Nature - USA (2020-01-23)

Nature | Vol 577 | 23 January 2020 | 541

decreased after TrB specification at 7 d.p.f. (Extended Data Fig. 8k),
given the absence of CDX2 expression in human peri-implantation
embryos and trophoblast stem cells^33.
Because the human placenta must secrete steroids or polypeptide
hormones to maintain pregnancy, we analysed the expression of
120 polypeptide hormone genes produced by TrBs^32 (Extended Data
Fig. 8n). STBs, which primarily produce placental hormones, expressed
31 hormone genes starting from 8 d.p.f. EVTs expressed 19 polypep-
tide hormone genes with upregulated expression over culture, which
indicates maturation. Expression of CGA, PGF and CGB family genes in
EVTs suggested pre-gastrulation EVTs can secrete hCG, progesterone
and oestrogen. However, these genes are significantly downregulated
in 8-week EVTs, and completely disappear in 24-week EVTs^32. Thus, the
ability of EVTs to secrete hormones gradually decreases over placenta
development.
Next, we identified genes corresponding to the different TrB sub-
types (Extended Data Fig. 8o). CTB-, STB- and EVT-specific genes closely
associated with their functions and specific signalling pathways accord-
ingly to their characteristics^33 (Supplementary Table 4). EVT-specific
genes helped to regulate the immune system and angiogenesis. These
results corroborate the finding that EVTs in human first-trimester pla-
centas are crucial for immunomodulatory and spiral artery remodelling
of the early maternal–fetal interface^34. We determined the top-ranked
transcription factors that control TrB development. Transcription
factors for CTBs, STBs and EVTs included well-documented TrB and
pluripotency factors and new potential transcription factors, such as
MYBL2, TCF7L1 and NR2F2 (Extended Data Fig. 8o).

Epiblast development and transition

We analysed EPI transcriptome dynamics across development, which

revealed four main clusters: ICM, pre-implantation EPI (pre-EPI), post-

EPI and PSA-EPI (Fig. 4a, b). When tracking naive and primed pluripotent

gene expression, we found that embryos lost naive genes TFCP2L1,

KLF17 and KLF4 and activated primed gene CD24 after implantation,

while maintaining general pluripotent genes (Extended Data Fig. 9a–d).

scRNA-seq data confirmed EPI pluripotent state transition (EPST)^35 ,^36

(Extended Data Fig. 9e, f ).

We speculated whether EPIs from different developmental stages

show distinct pluripotency regulatory networks by performing Pluri-

NetWork analysis using existing mouse databases^37 ,^38. Different com-

binations of key pluripotency regulators dominated and coordinated

EPST networks (Extended Data Fig. 9g–j). Naive pluripotency genes

(ESRRB, KLF4 and TFCP2L1) only occupied ICM networks, which sug-

gests that human EPIs quickly lose naive pluripotency after lineage

diversification, consistent with monkey EPIs that only transiently

express naive pluripotency before the late- and hatched-blastocyst

stages^38. Genes specific for different developmental stage EPIs revealed

that EPIs maintained a stable transcriptome from pre-implantation to

post-implantation with marked changes in gene expression occurring

during the pre-EPI transition and PSA initiation stage (Fig. 4c). Differ-

ent EPI pluripotency states were dominated by different transcription

factors and regulatory pathways (Fig. 4c, Supplementary Table 5).

Pairwise comparisons showed similar data (Extended Data Fig. 9k–n,

Supplementary Table 6).

t-SNE 1 t-SNE 1

–20 0020 –20 20

20

20

0 0

–20 –20

t-SNE 2ICM (52 cells) t-SNE 2

Pre-EPI (63 cells)

Post-EPI (64 cells)

PSA-EPI (43 cells)

a b

c

407

2,026

114

144

ICMPre-EPIPost-EPIPSA-EPI

PPAR, Hippo signalling pathway

Metabolic pathways, endocytosis

mTOR signalling pathway

Embryo development

Cell development, communication

Epithelium development

Oxidative phosphorylation

Cardiac muscle contraction

Glutathione, pyrimidine metabolism

PPAR, PI3K-Akt signalling pathway

Ribosome, mitochondrial part

Translational initiation

Respiratory electron transport chain

PSA-EPI

HES1 CREB3 MXD3

MAZ PITX1 ADNP

LIN28B CREB3L2

SOX11 ZNF746 NME2

ZNF585A NR4A1 ZEB2

ICM

TET2 DPF3 TRERF1

DLX3 CDX2 GRHL2

ZFHX3 CEBPA NR2F2

ESRRB SMAD5 SATB2

Pre-EPI

ARGFX KLF17 KLF4

OVOL2 ARX ZNF675

222 samples, 2,691 genes

Signalling pathways regulating PSCs

Glycolysis/gluconeogenesis

TNF, TGF-β signalling pathway

Post-EPI

PODXL TERF1 ZIC3 CBX2

ETS1 ZSCAN10 SKIL

NKXI-2 ZNF462

ZNF121 BCL11A PBX1

Wnt, VEGF signalling pathway

Glutathione metabolism

Regulation of neuron differentiation

Nervous system development

Vasculature development

Cell differentiation

68 10 12 14 d.p.f.

Basement membrane (laminin)

Primitive endoderm (GATA6)

Epiblast cells (OCT4)

Extra-embryonic mesenchyme

STB EVT

AME

PSA

CTB

STB EVT Squamous parietal endoderm Visceralendoderm

AC

Anterior

SYS

PY

Anterior

visceral

endorderm

AC

PYS

CTB

6 d.p.f. (60 cells)

7 d.p.f. (33 cells)

8 d.p.f. (11 cells)

9 d.p.f. (12 cells)

10 d.p.f. (14 cells)

12 d.p.f. (22 cells)

14 d.p.f. (70 cells)

222 cells, 994 genes

Time

d

Fig. 4 | Development of EPI lineage. a, b, t-SNE

analyses revealed four clusters, identified as ICM,

pre-EPI, post-EPI and PSA-EPI based on

developmental time. c, Heat map of genes specific

for every cell type. Representative transcriptional

factors and KEGG pathways are shown

(Supplementary Table 5). PSC, pluripotent stem

cells. d, Model of human pre-gastrulation embryo

development landmarks based on our results and

the Carnegie series. See also Extended Data Fig. 9.