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a

PS

NP
3.34%

3.94%

Runx1

HF
5.30%

10.20% 4SG
4SFG-

Flk1

80
60
40
20

4S

HF

NP

0
PS

Number of cells per embryo (x103)

4.27%

Supplementary Figure 1: Fluorescence activated cell sorting of cells with blood potential. (a)
Gating strategy for Flk1+ cells at PS, NP and HF stages, and Runx1-ires-GFP+ cells (4SG) or
Flk1+Runx1-ires-GFP- cells (4SFG-) at the 4S stage. (b) Total numbers of cells per embryo at
each stage, estimated from FACS data. Each point represents one embryo and lines indicate
the median.

Nature Biotechnology: doi:10.1038/nbt.3154

PC 2 scores
30 20 10 0
10

PC 2 scores
30 20 10 0
10 20

30

30 20 10 0
10
PC 1 scores

30

20

30

20

30

4SFG-

20

30

4SG

20

HF

30
30

PC 2 scores
30 20 10 0
10 20

20

30 20 10 0
10
PC 1 scores

20

30

Cluster

30 20 10 0
10
PC 1 scores
100
80

20

30

NP

30 20 10 0
10
PC 1 scores

30 20 10 0
10
PC 1 scores

% of cells in each cluster

PC 2 scores
30 20 10 0
10 20
30

30

PS

PC 2 scores
30 20 10 0
10

30

30 20 10 0
10 20
PC 1 scores

PC 2 scores
30 20 10 0
10 20

Stage

30

PC 2 scores
30 20 10 0
10 20

60
40
20

30 20 10 0
10
PC 1 scores

20

30

PS
NP
HF 4SG 4SFGCluster1 Cluster2 Cluster3

Supplementary Figure 2: Development is asynchronous. (a) PCA of the 3,934 cells, coloured
retrospectively according to the stage from which they were sorted. Blue, PS; green, NP; orange,
HF; red, 4SG; purple, 4SFG-. (b) For each stage, the cells from different embryos are shown on
the PCA as different colours (cells from other stages shown in grey). (c) PCA coloured according
to the clusters cells belong to in Figure 1e. Green, I; Blue, II; Pink, III. (d) For each embryo, the
percentage of cells in clusters I, II and III was calculated. The mean and standard deviation was
then calculated for each cluster in each stage. Number of embryos shown in Fig. 1C.

Nature Biotechnology: doi:10.1038/nbt.3154

30
20
10
0
10
20
30

20

10

10

20

30

30

20

10

10

20

30

30

20

10

10

20

30

PC 2 scores

30

PC 1 scores

Supplementary Figure 3: Flk1+Runx1+ cells are found


in all anatomical stages. Principal component analysis
coloured according to stage of sorting (top) and whether
cells express both Flk1 and Runx1 at the gene expression level (bottom). Cells not expressing both genes
shown in grey. Blue, PS; green, NP; orange, HF; red,
4SG; purple, 4SFG-.

Nature Biotechnology: doi:10.1038/nbt.3154

E7 - E7.75 Flk1+ Cells


Pluripotent
Epiblast

Mesoderm

a
Ectoderm

Endoderm

Meso-haemangioblasts

c
Somitic
muscle

d
Cardiac
muscle

40

Myod1
Myog
Myf5

20
10

PS

NP

FPKM

200
150

Hand1
Hand2
Mesp1

100
50

PS

NP

e
Smooth
muscle

Endothelium

20

Mixl1
Eomes

10

PS

NP

HF

30
20

Acta2
Myocd
Cald1

10
0

HF

PS

NP

HF

60
50

30

40

Cdh5
Kdr
Sox7

30
20
10
0

Blood
progenitors

40

HF

250

30

30

Haemangioblasts

PS

NP

HF

Runx1
Tal1
Gata1

20
10
0

PS

NP

HF

Supplementary Figure 4: RNA sequencing of populations of 50 cells. Schematic of early embryonic


development of the mesodermal lineage. The cardiac, smooth muscle, endothelial and blood lineages all
diverge from Flk1+ mesoderm, while somatic muscle diverges earlier. (a-f) Populations of 50 cells were
sorted from 5 embryos each at PS, NP and HF stages. FPKM values for three key genes are shown for
each of the lineage decisions in the schematic. Points are the mean and s.d. of the 5 replicates.

Nature Biotechnology: doi:10.1038/nbt.3154

Cbfa2t3h

Egfl7

Eif2b1

Ets1

Ets2

Etv6

Fli1

Foxh1

Foxo4

Gfi1

Gfi1b

Hhex

Hoxb2

Hoxd8

Ikzf1

Itga2b

Kit

Ldb1

Lmo2

Lyl1

Mecom

Meis1

Mitf

Nfe2

Notch1

Pecam1

Polr2a

Procr

Spi1

Sox17

Tbx3

Tbx20

Ubc

dCt

ND

-10

Mrpl19

-5

Myb

Supplementary Figure 5: Diffusion plots. Diffusion plots showing expression patterns of additional genes not shown in Figure 2.

