
2/30

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20

10

0

10

20

30

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30 20 10 0 10 20 3030

20

10

0

10

20

3

0

PC 1 scores

PC2scores

30 20 10 0 10 20 3030

20

10

0

10

2

0

30

PC 1 scores

PC2

scores

30 20 10 0 10 20 3030

20

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30

PC 1 scores

PC2

scores

30 20 10 0 10 20 3030

20

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PC 1 scores

PC2

scores

PC 1 scores

30 20 10 0 10 20 3030

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30

PC 1 scores

PC2

scores

30 20 10 0 10 20 3030

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30

PC 1 scores

PC2

scores

Stage

Cluster

PS

NP HF

4SG 4SFG-

0

20

40

60

80

100

PS NP HF 4SG 4SFG-

Cluster3Cluster2Cluster1

%o

fcellsineachcluster

a b

c d

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


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30 20 10 0 10 20 30

30

20

10

0

10

20

30

PC2

s

cores

30 20 10 0 10 20 30

30

20

10

0

10

20

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 expres-

sion level (bottom). Cells not expressing both genes

shown in grey. Blue, PS; green, NP; orange, HF; red,

4SG; purple, 4SFG-.


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PS NP HF

Runx1

Tal1

Gata1

0

50

100

150

200

250

PS NP HF

Hand1

Hand2

Mesp1

PS NP HF

Cdh5

Kdr

Sox7

0

10

20

30

40

PS NP HF

T

Mixl1

Eomes

PS NP HF

Acta2

Myocd

Cald1

0

10

20

30

40

PS NP HF

Myod1

Myog

Myf5

FPKM

Pluripotent

EpiblastMesoderm

Meso-haem-

angioblasts

Haem-

angioblasts

Blood

progenitors

Ectoderm Endoderm Somitic

muscle

Cardiac

muscle

Smooth

muscle

Endothelium

E7 - E7.75 Flk1+ Cells

a

b

c d e

f

a b

c d

e f

0

10

20

30

0

10

20

30

40

50

60

0

10

20

30

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.


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Cbfa2t3h Egfl7 Ets1Eif2b1 Ets2

Etv6 Fli1 Foxh1 Foxo4 Gfi1

Gfi1b Hhex Hoxb2 Hoxd8 Ikzf1

Itga2b Kit Ldb1 Lmo2 Lyl1

Mecom Meis1 Mitf Mrpl19 Myb

Nfe2 Notch1 Pecam1 Polr2a Procr

Spi1 Sox17 Tbx3 Tbx20 Ubc

dCt

ND -10 -5 0 5

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


6/30

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

D

iffusioncomponent1

Diffusion component 2

Diffusion

component 4

PS

NP HF

4SG 4SFG-

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

0.04 0.00 0.040.0

4

0.0

0

0.0

4

0.00

0.10

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-.


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Diffusion map PCA

ICA tSNE

Supplementary Figure 7:Comparison of multivariate analysis techniques. The data for all

3,934 cells were plotted using diffusion maps, principal component analysis, independentcomponent analysis and t-SNE. Blue, PS; green, NP; orange, HF; red, 4SG; purple, 4SFG-.


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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.


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NP

4SFG- HF simulated (black)

HF real (pink)

30

20

10

0

1

0

20

30

30

20

1

0

0

10

20

30

PC2

scores

30 20 10 0 10 20 30

30

20

10

0

10

20

30

PC 1 scores

30 20 10 0 10 20 30

PS

HF 4SG

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

f f ( )


10/30

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Calculate PhlyoP scores for

conservation

TFBS search

Extract all 595 mouse

ChIP-seq peaks

Merge peaks

Identify regions

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

Human :

Platypus :

Chicken :

Xenopus :

GGTG-AAACAATGCGGAG

GGAG-AAACAATGCAAAG

AGTG-AAACAATGCAAAG

GGCGAAAACAATGAAAAG

......

