Ephb4. Ephb3. Ephb2. Ephb1. Epha8. Epha7. Epha6. Epha5. Epha4. Epha3. Epha2. Epha10. Epha1. Csf3r. Csf2rb. Csf2ra. Csf1r. Ngfr. Ntrk3. Ntrk2. Ntrk1. Flt4.
Developmental Cell Supplemental Information
Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis Thorsten Boroviak, Remco Loos, Patrick Lombard, Junko Okahara, Rüdiger Behr, Erika Sasaki, Jennifer Nichols, Austin Smith, and Paul Bertone
Supplemental Figures
A
0.9
0.95
B
1
Primitive endoderm
E4.5 PrE 1 E4.5 PrE 2 E4.5 PrE 3 E4.5 EPI 3 E4.5 EPI 1 E4.5 EPI 2
ICM
Morula
E5.5 EPI 1 E5.5 EPI 2
Preimplantation epiblast
E5.5 EPI 3 E3.5 ICM 1 E3.5 ICM 2 E3.5 ICM 3 E2.5 MOR 2 E2.5 MOR 1 E2.5 MOR 3
Gata6
Pdgfra
Gata4
Foxa2
Pou5f1
Nanog
Fgf4
Bmp4
Fgfr2
Sox17
Sox7
Dab2
Sox2
Klf2
Tdgf1
Foxd3
Figure S1, related to Figure 1: (A) Global correlation of mouse embryonic samples. (B) Stream plot of 16 lineage markers for PrE (green) and preimplantation epiblast (blue). Track widths represent the relative expression of the gene normalized to the mean expression across the developmental stages displayed. Genes specifically expressed in PrE or preimplantation epiblast include all established lineage markers. The early PrE markers Gata6 and Fgfr2 were expressed from the morula stage, with upregulation of Pdgfra in the early ICM. Late PrE markers, including Gata4, Sox7, Foxa2 and Dab2, were specifically upregulated in PrE at E4.5, with Gata6, Fgfr2 and Pdgfra expression maintained, consistent with the proposed sequential activation of Gata6, Sox17, Gata4 and Sox7 during PrE specification (Artus et al., 2011) and expression patterns observed by others (Chazaud et al., 2006; Gerbe et al., 2008; Guo et al., 2010; Ohnishi et al., 2014). Conversely, preimplantation epiblast cells exhibited robust expression of epiblast-specific genes, including Sox2, Nanog, Klf2, Bmp4 and Fgf4 (Guo et al., 2010; Kurimoto et al., 2006; Tang et al., 2010a).
Term oxidation-reduction process
PrE
P-val. Adj (BH) 0.00796682
Term response to chemical stimulus cell proliferation organ morphogenesis positive regulation of cell development positive regulation of MAP kinase activity generation of neurons neural crest cell migration anatomical structure morphogenesis positive regulation of neurogenesis branching morphogenesis of a tube cell morphogenesis involved in differentiation cell death organ development cellular response to chemical stimulus programmed cell death small molecule metabolic process death response to superoxide response to oxygen radical negative regulation of cellular process regulation of developmental process response to organic substance neurogenesis apoptotic process
P-val. Adj (BH) 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00516425 0.00589999 0.00589999 0.00589999 0.00643218 0.00675768
Term nervous system development cell differentiation cell communication signaling anatomical structure development system development cellular developmental process multicellular organismal development positive regulation of biological process positive regulation of cellular process regulation of multicellular organismal development signal transduction developmental process neurogenesis regulation of response to stimulus multicellular organismal process cellular response to stimulus response to stimulus neuron differentiation cell surface receptor signaling pathway regulation of cell differentiation generation of neurons regulation of signaling regulation of nervous system development
P-val. Adj (BH) 7.0003E-05 7.0003E-05 7.0003E-05 7.0003E-05 7.0003E-05 9.567E-05 0.00015016 0.00015016 0.00016447 0.00016447 0.00021907 0.00021907 0.00022631 0.00022631 0.00032091 0.00032091 0.00043936 0.00063096 0.00078691 0.00079899 0.00095642 0.0016368 0.00197571 0.00253872
ig
en
et
ic