Nature Biotechnology: doi:10.1038/nbt.3154

0.00

0.00

0.04

0.04

PS

0.10

0.00
0.04

0.00

0.04

0.04

0.10

0.04

0.04

0.00

0.04

0.00

0.00

0.04

0.04

HF

0.10
0.00
0.04

0.00

0.10
0.04

0.04

Diffusion component 1

NP

0.00

0.04

4SG

0.00
0.04

0.00

0.04

0.00

0.00

0.04

0.04

4SFG-

0.00
0.04

0.00

0.04
Diffusion component 2

0.10
0.04

0.04

0.10
0.00
0.04

0.00

0.04
Diffusion
component 4

Supplementary Figure 6: Diffusion plots highlighting each stage. Top left plot shows all populations, other
plots were coloured according to embryonic stage with cells from other stages shown in grey. Blue, PS;
green, NP; orange, HF; red, 4SG; purple, 4SFG-.
Nature Biotechnology: doi:10.1038/nbt.3154

Diffusion map

ICA

PCA

tSNE

Supplementary Figure 7: Comparison of multivariate analysis techniques. The data for all
3,934 cells were plotted using diffusion maps, principal component analysis, independent
component analysis and t-SNE. Blue, PS; green, NP; orange, HF; red, 4SG; purple, 4SFG-.

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Figure 8: Some states occur in multiple cells. State transition graph coloured
by the number of times each binary state occurs in the 3,934 expression profiles. Grey, occurs
once; blue, occurs twice; red, occurs more than twice.

Nature Biotechnology: doi:10.1038/nbt.3154

30

NP

HF

4SG

4SFG-

HF simulated (black)
HF real (pink)

0
30

20

10

10

20

30

30

20

10

PC 2 scores

10

20

30

30

20

10

10

20

PS

30

20

10

10

20

30

30

20

10

10

20

30

PC 1 scores

Supplementary Figure 9: Populations mask variation and asynchrony. Pools of 20 cells were simulated
from single cell data and projected onto the principal component analysis for all 3,934 cells. Each plot
shows one embryonic stage and the simulated pools of 20 cells for that population in black (normalized in
the same way as single cells). Bottom right hand plot shows the head fold stage and simulated pools as
well as actual pools of 20 cells sorted from embryos (pink).

Nature Biotechnology: doi:10.1038/nbt.3154

Motif

AP1-like
Ets
Ebox
Gata
Gfi
HMG box
Homeobox
Homeobbox
Ikaros
Myb
Rbpj

Nfe2
Erg, Ets1, Etv2, Fli1, PU.1
Scl, Lyl1
Gata1
Gfi1, Gfi1b
Sox7, Sox17
Hhex
Hoxb4
Ikaros
Myb
Notch1

TCAY
GGAW
CANNTG
GATA
AATC
WWCAAWG
YWATTAAR
TAAT
HRGGAW
YAACNG
TTCCCAA

Extract all 595 mouse


ChIP-seq peaks
Merge peaks
Identify regions

TFBS search

Mouse

Nature Biotechnology: doi:10.1038/nbt.3154

: ...... ACAG-AAACAATGCAGAG ......


Calculate PhlyoP scores for
conservation

Human
Platypus
Chicken
Xenopus
. . . .

Supplementary Figure 10: Analysis of transcription factor binding sites in


network gene loci. (a) Motifs for DNA-binding TFs in the core network. Note
that Eto2 and Lmo2 do not bind directly to the DNA, and the binding site of the
Notch partner Rbpj was used for Notch1 as described previously1. (b) To
search for evidence that predicted regulatory interactions are direct, all
haematopoietic TF ChIP-seq data in Codex2 were searched for peaks within
the gene body or 50 kb up- and down-stream of the 20 network genes. Overlapping peaks were merged to identify putative regulatory sequences. Different colours indicate peaks from different experiments. A transcription factor
binding site search (TFBS) was performed using TFBSsearch3 on the mouse
genomic sequence of all regions to identify the consensus binding sites of the
network TFs. Evolutionary conservation of TF binding motifs is a wellrecognised feature of truly functional binding sites4. To identify conserved
binding sites, PhyloP scores were calculated for each motif across 60 species
using the tool provided by the UCSC Genome Browser. Evolutionarily conserved motifs previously validated in functional assays by us and other were
characterised by PhyloP scores greater than 1, which was therefore used as
the threshold. Results are summarised in Supplementary Table 2.

: ...... GGTG-AAACAATGCGGAG ......


: ...... GGAG-AAACAATGCAAAG ......
: ...... AGTG-AAACAATGCAAAG ......
: ...... GGCGAAAACAATGAAAAG ......
. . . .