......

......

......

......

......

......

......

Factor type

AP1-like

Ets

Ebox

Gata

Gfi

HMG box

Homeobox

Homeobbox

Ikaros

Myb

Rbpj

Motif

TCAY

GGAW

CANNTG

GATA

AATC

WWCAAWG

YWATTAAR

TAAT

HRGGAW

YAACNG

TTCCCAA

TFs

Nfe2

Erg, Ets1, Etv2, Fli1, PU.1

Scl, Lyl1

Gata1

Gfi1, Gfi1b

Sox7, Sox17

Hhex

Hoxb4

Ikaros

Myb

Notch1

a b

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 previously 1. (b)To

search for evidence that predicted regulatory interactions are direct, all

haematopoietic TF ChIP-seq data in Codex2were searched for peaks within

the gene body or 50 kb up- and down-stream of the 20 network genes. Over-

lapping peaks were merged to identify putative regulatory sequences. Differ-

ent colours indicate peaks from different experiments. A transcription factor

binding site search (TFBS) was performed using TFBSsearch3on 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 well-

recognised 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 con-served 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.


11/30

Erg

HoxB4 day6160 _

1 _

HoxB4 day16160 _

1 _

oxB4 day26160 _

1 _

mm10100 kba

Hsp/Venus

Hsp/Venus/Erg+85

Hsp/Venus/

Erg+85Hox

Hsp/Venus/

Erg+85Sox

Hsp/Venus/

Erg+85HoxSox

CD41

Flk1

Day 3 Day 4 Day 7Day 6Day 5b

11.3

22.3

32.1

34.3

1.47

0.11

53.0

45.5

1.67

0.12

56.4

41.8

1.26

0.16

61.8

36.8

0.50

0.06

40.5

58.9

2.02

0.15

64.0

33.8

5.71

5.95

46.9

41.4

8.78

6.51

50.7

34.1

3.09

6.38

38.8

51.8

5.88

4.66

49.5

40.0

8.13

8.38

43.0

40.5

7.13

21.6

33.2

38.1

5.86

22.1

29.5

42.5

8.70

19.0

29.2

43.0

6.51

19.7

28.5

45.3

10.5

46.0

18.7

24.8

17.4

47.5

14.3

20.8

8.63

41.9

14.6

34.9

14.1

45.7

13.3

26.8

7.50

48.4

22.4

21.7

15.8

53.2

10.8

20.2

21.1

57.0

6.49

15.4

13.1

51.2

9.78

25.9

22.6

42.8

5.22

29.4

10.3

55.6

17.8

16.4

Hsp/Venus

Hsp/Venus/Erg+85

Hsp/Venus/

Erg+85Hox

Hsp/Venus/

Erg+85Sox

Hsp/Venus/

Erg+85HoxSox

SSC

VenusYFP

Day 3 Day 4 Day 7Day 6Day 5c0.122

1.61

0.835

0.568

1.34

0.873

18.6

7.38

13.6

11.7

0.373

17.4

7.33

14.2

6.34

0.145

13.0

5.5

9.01

3.44

0.219

8.92

3.62

3.72

2.09

Supplementary Figure 11: The Erg+85enhancer is active during haematopoietic development. (a)UCSC Genome Browser tracks of re-analysed

ChIP-Seq data for HoxB45indicate that HoxB4 binds to theErg+85enhancer (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.