s
Term P-val. Adj (BH) multicellular organismal process 0.00029974 high-density lipoprotein particle clearance 0.01591473 developmental process 0.01591473 multicellular organismal development 0.02802533 anatomical structure development 0.03692507 trophoblast cell migration 0.03692507 regulation of trophoblast cell migration 0.03692507 negative regulation of trophoblast cell migration 0.03692507 negative regulation of cell migration 0.03692507 negative regulation of cell motility 0.04204448 negative regulation of locomotion 0.04775401 negative regulation of cellular component movement 0.04871758 trophectodermal cellular morphogenesis 0.05865734 negative regulation of platelet activation 0.05865734 chylomicron remnant clearance 0.05865734 triglyceride-rich lipoprotein particle clearance 0.05865734 blood vessel development 0.05865734 0.05865734 system development regulation of tube size 0.05865734 regulation of blood vessel size 0.05865734 embryo implantation 0.06594336
ep
tio rip sc an tr
si gn al lin g
n
Genes regulating E4.5 3
E4.5 2
E4.5 1
E4.5 3
E4.5 2
E3.5 3
E3.5 1
ICM ICM, ICM, preimpl. PrE EPI
preimpl. EPI preimpl. EPI, PrE
PrE
preimpl. EPI
ICM
E4.5 1
B
Term P-val. Adj (BH) response to chemical stimulus 0.00010514 multicellular organismal process 0.00013049 response to stimulus 0.00254199 cellular response to organic substance 0.00254199 response to organic substance 0.00254199 developmental process 0.00254199 anatomical structure development 0.00297988 cell migration 0.00323982 cellular response to chemical stimulus 0.00347489 cell motility 0.00585354 localization of cell 0.00585354 multicellular organismal development 0.00698981 cellular response to cytokine stimulus 0.00839442 anatomical structure morphogenesis 0.00839442 cell-cell signaling 0.00839442 immune system process 0.00839442 negative regulation of multicellular organismal process 0.00839442 secretion 0.00839442 segmentation 0.00839442 cytokine secretion 0.00839442 regulation of cell migration 0.00948099 secretion by cell 0.00948099 0.00991232 BMP signaling pathway tissue development 0.01001977 response to nutrient 0.0102139 polysaccharide metabolic process 0.01024069 regulation of cell motility 0.01129983 cell activation 0.01129983 locomotion 0.01129983 regulation of locomotion 0.01214547 regulation of multicellular organismal process 0.01214547
preimpl. EPI
P-val. Adj (BH) 0.01028629 0.01537011 0.01537011 0.01537011 0.02056179 0.02176294 0.02176294 0.02176294 0.02176294 0.02176294 0.02571741 0.03044577 0.04756802 0.04756802 0.04909514 0.04909514 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919 0.04940919
P-val. Adj (BH) 0.00052285 0.00175971 0.00175971 0.00175971 0.00412043 0.00412043 0.00461262 0.00461262 0.00764043 0.00865531 0.00865531 0.00977462 0.01234617 0.01375272 0.01375272 0.01375272 0.01458803 0.01458803 0.01458803 0.01642693 0.01642693 0.01642693 0.01642693 0.01646228 0.01714087 0.01857364 0.01999361
preimpl. EPI, postimpl. EPI
Term detoxification of copper ion nitric oxide mediated signal transduction regulation of humoral immune response response to copper ion regulation of autophagy regulation of acute inflammatory response regulation of developmental growth regulation of response to external stimulus humoral immune response response to external stimulus developmental growth negative regulation of autophagy cellular zinc ion homeostasis zinc ion homeostasis positive regulation of developmental process humoral immune response response to metal ion positive regulation of developmental growth autophagy acute inflammatory response cellular response to metal ion cellular divalent inorganic cation homeostasis response to toxin cellular response to inorganic substance divalent inorganic cation homeostasis cellular response to chemical stimulus immunoglobulin mediated immune response B cell mediated immunity cell projection assembly second-messenger-mediated signaling positive regulation of transmembrane transport growth developmental cell growth