TFs

. . . .

Factor type

. . . .

a
HoxB4 day6
HoxB4 day16
HoxB4 day26

Day 3

Day 4

53.0 1.47
45.5 0.11

46.9 5.71
41.4 5.95

Day 5
33.2 7.13
38.1 21.6

1_
160 _
1_
160 _
1_

Erg

Day 6
18.7 10.5
24.8 46.0

Day 7
10.8 15.8
20.2 53.2

Hsp/Venus

Day 3

Day 4

Day 5

Day 6

Day 7

0.122

0.873

0.373

0.145

0.219

1.61

18.6

17.4

13.0

8.92

0.835

7.38

7.33

5.5

3.62

0.568

13.6

14.2

9.01

3.72

1.34

11.7

6.34

3.44

2.09

Hsp/Venus

56.4 1.67
41.8 0.12

50.7 8.78
34.1 6.51

32.1 11.3
34.3 22.3

14.3 17.4
20.8 47.5

6.49 21.1
15.4 57.0

Hsp/Venus/
Erg+85

Hsp/Venus/
Erg+85Sox

Hsp/Venus/
Erg+85HoxSox

61.8 1.26
36.8 0.16

38.8 3.09
51.8 6.38

29.5 5.86
42.5 22.1

14.6 8.63
34.9 41.9

9.78 13.1
25.9 51.2

40.5 0.50
58.9 0.06

49.5 5.88
40.0 4.66

29.2 8.70
43.0 19.0

13.3 14.1
26.8 45.7

5.22 22.6
29.4 42.8

64.0 2.02
33.8 0.15

43.0 8.13
40.5 8.38

28.5 6.51
45.3 19.7

22.4 7.50
21.7 48.4

17.8 10.3
16.4 55.6

CD41

Hsp/Venus/
Erg+85Hox

Venus YFP

Hsp/Venus/
Erg+85

Flk1

Hsp/Venus/
Erg+85Hox

mm10

100 kb

160 _

Hsp/Venus/
Erg+85Sox

Hsp/Venus/
Erg+85HoxSox
SSC

Supplementary Figure 11: The Erg+85 enhancer is active during haematopoietic development. (a) UCSC Genome Browser tracks of re-analysed
ChIP-Seq data for HoxB45 indicate that HoxB4 binds to the Erg+85 enhancer (shown in grey) during a 26-day differentiation time course that produces
HSCs from ES cells, with HSCs able to contribute to long-term engraftment in recipient mice. Binding is absent at day 6, but becomes apparent by
day 16. (b) Flow cytometric analysis of Flk1 and CD41 expression across a time-course of EB differentiation from Figure 4B indicates that different
clones have similar kinetics of differentiation, with Flk1+ cells giving rise to Flk1+CD41+ and then Flk1-CD41+ cells. (c) Flow cytometric analysis of YFP
expression in all live cells across a time-course of EB differentiation from Figure 4b illustrates reduction of YFP positive cells in ES cell clones where
YFP expression is driven by mutant enhancer constructs. In (b) and (c), a representative experiment is shown from three biological repeats of 2-3
clones per construct.
Nature Biotechnology: doi:10.1038/nbt.3154

b
Meis1

Sox7

Tal1

Etv2

Etv6

Correlation

Tbx3

FoxO4

Myb
Runx1
Ikaros
Gata1
Gfi1b

Nfe2
Lyl1

Hoxb2

Sox17

Sox7
Tal1

Ldb1
Mecom

Etv2
Hhex
Lmo2

Hoxb2

Gfi1

Cbfa2t3h
Mitf

Hoxd8

Nfe2
Lyl1

Hoxb4

Foxh1
Foxo4

Tbx3
Tbx20

Etv6
Meis1
Spi1
Myb
Runx1
Ikaros
Gata1
Gfi1b

Cbfa2t3h
Mitf

0.5 1.0

Ldb1
Mecom

Notch1

Ets2
Fli1
Ets1
Erg
Notch1

-1.0 -0.5

Sox17

Tbx20

Fli1

FoxH1

Lyl1

Eto2

Ets2

Gfi1

Sox17

Ets2
Fli1
Ets1
Erg
Notch1

Sox7
Tal1

Etv2
Hhex
Lmo2

Hoxd8

Gfi1

Hoxb4

Tbx3
Tbx20

Foxh1
Foxo4

Etv6
Meis1
Spi1

Erg

Gfi1b

Ets1

Myb

HoxB4

Ikaros
Hhex

Gata1
Mecom

Ldb1

Runx1

PU.1

Nfe2

Supplementary Figure 12: Partial correlation analysis. (a) Hierarchical clustering of partial correlation coefficients between pairs of transcription
factors for all 3,934 expression profiles. Pairwise coefficients were calculated while controlling for the effect of all other transcription factors. Erg does
not correlate with Sox or Hox factors. (b) Network diagram showing putative undirected activating (red) and inhibiting (blue) relationships suggested
by significant correlations (p-value < 1e-10).