12/30

Erg

Ets2

Ets1

Lyl1

Fli1

HoxB4

FoxO4

Notch1

FoxH1

Gata1

Tbx20

Gfi1

Gfi1b

Myb

Ikaros

Tbx3

Eto2

Mecom

Runx1 PU.1

Nfe2

Hhex

Ldb1

Tal1Etv2

Sox17

Meis1Sox7

Etv6

a b

Spi1Meis1Etv6Foxo4Foxh1Tbx20Tbx3Hoxb4Gfi1

Hoxd8Lmo2HhexEtv2Tal1Sox7

Notch1ErgEts1Fli1Ets2Sox17MecomLdb1Hoxb2Mitf

Cbfa2t3h

Lyl1Nfe2Gfi1bGata1IkarosRunx1Myb

-1.0 -0.5 0 0.5 1.0Spi1

Meis1

Etv6

Foxo4

Foxh1

Tbx20

Tbx3

Hoxb4

Gfi1

Hoxd8

Lmo2

Hhex

Etv2

Tal1

Sox7

Notch1Erg

Ets1Fli1

Ets2

Sox17

Mecom

Ldb1

Hoxb2

Mitf

Cbfa2t3h

Lyl1

Nfe2

Gfi1b

Gata1

Ikaros

Runx1

Myb

Correlation

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. Ergdoes

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).


13/30

Supplementary Table 1: List of TaqMan assays used for single cell gene expression analysis.

Gene name Protein name Assay ID

Cbfa2t3h Eto2 Mm00486780 m1

Cdh1 E-cadherin Mm01247357 m1

Cdh5 VE-cadherin Mm00486938 m1Egfl7 Egfl7 Mm00618004 m1

Eif2b1 Eif2b1 Mm01199614 m1

Erg Erg Mm01214246 m1

Ets1 Ets1 Mm01175819 m1

Ets2 Ets2 Mm00468977 m1

Etv2 Etv2 Mm00468389 m1

Etv6 Tel Mm01261325 m1

Fli1 Fli1 Mm00484409 m1

FoxH1 FoxH1 AJD1TLL (custom assay)

FoxO4 FoxO4 AJLJIMX (custom assay)

Gata1 Gata1 Mm00484678 m1

Gata2 Gata2 Mm00492300 m1Gfi1 Gfi1 Mm00515855 m1

Gfi1b Gfi1b Mm00492318 m1

Hbb-bH1 Hbb-bH1 Mm00756487 mH

Hhex Hhex Mm00433954 m1

HoxB2 HoxB2 Mm04209931 m1



HoxD8 HoxD8 AJFARRT (custom assay)

Ikzf1 Ikaros Mm01187882 m1

Itga2b CD41 Mm00439768 m1

Kdr Flk1 Mm01222421 m1

Kit c-Kit Mm00445212 m1Ldb1 Ldb1 Mm00440156 m1

Lmo2 Lmo2 Mm01281680 m1

Lyl1 Lyl1 Mm01247198 m1

Mecom MDS1/Evi1 Mm01289155 m1

Meis1 Meis1 Mm00487659 m1

Mitf Mitf Mm01182480 m1

Mrpl19 Mrpl19 Mm03048937 m1

Myb Myb Mm00501741 m1

Nfe2 Nfe2 Mm00801891 m1

Notch1 Notch1 Mm00435249 m1

Pecam1 CD31 Mm01242584 m1

Polr2a Polr2a Mm00839493 m1Procr Epcr Mm00440993 mH

Runx1 Runx1 Mm01213405 m1

Spi1 PU.1 Mm00488142 m1

Sox17 Sox17 Mm04208182 m1

Sox7 Sox7 Mm00776876 m1

Tal1 Scl Mm01187033 m1

Tbx20 Tbx20 Mm00451517 m1

Tbx3 Tbx3 Mm01195726 m1

Ubc Ubc Mm01201237 m1


14/30

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

Gene Synthesised update functions % Non-observed Motifs presenttransitions disallowed (Ni)

Scl Fli1 98 Yes

Etv2 Notch1 96 Yes

Fli1 Etv2 96 YesSox7 97 Yes

Lyl1 Sox7 92 Yes

Sox7 Sox17HoxB4 82 No (Sox missing)

