Extended Data Fig. 4 | AME separation from EPI. a, E-cadherin expression in
amnion from a 12-d.p.f. embryo. Right panels show magnified squares (3 out of
3 embryos). White and red lines indicate nuclei and apical orientation,
respectively. b, Quantification of expression of E-cadherin (AME = 33 cells,
EPI = 44 cells) and OCT4 (AME = 22 cells, EPI = 44 cells) in columnar EPIs and
squamous AME. Data are mean ± s.d from 3 embryos. *P < 0.001, two-sided t-
test. c, d, Representative staining of laminin at 12 and 14 d.p.f. (3 out of 3
embryos). Right panels show magnified squares from d. e, f, Representative
staining and quantification of NANOG expression (AME = 33 cells, EPI = 30 cells,
and SOX2 expression (AME = 34 cells, EPI = 30 cells) in 14-d.p.f. EPIs and AME (3
out of 3 embryos). Data are mean ± s.d. *P < 0.01, P < 0.001, two-tailed t-test.
Right panels show magnified squares from e. g, h, Representative staining and
quantification of laminin and E-cadherin in 6-d.p.f. embryos (3 out of 3
embryos). i, j, Representative staining and quantification of laminin and
E-cadherin in 8-d.p.f. embryos (3 embryos). White long lines in h and j show
positions used to plot intensity profiles (right). k, l, Representative staining of
laminin and E-cadherin in 10-d.p.f. embryos (3 out of 3 embryos). Together, we
conclude that AME separation from EPIs correlates with asymmetrical
distributions of E-cadherin and laminin (a–l). m, Representative staining of
EZRIN in 14-d.p.f amnion (2 out of 2 embryos). White arrows indicate EZRIN
expression in apical surface in EPIs and AME. n, Representative staining of WGA
in 14-d.p.f. embryos (2 out of 2 embryos). Red arrowheads indicate WGA
expression in extra-embryonic cells. o, t-SNE analyses revealed three clusters of
12- and 14-d.p.f. embryos—AME, intermediate state cells and EPIs. p, Compared
to EPIs, the violin plots show AME significantly downregulated pluripotency
genes and upregulated genes that specifically expressed in the AME of
12–17-d.p.f monkey embryos or self-organized amnion from human pluripotent
stem cells. All violins have the same maximum width; black dot denotes
the mean. o, p, AME, n = 13 cells; intermediate state cells, n = 26 cells; EPI, 53
cells. q–t, Gene expression profiles of AME and EPI in the 12- and 14-d.p.f.
embryos (Supplementary Table 2). AME, n = 12 cells (one single cell with high
NANOG expression was not included); EPI, 53 cells. q, Volcano map of
differentially expressed genes (DEGs) between AME and EPI in the 12- and
14-d.p.f. embryos. DEGs were defined with uncorrected P < 0.01 (two-sided
likelihood ratio tests) and log 2 -transformed fold change > 1 or <−1, and median
FPKM > 1 in one group. r, Heat map of DEGs between the AME and EPI. Right
panel presents representative transcription factors. s, Compared to the EPIs,
GO terms of upregulated genes in the AME. t, Compared to EPI, KEGG pathways
of genes enriched in the AME. u, hCG was expressed in the AMEs, but not in the
EPIs (2 out of 2 embryos). White arrows indicate the AMEs have squamous
nuclear shape, expressed hCG, but downregulated the pluripotent gene, O C T4.
Scale bars, 15 μm (a, l), 20 μm (c, e), 25 μm (d, g, h, k, m, n, u) or 50 μm (i).