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 1: List of TaqMan assays used for single cell gene expression analysis.
Gene name
Cbfa2t3h
Cdh1
Cdh5
Egfl7
Eif2b1
Erg
Ets1
Ets2
Etv2
Etv6
Fli1
FoxH1
FoxO4
Gata1
Gata2
Gfi1
Gfi1b
Hbb-bH1
Hhex
HoxB2
HoxB3
HoxB4
HoxD8
Ikzf1
Itga2b
Kdr
Kit
Ldb1
Lmo2
Lyl1
Mecom
Meis1
Mitf
Mrpl19
Myb
Nfe2
Notch1
Pecam1
Polr2a
Procr
Runx1
Spi1
Sox17
Sox7
Tal1
Tbx20
Tbx3
Ubc

Nature Biotechnology: doi:10.1038/nbt.3154

Protein name
Eto2
E-cadherin
VE-cadherin
Egfl7
Eif2b1
Erg
Ets1
Ets2
Etv2
Tel
Fli1
FoxH1
FoxO4
Gata1
Gata2
Gfi1
Gfi1b
Hbb-bH1
Hhex
HoxB2
HoxB3
HoxB4
HoxD8
Ikaros
CD41
Flk1
c-Kit
Ldb1
Lmo2
Lyl1
MDS1/Evi1
Meis1
Mitf
Mrpl19
Myb
Nfe2
Notch1
CD31
Polr2a
Epcr
Runx1
PU.1
Sox17
Sox7
Scl
Tbx20
Tbx3
Ubc

Assay ID
Mm00486780 m1
Mm01247357 m1
Mm00486938 m1
Mm00618004 m1
Mm01199614 m1
Mm01214246 m1
Mm01175819 m1
Mm00468977 m1
Mm00468389 m1
Mm01261325 m1
Mm00484409 m1
AJD1TLL (custom assay)
AJLJIMX (custom assay)
Mm00484678 m1
Mm00492300 m1
Mm00515855 m1
Mm00492318 m1
Mm00756487 mH
Mm00433954 m1
Mm04209931 m1
Mm00650701 m1
Mm00657964 m1
AJFARRT (custom assay)
Mm01187882 m1
Mm00439768 m1
Mm01222421 m1
Mm00445212 m1
Mm00440156 m1
Mm01281680 m1
Mm01247198 m1
Mm01289155 m1
Mm00487659 m1
Mm01182480 m1
Mm03048937 m1
Mm00501741 m1
Mm00801891 m1
Mm00435249 m1
Mm01242584 m1
Mm00839493 m1
Mm00440993 mH
Mm01213405 m1
Mm00488142 m1
Mm04208182 m1
Mm00776876 m1
Mm01187033 m1
Mm00451517 m1
Mm01195726 m1
Mm01201237 m1

Supplementary Table 2: Boolean update rules for the network in Figure 3c, synthesized from
the state graph in Figure 3b. The final column indicates whether the DNA binding motifs of the
predicted regulators are present in the locus of the target gene (see Supplementary Figure 10).
Gene

Synthesised update functions

Scl
Etv2
Fli1

Fli1
Notch1
Etv2
Sox7
Sox7
Sox17 HoxB4
(HoxB4 Lyl1) Sox17
(HoxB4 Tal1) Sox17
Sox7
Gfi1b Lmo2
Gfi1b Hhex
Gfi1b Ets1
(Lyl1 Ets1) Gata1
(Lyl1 Nfe2) Gata1
(Lyl1 Ikaros) Gata1
Lyl1 Gfi1b
(Eto2 Sox7) Gfi1b
(Eto2 Tal1) Gfi1b
Notch1
Gata1 Sox17
Nfe2 Sox17
Nfe2 Myb
Pu.1 Ikaros
Pu.1 Nfe2
Pu.1 Myb
Sox7
Hhex
Ets1 Fli1
Sox7
Notch1
Nfe2 Gfi1b
Nfe2 Gata1
Nfe2 Gfi1
Sox7 Gfi1
Sox7 Erg
Sox7 HoxB4
Ikaros
Gfi1 Erg
HoxB4

Lyl1
Sox7
Erg
Notch1
Gata1

HoxB4

Sox17

Ets1
Gfi1
Gfi1b

Eto2

Hhex
Ikaros

Lmo2

Nfe2
Pu.1
Myb

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed
transitions disallowed (Ni )
98
96
96
97
92
82
84
83
94
86
84
84
65
65
65
77
76
75
96
88
88
87
86
86
86
93
92
94
97
93
84
83
82
79
79
77
78
67
64