Erg (HoxB4 Lyl1) Sox17 84 Yes(HoxB4 Tal1) Sox17 83 Yes

Notch1 Sox7 94 Yes

Gata1 Gfi1b Lmo2 86 YesGfi1bHhex 84 No (Hhex missing)Gfi1b Ets1 84 Yes

HoxB4 (Lyl1 Ets1) Gata1 65 Yes(Lyl1 Nfe2) Gata1 65 Yes(Lyl1 Ikaros) Gata1 65 No (Ikaros missing)

Sox17 Lyl1 Gfi1b 77 No (Gfi missing)(Eto2 Sox7) Gfi1b 76 No (Gfi missing)(Eto2 Tal1) Gfi1b 75 No (Gfi missing)

Ets1 Notch1 96 Yes

Gfi1 Gata1 Sox17 88 YesNfe2 Sox17 88 Yes

Gfi1b Nfe2Myb 87 YesPu.1 Ikaros 86 No (Ikaros missing)

Pu.1 Nfe2 86 YesPu.1 Myb 86 Yes

Eto2 Sox7 93 No (Sox missing)Hhex 92 No (Hhex missing)Ets1 Fli1 94 No (Ets missing)

Hhex Sox7 97 No (Sox missing)Notch1 93 No (Rbpj missing)

Ikaros Nfe2Gfi1b 84 YesNfe2Gata1 83 YesNfe2Gfi1 82 Yes

Lmo2 Sox7Gfi1 79 YesSox7 Erg 79 Yes

Sox7HoxB4 77 YesNfe2 Ikaros 78 Yes

Pu.1 Gfi1 Erg 67 Yes

Myb HoxB4 64 Yes


15/30

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 ofdata used is decreased, and including the full data set narrows these possibilities.

Supplementary Table 3a

Gene Synthesised update functions % Non-observed transitions disallowed (Ni)

Scl Hhex 96Sox7 98Etv2 98Fli1 99Ets1 100Scl 100

Etv2 No solution

Fli1 Etv2 98Sox7 98Notch1 98

Lyl1 Etv2 90Notch1 91Sox7 92

Sox7 Sox17HoxB4 85

Erg Sox7HoxB4 90Sox7Notch1 89Sox7 89Notch1 89Sox7Notch1 88Sox17HoxB4 84

Notch1 No solution

Gata1 Gfi1 89

HoxB4 Lyl1 76

Sox17 Lyl1 Gfi1b 78

Ets1 Sox7 98Notch1 99

Gfi1 Gfi1b Sox17 88Nfe2 Sox17 90Gata1 90Gata1 Sox17 91

Gfi1b Gata1 87Nfe2Myb 87Nfe2 Ikaros 86Pu.1 Nfe2 86Sox7Gata1 86

Pu.1 Myb 85Eto2 Ets1 95

Sox7 94Hhex 93Notch1 93Etv2 93

Hhex Sox7 98Notch1 98

Ikaros Nfe2Gfi1b 81

Lmo2 Notch1 79Sox7 79

Nfe2 Ikaros 75

Gfi1 81Continued on next page


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continued from previous page


Gfi1b 83Gata1 84

Pu.1 Erg 71

Sox7 Eto2 71Lyl1 Erg 71Notch1 Eto2 71Gfi1 Erg 72HoxB4 Erg 72Erg Eto2 73Ikaros Erg 73Notch1 Ikaros 75Sox7 Ikaros 75

Myb HoxB4 60Gfi1 67


17/30

Supplementary Table 3b


Scl Hhex 96Notch1 96

Etv2 96Ets1 97Sox7 98Fli1 98

Etv2 No solution

Fli1 Notch1 96Etv2 96Sox7 98

Lyl1 Erg 84Notch1 87Etv2 87Sox7 91

Sox7 Sox17 Erg 90HoxB4 Erg 88Gfi1 Erg 85Sox17HoxB4 84Scl Erg 82Fli1 Erg 82Erg 82

Erg Notch1 Etv2 81Sox17HoxB4 83Sox7Notch1 85

Notch1 No solution

Gata1 Gfi1 84Gfi1b Ets1 85Lmo2Gfi1b 87

HoxB4 Lyl1 75

Sox17 Lyl1 Gfi1b 78


Gfi1 Gata1 86Nfe2 Sox17 88Gfi1b Sox17 88Gata1 Sox17 88Gfi1b Erg 87Gata1 Erg 86Nfe2 Hhex 86

