 12-d.p.f. embryos, which significantly increased in 14-d.p.f.
embryos (Extended Data Fig. 8f, g). Mutually exclusive expression of
hCG, TEAD4 and HLA-G in most cells showed molecular and physical
delineation of three TrB types at 12–14 d.p.f. (Extended Data Fig. 8h, i).
We identified the top 2,603 most variable genes across the ICM and
all TrBs using scRNA-seq data. On the basis of developmental time and
markers, t-SNE analysis categorized them into six populations: pre-
CTBs (TEAD4+HLA-G−), post-CTBs, early STBs (CGB+CSH1−HLA-Glow),
STBs, early EVTs (HLG-A+CSH1+MMP2+ERBB2+) and EVTs^32 ,^33 (Extended
Data Fig. 8j–l). Continuous transcriptome shifts from 6 to 14 d.p.f.
revealed a stepwise developmental progression, in which CTBs produce
EVTs and STBs, and segregation initiated at 9 d.p.f. and completed at
12 d.p.f. (Extended Data Fig. 8l, m). Notably, CDX2 expression quickly12 d.p.f.AMEa
GATA6 OCT4 N-cadherin DAPIGATA6 OCT4
N-cadherin DAPIGATA6 OCT4
DAPIb14 d.p.f.TTTGATA6 OCT4
T DAPILEFTY1 OCT4
T DAPILEFTY1 OCT4
DAPId14 d.p.f. 14 d.p.f.AMEce T OCT4 CER1 MergeTTOCT4 HESX1 DAPI MergeMergeMagni cationAME AME
14 d.p.f.AMEN-cadherinAMEfAME14 d.p.f.
g OCT4 N-cadherin Merge Magni cation14 d.p.f. AME
h GATA6 OCT4AMEFig. 3 | Human embryos display anterior–posterior polarity and formation
of PSA. a, b, Staining of embryo sections at 12 d.p.f. (a, 3 out of 3 embryos) and
14 d.p.f. (b, 4 out of 5 embryos) with N-cadherin. Red arrows denote N-cadherin+
PrEs; white arrows denote N-cadherin+ EPIs migrating out from the disc (b).
c–f, Staining of embryo sections at 14 d.p.f with T, LEFTY1, CER1 and HESX1.
In c, red arrows denote T+ cells (5 out of 8 embryos). In d, white arrows denote
LEFTY1+ and red arrows denote T+ cells (3 out of 4 embryos). In e, white arrows
denote CER1+ cells; red arrows denote T+ cells (e, 2 out of 2 embryos). In f, white
arrows denote T−HESX1+ EPIs; red arrows denote T+ HESX1− EPIs (2 out of 2
embryos). g, h, Staining of human embryo sections at 14 d.p.f. In g, white arrows
denote T+ cells; red arrows denote N-cadherin+ cells (4 out of 6 embryos). In h,
white arrows denote T+O C T4+G ATA6+ cells; asterisks denote T+O C T4+G ATA6−
cells (3 out of 4 embryos). Scale bars, 50 μm (a–e, g) or 25 μm (f, h). See also
Extended Data Figs. 5–7.