Term response to chemical stimulus response to wounding intracellular protein kinase cascade cellular response to chemical stimulus cell adhesion biological adhesion response to stimulus odontogenesis cellular response to organic substance regulation of multicellular organismal process regulation of intracellular protein kinase cascade multicellular organismal process organ development cell communication phospholipid homeostasis defense response response to external stimulus response to organic substance inflammatory response regulation of response to stress cell-cell adhesion gland development odontogenesis of dentin-containing tooth response to metal ion MAPK cascade cytokine production system development
postimpl. EPI
P-val. Adj (BH) 5.065E-05 0.0004266 0.0004266 0.00494365 0.00641332 0.00641332 0.00641332 0.01695561 0.02443758 0.02450709 0.02478382 0.04258611 0.0484731 0.04881227 0.05219916 0.05219916
ICM
Term organ development multicellular organismal process system development regulation of cell differentiation tissue development regulation of multicellular organismal process multicellular organismal development regulation of developmental process cell differentiation cell development anatomical structure development regulation of multicellular organismal development positive regulation of developmental process small molecule biosynthetic process response to external stimulus cellular developmental process
ICM, preiml. EPI
P-val. Adj (BH) 0.02269575 0.02269575 0.0292063 0.04885411
ICM, pre-EPI, postEPI
Term amine metabolic process cellular biogenic amine metabolic process cellular amine metabolic process in utero embryonic development
E3.5 2
MOR, ICM, pre-EPI
MOR, ICM
MOR
A
Hand1 Tfap2c Tfcp2l1 Tbx15 Sox21 Sox15 Nkx6-2 Pou2f3 Dppa2 Gsc
Lama5 Fgfr1 Fgf10 Fos Esrra Wnt6
Satb1 Med24 Hmgn5
Klf2 Nanog Gata6 Akt1
Bmp4 Itgb7 Pdgfra Krt18
Tet1 Jarid2 Tada3
Tdgf1 Sox2 Foxd3 Zic5
Fgf4 Pdgfa Etv5 Nodal
Hmga1 Kdm4c Kdm5b Kdm3b
Nr0b1 Zic3 Dnmt3l
Tcf7l1 Stmn1
Apobec2
Sox17 Gata4 Sox7 Foxa2 Dab2 Hes6 Runx1 Meis3 Foxq1
Fgfr2 Fgfr3 Fgfr4 Dkk1 Lama1 Lamb1 Igf2 Col4a1 Col4a2
Dnmt3a Dnmt3b Hdac7
Figure S2, related to Figure 2: (A) GO enrichment for stage-specific expression in early mouse development. (B) Dynamically expressed genes during epiblast and PrE specification. Selected transcription factors, signaling pathway components and epigenetic regulators are shown to the right, with a subset of lineage markers in bold.
C
A
Dynamically expressed genes (1857) 0 1 2 3 4 5 6
DC3
−0.20 −0.15 −0.10 −0.05 0.00 0.05 0.10 0.15 0.20
ESC (2i/LIF)
−0.4
Morula postimpl. EPI
early ICM preimpl. EPI
0.3 0.2 0.1 0.0 −0.1 −0.2 −0.3
−0.2
0.0
0.2
0.4
0.6
0.8
DC1
B
0
50
100 150
E4.5 EPI 3 E4.5 EPI 2 E4.5 EPI 1 E5.5 EPI 2 E5.5 EPI 1 E5.5 EPI 3 ESC (2i/LIF) 2 ESC (2i/LIF) 1 E2.5 MOR 2 E2.5 MOR 1 E2.5 MOR 3 E3.5 ICM 2 E3.5 ICM 1
E4.5-preimpl. EPI 1
E4.5-preimpl. EPI 3
E4.5-preimpl. EPI 2
ESC (2i/LIF) 1
ESC (2i/LIF) 2
E5.5-postimpl.EPI 1
E5.5-postimpl.EPI 2
E3.5-ICM 2
E5.5-postimpl.EPI 3
E3.5-ICM 1
E3.5-ICM 3
E2.5-MOR 2
E2.5-MOR 1
E2.5-MOR 3
E3.5 ICM 3
Figure S3, related to Figure 3: (A) Diffusion map of mouse embryonic samples and ESC cultured in 2i/LIF, based on the full transcriptome; DC = diffusion coefficient. (B) Global gene expression and hierarchical clustering of mouse embryonic samples and ESC in 2i/LIF. (C) Heatmap and hierarchical clustering based on the expression of dynamically regulated genes. The subgroup shared by ESC and E4.5 epiblast is marked in orange.