Motifs present
Yes
Yes
Yes
Yes
Yes
No (Sox missing)
Yes
Yes
Yes
Yes
No (Hhex missing)
Yes
Yes
Yes
No (Ikaros missing)
No (Gfi missing)
No (Gfi missing)
No (Gfi missing)
Yes
Yes
Yes
Yes
No (Ikaros missing)
Yes
Yes
No (Sox missing)
No (Hhex missing)
No (Ets missing)
No (Sox missing)
No (Rbpj missing)
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Supplementary Table 3: Boolean update rules after multiple rounds of bootstrapping (a-e).
In general, the number of possible solutions for a genes update function grows as the amount of
data used is decreased, and including the full data set narrows these possibilities.
Supplementary Table 3a
Gene
Scl

Etv2
Fli1

Lyl1

Sox7
Erg

Notch1
Gata1
HoxB4
Sox17
Ets1
Gfi1

Gfi1b

Eto2

Hhex
Ikaros
Lmo2
Nfe2

Synthesised update functions


Hhex
Sox7
Etv2
Fli1
Ets1
Scl
No solution
Etv2
Sox7
Notch1
Etv2
Notch1
Sox7
Sox17 HoxB4
Sox7 HoxB4
Sox7 Notch1
Sox7
Notch1
Sox7 Notch1
Sox17 HoxB4
No solution
Gfi1
Lyl1
Lyl1 Gfi1b
Sox7
Notch1
Gfi1b Sox17
Nfe2 Sox17
Gata1
Gata1 Sox17
Gata1
Nfe2 Myb
Nfe2 Ikaros
Pu.1 Nfe2
Sox7 Gata1
Pu.1 Myb
Ets1
Sox7
Hhex
Notch1
Etv2
Sox7
Notch1
Nfe2 Gfi1b
Notch1
Sox7
Ikaros
Gfi1

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


96
98
98
99
100
100
98
98
98
90
91
92
85
90
89
89
89
88
84
89
76
78
98
99
88
90
90
91
87
87
86
86
86
85
95
94
93
93
93
98
98
81
79
79
75
81
Continued on next page

Gene

Pu.1

Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Gfi1b
83
Gata1
84
Erg
71
Sox7 Eto2
71
Lyl1 Erg
71
Notch1 Eto2
71
Gfi1 Erg
72
HoxB4 Erg
72
Erg Eto2
73
Ikaros Erg
73
Notch1 Ikaros
75
Sox7 Ikaros
75
HoxB4
60
Gfi1
67

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 3b
Gene
Scl

Etv2
Fli1

Lyl1

Sox7

Erg

Notch1
Gata1

HoxB4
Sox17
Ets1
Gfi1

Gfi1b

Eto2

Hhex

Synthesised update functions


Hhex
Notch1
Etv2
Ets1
Sox7
Fli1
No solution
Notch1
Etv2
Sox7
Erg
Notch1
Etv2
Sox7
Sox17 Erg
HoxB4 Erg
Gfi1 Erg
Sox17 HoxB4
Scl Erg
Fli1 Erg
Erg
Notch1 Etv2
Sox17 HoxB4
Sox7 Notch1
No solution
Gfi1
Gfi1b Ets1
Lmo2 Gfi1b
Lyl1
Lyl1 Gfi1b
Sox7
Notch1
Gata1
Nfe2 Sox17
Gfi1b Sox17
Gata1 Sox17
Gfi1b Erg
Gata1 Erg
Nfe2 Hhex
Gata1 Hhex
Ikaros Hhex
Nfe2 Erg
Gfi1
Pu.1 Nfe2
Nfe2 Myb
Sox7
Hhex
Ets1
Etv2
Notch1
Notch1

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


96
96
96
97
98
98
96
96
98
84
87
87
91
90
88
85
84
82
82
82
81
83
85
84
85
87
75
78
95
97
86
88
88
88
87
86
86
86
86
86
81
88
89
94
93
92
91
91
94
Continued on next page

Gene
Ikaros

Lmo2
Nfe2

Pu.1

Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Sox7
97
Myb Eto2
80
Nfe2 Gata1
81
Nfe2 Gfi1
82
Nfe2 Gfi1b
83
Sox7
78
Ikaros
77
Gfi1
80
Gata1
83
Gfi1b
86
Nfe2 Erg
67
HoxB4 Gfi1b
67
Myb Erg
67
Sox7 Myb
67
Erg Eto2
68
Gfi1 Erg
68
Lyl1 Erg
68
Notch1 Nfe2
68
Tbx20 Gfi1b
68
Sox7 Nfe2
69
Gfi1b Erg
72
Ikaros Erg
73
Notch1 Gfi1b
74
Sox7 Gfi1b
75
Notch1 Ikaros
75
Sox7 Ikaros
75
HoxB4
65
Gfi1
66