Gata1 Hhex 86Ikaros Hhex 86Nfe2 Erg 86

Gfi1b Gfi1 81Pu.1 Nfe2 88Nfe2Myb 89

Eto2 Sox7 94Hhex 93Ets1 92Etv2 91Notch1 91

Hhex Notch1 94

Continued on next page


18/30



Sox7 97

Ikaros Myb Eto2 80Nfe2Gata1 81

Nfe2Gfi1 82Nfe2Gfi1b 83

Lmo2 Sox7 78

Nfe2 Ikaros 77Gfi1 80Gata1 83Gfi1b 86

Pu.1 Nfe2 Erg 67HoxB4Gfi1b 67Myb Erg 67Sox7Myb 67Erg Eto2 68

Gfi1 Erg 68Lyl1 Erg 68Notch1 Nfe2 68Tbx20Gfi1b 68Sox7Nfe2 69Gfi1b Erg 72Ikaros Erg 73Notch1 Gfi1b 74Sox7Gfi1b 75Notch1 Ikaros 75Sox7 Ikaros 75

Myb HoxB4 65Gfi1 66


19/30

Supplementary Table 3c


Scl Hhex 97Notch1 97

Sox7 98Etv2 98Fli1 99Ets1 100Scl 100

Etv2 No solution


Lyl1 Notch1 90Sox7 92

Sox7 Sox17HoxB4 85

Erg Sox7 88Notch1 89Sox17HoxB4 85

Notch1 No solution

HoxB4 Lyl1 76

Sox17 Lyl1 Gfi1b 75Erg Gfi1b 74Fli1 Gfi1b 74Eto2 Gfi1b 74Sox7 Gfi1b 74Lyl1 Gata1 73

Ets1 Notch1 98Sox7 98

Gfi1 Gfi1b Sox17 86Gata1 87Nfe2 Sox17 87Gata1 Sox17 89

Gfi1b Gfi1 Eto2 80IkarosGfi1 80Nfe2 Ets1 82Notch1 Gata1 82Myb Ikaros 83Gata1 Etv2 83Pu.1 Nfe2 83Sox7Gata1 83

Pu.1 Gata1 84Pu.1 Ikaros 84Pu.1 Myb 84Gata1 Ets1 85MybGata1 85Nfe2Myb 85

Eto2 Ets1 96Sox7 95Hhex 94Etv2 94Notch1 93

Hhex Notch1 96



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Sox7 97

Ikaros Myb 80Myb Ets1 80

Myb Fli1 80SclMyb 80Myb Lyl1 80MybGfi1 80MybGata1 81MybGfi1b 81Nfe2Myb 81Myb Eto2 82Nfe2Gata1 82Nfe2Gfi1 82Nfe2Gfi1b 83

Lmo2 Sox7 79

Notch1 78Nfe2 Ikaros 77

Gfi1 79Gfi1b 83Gata1 84

Pu.1 Erg 67Scl Erg 67Ets1 Erg 67Fli1 Erg 67Notch1 Eto2 68HoxB4 Erg 68Sox7 Eto2 68Erg Eto2 69Gfi1b Erg 69Notch1 Lyl1 69Gfi1 Erg 70Ikaros Erg 70Lyl1 Erg 70Notch1 Gfi1b 70Sox7 Lyl1 70Sox7Gfi1b 71Notch1 Ikaros 72Sox7 Ikaros 73