A E4.5 PrE 3 E4.5 PrE 2
E4.5 EPI 2 E4.5 EPI 3 E4.5 EPI 1 E5.5 EPI 2 E5.5 EPI 1 E5.5 EPI 3 E2.5 MOR 2 E2.5 MOR 1
DOWNREGULATED
E4.5 PrE 1
Term
Count ExpCount
Size P-val.Adj (BH)
1.
cellular metabolic process
191
109
7688
4.4926E-17
2.
primary metabolic process
193
111
7865
4.9243E-17
3.
metabolic process
214
133
9440
4.8979E-16
4.
cellular process
255
188
13292 1.6293E-11
5.
cellular protein metabolic process
92
41
2908
2.0459E-11
6.
protein metabolic process
101
51
3574
6.8434E-10
7.
catabolic process
66
26
1845
8.
cellular macromolecule metabolic process
137
79
5619
6.8434E-10
9.
macromolecule metabolic process
149
91
6434
1.9745E-09
10. cellular component organization or biogenesis
99
51
3578
2.905E-09
11. cellular component organization
94
49
3447
2.4584E-08
12. translation
32
8
598
3.0756E-08 1.1601E-07
13. cellular catabolic process
55
22
1584
14. cellular component biogenesis
50
21
1451
6.8434E-10
9.455E-07
15. small molecule metabolic process
60
27
1937
1.1433E-06
16. cell cycle
42
16
1132
2.0568E-06
E2.5 MOR 3
17. cellular component organization or biogenesis at cellular level
77
40
2851
2.1182E-06
18. biosynthetic process
101
60
4235
3.8393E-06
E3.5 ICM 2
19. macromolecular complex subunit organization
35
13
944
3.1918E-05
20. cellular biosynthetic process
95
58
4093
3.9861E-05
E3.5 ICM 1 E3.5 ICM 3 DIA EPI 2 DIA EPI 1
C
GO enrichment Rank
50 100 150 200
0
21. organophosphate metabolic process
40
17
1183
4.1803E-05
22. cellular component organization at cellular level
70
39
2731
6.6093E-05
23. cellular component assembly
42
19
1312
8.0252E-05
24. cell cycle process
30
11
776
8.2357E-05
25. heterocycle metabolic process
37
16
1127
0.00020108
GO enrichment Rank
ES C( 2i/ LI
F) ES 1 C( 2i/ LIF )2
AU SE 1 AP AU SE 2
AP
DI
preimpl. EPI postimpl. EPI
DI
2
1
3 .5 E5
.5
.5
E5
E5
2
1
.5 3
E4
.5 E4
2
3
E4 .5
.5
.5
ICM
E3
E3
3
1 .5
.5
E3
E2
1
2
E2 .5
MORULA
UPREGULATED
0 1 2 3 4 5 6
Pou5f1 Nanog Sox2 Tdgf1 Gdf3 Sall4 Utf1 Pecam1 Tfap2c Cdh1 Fgf4 Klf2 Klf4 Klf5 Dppa3 Zfp42 Nr0b1 Esrrb Tfcp2l1 Tbx3 Gbx2 E2 .5
ESC(2i/LIF) / preimpl. EPI
B
Term
Count ExpCount
Size P-val.Adj (BH)
1.
cellular biosynthetic process
31
12
4093
0.00028531
2.
biosynthetic process
31
13
4235
0.00028531
3.
cellular process
60
40
13292 0.00028531
4.
melanosome organization
3
0
11
0.0017611
5.
pigment granule organization
3
0
12
0.00187446
6.
positive regulation of aldosterone metabolic process
7.
positive regulation of aldosterone biosynthetic process
2
0
2
0.0021249
8.
cellular metabolic process
41
23
7688
0.00239135
9.