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 3c
Gene
Scl

Etv2
Fli1

Lyl1
Sox7
Erg

Notch1
HoxB4
Sox17

Ets1
Gfi1

Gfi1b

Eto2

Hhex

Synthesised update functions


Hhex
Notch1
Sox7
Etv2
Fli1
Ets1
Scl
No solution
Notch1
Etv2
Sox7
Notch1
Sox7
Sox17 HoxB4
Sox7
Notch1
Sox17 HoxB4
No solution
Lyl1
Lyl1 Gfi1b
Erg Gfi1b
Fli1 Gfi1b
Eto2 Gfi1b
Sox7 Gfi1b
Lyl1 Gata1
Notch1
Sox7
Gfi1b Sox17
Gata1
Nfe2 Sox17
Gata1 Sox17
Gfi1 Eto2
Ikaros Gfi1
Nfe2 Ets1
Notch1 Gata1
Myb Ikaros
Gata1 Etv2
Pu.1 Nfe2
Sox7 Gata1
Pu.1 Gata1
Pu.1 Ikaros
Pu.1 Myb
Gata1 Ets1
Myb Gata1
Nfe2 Myb
Ets1
Sox7
Hhex
Etv2
Notch1
Notch1

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


97
97
98
98
99
100
100
97
97
98
90
92
85
88
89
85
76
75
74
74
74
74
73
98
98
86
87
87
89
80
80
82
82
83
83
83
83
84
84
84
85
85
85
96
95
94
94
93
96
Continued on next page

Gene
Ikaros

Lmo2
Nfe2

Pu.1

Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Sox7
97
Myb
80
Myb Ets1
80
Myb Fli1
80
Scl Myb
80
Myb Lyl1
80
Myb Gfi1
80
Myb Gata1
81
Myb Gfi1b
81
Nfe2 Myb
81
Myb Eto2
82
Nfe2 Gata1
82
Nfe2 Gfi1
82
Nfe2 Gfi1b
83
Sox7
79
Notch1
78
Ikaros
77
Gfi1
79
Gfi1b
83
Gata1
84
Erg
67
Scl Erg
67
Ets1 Erg
67
Fli1 Erg
67
Notch1 Eto2
68
HoxB4 Erg
68
Sox7 Eto2
68
Erg Eto2
69
Gfi1b Erg
69
Notch1 Lyl1
69
Gfi1 Erg
70
Ikaros Erg
70
Lyl1 Erg
70
Notch1 Gfi1b
70
Sox7 Lyl1
70
Sox7 Gfi1b
71
Notch1 Ikaros
72
Sox7 Ikaros
73
Gfi1
64
HoxB4
62
Erg
62

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 3d
Gene
Scl

Etv2
Fli1

Lyl1

Sox7

Erg

Notch1
Gata1
HoxB4
Sox17

Ets1
Gfi1
Gfi1b

Synthesised update functions


Etv2
Notch1
Ets1
Hhex
Fli1
Sox7
No solution
Notch1
Etv2
Sox7
Notch1
Erg
Sox7
Lmo2 HoxB4
Sox17 Erg
HoxB4 Erg
Gfi1 Erg
Sox17 Lmo2
Scl Erg
Fli1 Erg
Erg
Sox17 HoxB4
Sox7
Notch1
Sox7 Gfi1
Sox17 HoxB4
Sox7 Fli1
Notch1 Eto2
Sox7 Eto2
Notch1 Lyl1
Sox7 Lyl1
No solution
Gfi1b
Gfi1
Lyl1 Gfi1
Lyl1 Gfi1b
Erg Gfi1b
Lyl1 Gata1
Erg Gata1
Eto2 Gfi1b
Lyl1 Myb
Hhex Gfi1b
Sox7 Gfi1b
Erg Gfi1
Lyl1 Gfi1
Sox7
Notch1
Gata1
Gata1 Sox17
Gata1
Nfe2

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


96
96
96
96
97
97
96
96
98
86
88
91
89
89
87
85
85
83
84
83
83
86
82
86
87
87
88
89
90
90
85
88
64
78
76
75
74
73
72
72
72
71
71
94
97
88
89
85
84
Continued on next page

Gene

Eto2

Hhex
Ikaros

Lmo2
Nfe2

Pu.1

Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Nfe2 Gfi1
85
Nfe2 Gata1
86
Pu.1 Nfe2
86
Myb Gata1
86
Pu.1 Ikaros
86
Nfe2 Ikaros
87
Pu.1 Myb
87
Nfe2 Myb
88
Gfi1 Gata1
88
Fli1
93
Sox7
92
Scl
92
Hhex
92
Lyl1
92
Ets1
90
Notch1
94
Sox7
97
Myb Gfi1b
80
Myb Lyl1
81
Nfe2 Gata1
81
Myb Eto2
82
Nfe2 Gfi1b
83
Sox7
76
Erg
72
Myb
70
Ikaros
76
Gfi1
81
Gata1
85
Gfi1b
86
Erg
69
Gfi1b Erg
74
Notch1 Gfi1b
75
Sox7 Gfi1b
75
Erg
62
Gfi1
64
HoxB4
66