Myb Gfi1 64HoxB4 62

Erg 62


21/30

Supplementary Table 3d


Scl Etv2 96Notch1 96

Ets1 96Hhex 96Fli1 97Sox7 97

Etv2 No solution


Lyl1 Notch1 86Erg 88Sox7 91

Sox7 Lmo2HoxB4 89

Sox17 Erg 89HoxB4 Erg 87Gfi1 Erg 85Sox17 Lmo2 85Scl Erg 83Fli1 Erg 84Erg 83Sox17HoxB4 83

Erg Sox7 86Notch1 82Sox7Gfi1 86Sox17HoxB4 87Sox7 Fli1 87

Notch1 Eto2 88Sox7 Eto2 89Notch1 Lyl1 90Sox7 Lyl1 90

Notch1 No solution

Gata1 Gfi1b 85Gfi1 88

HoxB4 Lyl1 Gfi1 64

Sox17 Lyl1 Gfi1b 78Erg Gfi1b 76Lyl1 Gata1 75Erg Gata1 74Eto2 Gfi1b 73Lyl1 Myb 72Hhex Gfi1b 72Sox7 Gfi1b 72Erg Gfi1 71Lyl1 Gfi1 71


Gfi1 Gata1 88Gata1 Sox17 89

Gfi1b Gata1 85Nfe2 84



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Nfe2Gfi1 85Nfe2Gata1 86Pu.1 Nfe2 86

MybGata1 86Pu.1 Ikaros 86Nfe2 Ikaros 87Pu.1 Myb 87Nfe2Myb 88Gfi1 Gata1 88

Eto2 Fli1 93Sox7 92Scl 92Hhex 92Lyl1 92Ets1 90

Hhex Notch1 94Sox7 97

Ikaros MybGfi1b 80Myb Lyl1 81Nfe2Gata1 81Myb Eto2 82Nfe2Gfi1b 83

Lmo2 Sox7 76Erg 72

Nfe2 Myb 70Ikaros 76Gfi1 81Gata1 85Gfi1b 86

Pu.1 Erg 69Gfi1b Erg 74Notch1 Gfi1b 75Sox7Gfi1b 75

Myb Erg 62Gfi1 64HoxB4 66


23/30

Supplementary Table 3e


Scl Lyl1 96Hhex 97

Eto2 97Notch1 97Etv2 98Sox7 98Ets1 100Fli1 100Scl 100

Etv2 Notch1 96Sox7 98

Fli1 Meis1 90Notch1 97Etv2 98

Sox7 98Lyl1 Hhex 95Notch1 95Sox7 97

Sox7 Sox17HoxB4 88

Erg Notch1 91Sox7 90

Notch1 No solution

Gata1 Gfi1 85

HoxB4 Sox17 Lmo2 67Eto2 Gfi1 59Lyl1 Gfi1 59

Fli1 Gfi1 59Scl Gfi1 59Lmo2 57

Sox17 Fli1 Gfi1b 79Sox7 Gfi1b 79Scl Gfi1b 79Ets1 Gfi1b 79Etv2 Gfi1b 79Notch1 Gfi1b 79Hhex Gfi1b 78Eto2 Gfi1b 77Lyl1 Gfi1b 75

Ets1 Notch1 97Sox7 98

Gfi1 Gata1 87Nfe2 Sox17 87Gata1 Sox17 88

Gfi1b Notch1 Gata1 81Nfe2 Etv2 81Sox7Nfe2 81Gata1 Etv2 82Sox7Gata1 82Pu.1 Gata1 82Nfe2 Ets1 83Gata1 Ets1 84



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Myb Ikaros 84MybGata1 84Pu.1 Ikaros 84