establishment of localization
2
0
2
0.0021249
24
10
3277
0.00317487
10. regulation of cellular biosynthetic process
22
9
2903
0.0037436
11. negative regulation of cellular biosynthetic process
12
3
959
0.0037436
12. cellular pigmentation
3
0
20
0.0037436
13. negative regulation of biosynthetic process
12
3
976
14. regulation of biosynthetic process
22
9
2938
0.0037436
15. cellular nitrogen compound metabolic process
29
14
4674
0.00391938
16. positive regulation of transcription, DNA-dependent
12
3
998
0.00391938
17. cellular component organization
24
10
3447
0.00391938
18. nitrogen compound metabolic process
29
14
4716
0.00422368
19. positive regulation of RNA metabolic process
12
3
1020
0.00422368
20. nucleobase-containing compound metabolic process
28
13
4490
0.00433432
21. negative regulation of cellular macromolecule biosynthetic process
11
3
893
0.00525252
22. cellular component organization or biogenesis
0.0037436
24
11
3578
23. cellular macromolecule biosynthetic process
23
10
3345
0.00525252
24. negative regulation of nucleobase-containing compound metabolic p
11
3
903
0.00525252
0.00525252
25. negative regulation of nitrogen compound metabolic process
11
3
916
0.00566842
Figure S4, related to Figure 4: (A) Global expression and hierarchical clustering of mouse embryonic samples, ESC cultured in 2i/LIF and diapaused preimplantation epiblast. (B) GO term enrichment analysis based on up- and downregulated genes in normal preimplantation versus diapaused epiblast. (C) Expression of pluripotency markers in mouse embryonic samples, ESC in 2i/LIF and diapaused epiblast.
B
D
C
Late blastocyst
60
n=4
40 30 20 10 0
E
n=3
n=6
50
Average cells/embryo
A
NANOG
OCT4
GATA4
Ensembl Callithrix jacchus version 73.321 (C_jacchus3.2.1) Chromosome 15: 3,408,647 - 3,414,298 5.65 kb
Marmoset late blastocyst
3,409,000
332.40
3,410,000
Forward strand
3,411,000
3,412,000
3,413,000
3,414,000
3,412,000
3,413,000
3,414,000
0.00
Ensembl genes
SOX2 >
protein coding 3,409,000
3,410,000
Reverse strand
F
3,411,000
5.65 kb
Ensembl Callithrix (C_jacchus3.2.1) Chromosome 3: 1,578,509 - 1,582,868 4.36 kb 1.579Mb
Forward strand
1.580Mb
1.581Mb
1.582Mb
681.50
Marmoset early blastocyst
0.00
Ensembl genes
< ZFP42-201
protein coding 1.579Mb
Reverse strand
1.582Mb
0 1 2 3 4 5 6
MOUSE
MARMOSET
100
Cd9 Nanog Pou5f1 Gdf3 Gata6 Brix1 Eef1a1 Eef1a1 Actb Lamb1 Lamc1 Dnmt3b Ctnnb1 Raf1 Tdgf1 Utf1 Nr6a1 Eomes Lifr Grb7 Rest Fgf4 Sox17 Fn1 Sox2 Il6st Commd3 Pten Tfcp2l1 Gata4 Lama1 Eras Igfbp2 Foxa2 Podxl Syp Pax6 Pdx1 T Gfap Gcg Neurod1 Pax4 Myf5 Afp Runx2 Cd34 Ptf1a Krt1 Actc1 Mnx1 Th Col2a1 Nppa Col1a1 Sfrp2 Sema3a Cdh5 Gal Sst Isl1 Cdx2 Kit Nes Gbx2 Wt1 Des Crabp2 Fgf5 Flt1 Olig2 Tat Myod1 Gabrb3 Tert Ddx4 Gcm1 Nr5a2 Nodal Pecam1 Sycp3
50
Average read coverage
1.581Mb
4.36 kb
J
150
G
1.580Mb
−5000
−4000
−3000
−2000