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 3e
Gene
Scl

Etv2
Fli1

Lyl1

Sox7
Erg
Notch1
Gata1
HoxB4

Sox17

Ets1
Gfi1

Gfi1b

Synthesised update functions


Lyl1
Hhex
Eto2
Notch1
Etv2
Sox7
Ets1
Fli1
Scl
Notch1
Sox7
Meis1
Notch1
Etv2
Sox7
Hhex
Notch1
Sox7
Sox17 HoxB4
Notch1
Sox7
No solution
Gfi1
Sox17 Lmo2
Eto2 Gfi1
Lyl1 Gfi1
Fli1 Gfi1
Scl Gfi1
Lmo2
Fli1 Gfi1b
Sox7 Gfi1b
Scl Gfi1b
Ets1 Gfi1b
Etv2 Gfi1b
Notch1 Gfi1b
Hhex Gfi1b
Eto2 Gfi1b
Lyl1 Gfi1b
Notch1
Sox7
Gata1
Nfe2 Sox17
Gata1 Sox17
Notch1 Gata1
Nfe2 Etv2
Sox7 Nfe2
Gata1 Etv2
Sox7 Gata1
Pu.1 Gata1
Nfe2 Ets1
Gata1 Ets1

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


96
97
97
97
98
98
100
100
100
96
98
90
97
98
98
95
95
97
88
91
90
85
67
59
59
59
59
57
79
79
79
79
79
79
78
77
75
97
98
87
87
88
81
81
81
82
82
82
83
84
Continued on next page

Gene

Eto2

Hhex
Ikaros

Lmo2

Nfe2

Pu.1
Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Myb Ikaros
84
Myb Gata1
84
Pu.1 Ikaros
84
Pu.1 Myb
85
Pu.1 Nfe2
85
Nfe2 Myb
86
Ets1
99
Sox7
97
Etv2
97
Notch1
96
Hhex
96
Notch1
96
Sox7
98
Myb Lyl1
80
Myb Gata1
80
Myb Gfi1
80
Myb Mitf
80
Myb Gfi1b
81
Nfe2 Myb
81
Nfe2 Gfi1b
82
Sox7
78
Notch1
78
Notch1 Erg
79
Sox7 Gfi1
79
Sox7 HoxB4
79
Sox7 Erg
79
Sox7 Notch1
79
Myb
71
Ikaros
74
Gfi1
80
Gata1
82
Gfi1b
84
Erg
68
Sox7
67
Erg
62
Gfi1
63
HoxB4
65

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 4: Boolean update rules synthesised from data filtered according to a
more stringent limit of detection (Ct 23).
Gene
Scl

Etv2
Fli1

Lyl1

Sox7
Erg

Notch1

Gata1
HoxB4
Sox17
Ets1
Gfi1

Gfi1b

Eto2

Hhex

Synthesised update functions


Scl
Fli1
Sox7
Sox7
Notch1
Scl
Ets1
Sox7
Etv2
Hhex
Fli1
Eto2
Scl
Ets1
Sox7
Hhex
Etv2
Notch1
Sox17 HoxB4
Sox7
Notch1
HoxB4 Sox17
Sox7
Scl
Ets1
Fli1
Etv2
Gfi1b
Gfi1
Lyl1
Erg
Lyl1 Gfi1b
Sox7
Notch1
Gata1
Gata1 Sox17
Nfe2 Sox17
HoxB4 Notch1
Gfi1b Sox17
Gata1
Nfe2
Gfi1
Fli1
Tal1
Ets1
Sox7
Hhex
Etv2
Scl
Fli1
Ets1

Nature Biotechnology: doi:10.1038/nbt.3154

% Non-observed transitions disallowed (Ni )


100
99
99
98
94
99
99
98
98
97
93
93
93
93
91
91
91
95
96
87
84
85
96
96
96
96
96
86
87
74
74
75
99
94
87
87
87
87
86
86
82
81
97
96
96
94
94
94
99
99
99
Continued on next page

Gene

Ikaros

Lmo2
Nfe2

Pu.1
Myb

continued from previous page


Synthesised update functions % Non-observed transitions disallowed (Ni )
Sox7
98
Etv2
97
Nfe2 Gfi1b
83
Gata1 Gfi1b
80
Myb Gfi1b
80
Gfi1 Gfi1b
80
Myb Eto2
80
Myb Lyl1
81
Sox7
72
Gata1
84
Gfi1b
84
Gfi1
80
Ikaros
73
Ikaros
75
Gfi1b
74
Ikaros
79
Gfi1b
75

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Table 6: Statistical analysis of Erg+85 enhancer assays.