−1000
TTS
Distance from transcription termination site
H
MARMOSET early-late ICM
0.4
0.6
0.8
late ICM
0.0 −0.6 −0.4
−0.2
PC3
0.2
mid ICM early ICM
0.2
−0.4
−0.8 0.315
0.4
0.0 −0.2 −0.6 0.320
0.325
0.330
0.335
0.340
0.345
PC1 early ICM MOUSE MARMOSET
Ascorbate and aldarate metabolism Inositol phosphate metabolism Lipoic acid metabolism Butirosin and neomycin biosynthesis Pentose and glucuronate interconversions Lysine biosynthesis Glycosphingolipid biosynth. − lacto and neolacto PPAR signaling pathway Biotin metabolism Mismatch repair
2
1
IC M lat e
M IC lat e
M IC
IC M rly ea
rly ea
5
4
3
2
M IC rly ea
1 ICM
)3
ICM rly ea
rly ea
ole dd at a
)2 lat e
IC M
(po
(po ole dd a ta
3
)1 dd ata lat e
ICM
(po ole
ICM rly ea
ICM lat e
M
ICM
IC rly
rly
Pathway score
ea
Pathway score
ea
−50 0 0 200 400 600 800 1000 1200 1400
−150
−250
−100
1
2
Estrogen signaling pathway Fructose and mannose metabolism Drug metabolism − other enzymes Arginine and proline metabolism Degradation of aromatic compounds mTOR signaling pathway D−Glutamine and D−glutamate metabolism Vitamin B6 metabolism Proteasome NOD−like receptor signaling pathway −200
I
Figure S5, related to Figure 5: (A–C) Immunosurgery on marmoset blastocysts. Phase contrast images of (A) marmoset blastocyst after complement reaction, (B) roughly separated ICM and trophectoderm and (C) cleanly separated ICM after dissociation with a fine Pasteur pipette. (D) Manual quantification of NANOG, OCT4 and GATA4-positive cells from whole-mount immunostainings of in vivo derived, late marmoset blastocysts; n = number of embryos. (E) SOX2 locus in the marmoset reference genome. Gene boundaries are annotated incorrectly with respect to transcribed sequence determined by RNA-seq. (F) A minority of genes such as ZFP42 (REX1) were not accurately quantified due to pronounced discrepancies between marmoset gene loci and sequencing data that remained unreconciled by automated reannotation. (G) Read coverage for genes >3 kb illustrating 3ʹ′ bias and threshold implemented to restrict transcript length (dashed line). (H) Principal component analysis of marmoset embryonic samples. (I) Pathway expression scores for the 10 most up- and downregulated signaling pathways (KEGG). Scores were calculated by summing FPKM values of individual pathway components followed by normalization for pathway size. (J) Expression of pluripotency and lineage markers in mouse and marmoset early and late ICM samples. The mouse late ICM sample was generated by in silico pooling of RNA-seq reads from individual E4.5 EPI and E4.5 PrE samples at a ratio of 1:1. This reflects the ratio of epiblast to PrE cells in the late marmoset ICM (D) to allow comparison of late ICM in both species.
A
C
B MOUSE
MARMOSET
MOUSE
MARMOSET
IC M mi dI CM lat eI CM
rly ea
E po sti mp l.E PI
EP I
Pr
M IC
pl.
pr
eim
MO R
eI
CM
Fgf1 Fgf10 Fgf11 Fgf12 Fgf13 Fgf14 Fgf15 Fgf16 Fgf17 Fgf18 Fgf2 Fgf20 Fgf21 Fgf22 Fgf23 Fgf3 Fgf4 Fgf5 Fgf6 Fgf7 Fgf8 Fgf9 Egf Vegfa Vegfb Vegfc Pdgfa Pdgfb Pdgfc Pdgfd Fgfr1 Fgfr2 Fgfr3 Fgfr4 Egfr Pdgfra Pdgfrb Insr Igf1r Igf2r Flt1 Kdr Flt4 Ntrk1 Ntrk2 Ntrk3 Ngfr Csf1r Csf2ra Csf2rb Csf3r Epha1 Epha10 Epha2 Epha3 Epha4 Epha5 Epha6 Epha7 Epha8 Ephb1 Ephb2 Ephb3 Ephb4 Ephb6 Grb2 Sos1 Sos2 Mapk9 Mapk10 Mapk11 Mapk12 Mapk13 Mapk14 Mapk15 Mapk3 Mapk1 Mapk4 Mapk7 Mapk6 Map2k1 Map2k2 Map2k4 Map2k5 Map2k6 Map2k7 Map3k1 Map3k10 Map3k11 Map3k12 Map3k13 Map3k14 Map3k15 Map3k2 Map3k3 Map3k5 Map3k6 Map3k7 Map3k8 Map3k9 Map4k1 Map4k2 Map4k3 Map4k4 Map4k5 Pak1 Pak2 Braf Raf1 Mos Myc Mycl1 Mycn Fos Elk1 Junb Spry2
lat
M
CM
IC
dI mi
rly ea
po sti mp l.E PI
I
E Pr
M
EP
IC
pl. eim
pr
Bmp1 Bmp10 Bmp15 Bmp2 Bmp2k Bmp3 Bmp4 Bmp5 Bmp6 Bmp7 Gdf1 Gdf10 Gdf11 Gdf15 Gdf2 Gdf3 Gdf5 Gdf6 Gdf7 Gdf9 Sostdc1 Grem1 Grem2 Nbl1 Chrd Chrdl1 Chrdl2 Dand5 Fst Fstl1 Fstl3 Fstl4 Fstl5 Cer1 Bmpr1a Bmpr1b Acvr1 Acvrl1 Bmpr2 Acvr2a Acvr2b Smad1 Smad4 Smad5 Smad7 Id1 Id2 Id3
MARMOSET
mi dI CM lat eI CM
M IC rly ea
E po sti mp l.E PI
EP I
Pr
IC M
pl.
pr
eim
MO R
MARMOSET
MO R
D MOUSE
MOUSE Wnt1 Wnt10a Wnt10b Wnt11 Wnt16 Wnt2 Wnt2b Wnt3 Wnt3a Wnt4 Wnt5a Wnt5b Wnt6 Wnt7a Wnt7b Wnt8a Wnt8b Wnt9b Sfrp1 Sfrp2 Sfrp4 Sfrp5 Wif1 Cer1 Dkk1 Dkk2 Dkk3 Dkk4 Dkkl1 Lrp1 Lrp10 Lrp11 Lrp12 Lrp1b Lrp2 Lrp2bp Lrp3 Lrp4 Lrp5 Lrp6 Lrp8 Fzd10 Fzd3 Fzd4 Fzd5 Fzd6 Fzd7 Fzd8 Tcf15 Tcf19 Tcf20 Tcf21 Tcf23 Tcf7l1 Tcf7l2 Tcf7 Dvl1 Dvl2 Dvl3 Gsk3a Gsk3b Ctnnb1 Apc Apc2 Axin2 Cdx1 Lef1
Inha Inhba Inhbc Inhbe Nodal Tgfa Tgfb1 Tgfb2 Tgfb3 Tgfbr1 Tgfbr2 Tgfbr3 Acvr1b Acvr1c Acvr2a Acvr2b Smad2 Smad3 Smad4 Smad7 Furin Pcsk1 Pcsk2 Pcsk4 Pcsk5 Pcsk6 Pcsk7 Pcsk9 Sp1 Nodal Cer1 Pitx2
CM
IC M
ICM
lat eI
mi d
rly ea
Pr E po sti mp l.E PI
IC M mp l.E PI pr ei
MO R
0 1 2 3 4 5 6
Figure S6, related to Figure 6: Expression data for (A) TGFß/NODAL, (B) WNT, (C) FGF and (D) BMP pathways.
A
Nanog
Gata6
Cdx2
DAPI
C
D
manual
automated
manual
180 160
cells / embryo
140
automated
B
120 100 80 60 40 20 0
E
Nanog Gata6 Cdx2
G
DAPI
Total cells (DAPI) n=12
600
NANOG
GATA6
CDX2
DAPI
F
cells / embryo
500 400
n=5
n=16 n=5
300 200 100
DMSO
A8301
IWP2
1 30
A8
P2
03
IW
229 cells
PD
153 cells
PD03
Single plane Nanog / Cdx2 / Gata6
H
179 cells
DM
49 cells
SO
0
Lineage segregation
I DMSO PD03
Fluorescence (AU)
IWP2
Fluorescence (AU)
Fluorescence (AU)
GATA6-only (NANOG