Results of statistical analysis for Figure 4B, comparing each construct to wild
type on each day. P values were generated for each differentiation with ttests, and differentiations were combined by the Fishers method to calculate
overall significance. Where replicates differed in whether the percentage of
YFP+ cells was higher or lower than wild type, Stouffers Z trend was used
rather than Fishers method. *, p <0.05; **, p<0.01; ***, p<0.001; ns, not
significant (p >0.05).

Population

Mutant

Day3

Day4

Day5

Day6

Day7

*
ns

ns
ns

ns
ns

ns
ns

ns
ns

ns

ns

ns

**

ns

**

Sox

ns

ns

ns

ns

HoxSox

ns

**

Flk1-CD41+ Hox

ns

***

**

ns

ns

Sox

ns

ns

ns

ns

ns

HoxSox

ns

**

ns

ns

Flk1+CD41- Hox
Sox
HoxSox
Flk1+CD41+ Hox

Nature Biotechnology: doi:10.1038/nbt.3154

Supplementary Note: Reconstructing an existing Boolean network


To assess the ability of the synthesis method to reconstruct existing asynchrnonous Boolean
networks from their state spaces, we applied the method to a Boolean network model of the core
regulatory network active in common myeloid progenitor cells9 . We took the existing Boolean
network, constructed its associated state space (shown in the figure below), and then used this
state space as input to our synthesis method in order to try to reconstruct the Boolean network
that we started with.
Table 1: Original Boolean rules.
Gene
Gata2
Gata1
Fog1
EKLF
Fli1
Scl
Cebpa
Pu.1
cJun
EgrNab
Gfi1

Update function
Gata2 (Pu.1 (Gata1 Fog1))
(Gata1 Gata2 Fli1) Pu.1
Gata1
Gata1 Fli1
Gata1 EKLF
Gata1 Pu.1
Cebpa (Scl (Fog1 Gata1))
(Cebpa Pu.1) (Gata1 Gata2)
Pu.1 Gfi1
(Pu.1 cJun) Gfi1
Cebpa EgrNab

Figure 1: Network state space

Nature Biotechnology: doi:10.1038/nbt.3154

The results of applying our synthesis method are shown in the table below. The method successfully reconstructs the Boolean update function for all but one gene (EgrNab), in some cases
uniquely identifying the correct function. We note that when multiple solutions are found for an
update function, these solutions, while not exact, all provide useful regulatory information that
could be verified in an experimental scenario. For example, both solutions for Scl successfully
predict Scls activation by Gata1, although one of the two solutions omits its repression by Pu.1.
We found that these results were robust when performing bootstrapping, randomly discarding
10% of the state space data and rerunning the analysis.
Table 2: Synthesised Boolean functions
Gene
Gata2

Gata1

Fog1
EKLF
Fli1
Scl
Cebpa

Pu.1

cJun
EgrNab
Gfi1

Synthesised update functions


Gata2 (Fog1 Pu.1)
Gata2 (Fog1 (Pu.1 Cebpa))
Gata2 (Fog1 (Pu.1 Gata2))
Gata2 (Gata2 (Pu.1 Fog1)
Gata2 (Pu.1 (Gata1 Fog1))
Gata2 (Pu.1 (Gata2 Fog1))
(Gata1 Cebpa) Pu.1
(Gata2 Fog1) Pu.1
(Gata1 Gata2) Pu.1
(Gata1 Gata2 Fli1) Pu.1
Other functions of the form (X Y Z) Pu.1
Gata1
Gata1 Fli1
Gata1 EKLF
Gata1
Gata1 Pu.1
Cebpa (Fog1 Scl)
Cebpa (Cebpa (Scl Fog1))
Cebpa (Fog1 (Scl Cebpa))
Cebpa (Fog1 (Scl Gata1))
Cebpa (Fog1 (Scl Gata2))
Cebpa (Gata1 (Fog1 Scl)
Cebpa (Scl (Fog1 Cebpa)
Cebpa (Scl (Fog1 Gata1)
Pu.1 Gata2
(Pu.1 Cebpa) Gata2
Pu.1 (Gata1 Gata2)
Other functions of the form Pu.1 (Gata2 X)
Pu.1 (Gata2 Cepba)
Pu.1 (Gata2 Pu.1)
Cebpa (Gata1 Gata2)
Cebpa (Gata2 Fog1)
(Cebpa Pu.1) (Gata1 Gata2)
(Cebpa Pu.1) (Gata1 Gata2)
Other functions of the form (Cebpa X) (Gata2 Y )
Other functions of the form (Pu.1 X) (Gata2 Y )
Other functions of the form (Cebpa Pu.1) (Gata2 X)
Pu.1 Gfi1
(cJun Gata1) Gfi1
Cebpa EgrNab

Nature Biotechnology: doi:10.1038/nbt.3154

Comments

Unique
Unique
Unique

Unique
Incorrect
Unique

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