Pu.1 Myb 85Pu.1 Nfe2 85Nfe2Myb 86

Eto2 Ets1 99Sox7 97Etv2 97Notch1 96Hhex 96

Hhex Notch1 96Sox7 98

Ikaros Myb Lyl1 80MybGata1 80

MybGfi1 80MybMitf 80MybGfi1b 81Nfe2Myb 81Nfe2Gfi1b 82

Lmo2 Sox7 78Notch1 78Notch1 Erg 79Sox7Gfi1 79Sox7HoxB4 79Sox7 Erg 79Sox7Notch1 79

Nfe2 Myb 71Ikaros 74Gfi1 80Gata1 82Gfi1b 84

Pu.1 Erg 68Sox7 67

Myb Erg 62Gfi1 63HoxB4 65


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


Scl Scl 100

Fli1 99Sox7 99

Etv2 Sox7 98Notch1 94

Fli1 Scl 99Ets1 99Sox7 98Etv2 98Hhex 97

Lyl1 Fli1 93Eto2 93Scl 93

Ets1 93Sox7 91Hhex 91Etv2 91

Sox7 Notch1 95Sox17HoxB4 96

Erg Sox7 87Notch1 84HoxB4 Sox17 85

Notch1 Sox7 96Scl 96Ets1 96Fli1 96Etv2 96

Gata1 Gfi1b 86Gfi1 87

HoxB4 Lyl1 74Erg 74

Sox17 Lyl1 Gfi1b 75


Gfi1 Gata1 87Gata1 Sox17 87Nfe2 Sox17 87HoxB4 Notch1 87

Gfi1b Sox17 86Gfi1b Gata1 86

Nfe2 82Gfi1 81

Eto2 Fli1 97Tal1 96Ets1 96Sox7 94Hhex 94Etv2 94

Hhex Scl 99Fli1 99

Ets1 99Continued on next page


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Sox7 98Etv2 97

Ikaros Nfe2Gfi1b 83

Gata1 Gfi1b 80MybGfi1b 80Gfi1 Gfi1b 80Myb Eto2 80Myb Lyl1 81

Lmo2 Sox7 72

Nfe2 Gata1 84Gfi1b 84Gfi1 80Ikaros 73

Pu.1 Ikaros 75Gfi1b 74

Myb Ikaros 79Gfi1b 75


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Supplementary Table 6: Statistical analysis of Erg+85 enhancer assays.Results of statistical analysis for Figure 4B, comparing each construct to wildtype on each day. P values were generated for each differentiation with t-tests, and differentiations were combined by the Fishers method to calculateoverall significance. Where replicates differed in whether the percentage of

YFP+ cells was higher or lower than wild type, Stouffers Z trend was usedrather than Fishers method. *, p


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Supplementary Note: Reconstructing an existing Boolean net-

work

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 coreregulatory network active in common myeloid progenitor cells9. We took the existing Booleannetwork, constructed its associated state space (shown in the figure below), and then used thisstate space as input to our synthesis method in order to try to reconstruct the Boolean networkthat we started with.

Table 1: Original Boolean rules.

Gene Update function

Gata2 Gata2 (Pu.1 (Gata1 Fog1))Gata1 (Gata1 Gata2 Fli1) Pu.1Fog1 Gata1

EKLF Gata1 Fli1Fli1 Gata1 EKLF

Scl Gata1 Pu.1

Cebpa Cebpa (Scl (Fog1 Gata1))Pu.1 (Cebpa Pu.1) (Gata1 Gata2)cJun Pu.1 Gfi1

EgrNab (Pu.1 cJun) Gfi1Gfi1 Cebpa EgrNab

Figure 1: Network state space


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The results of applying our synthesis method are shown in the table below. The method suc-cessfully reconstructs the Boolean update function for all but one gene (EgrNab), in some casesuniquely identifying the correct function. We note that when multiple solutions are found for anupdate function, these solutions, while not exact, all provide useful regulatory information thatcould be verified in an experimental scenario. For example, both solutions for Sclsuccessfully

predictScls activation byGata1, although one of the two solutions omits its repression by Pu.1.We found that these results were robust when performing bootstrapping, randomly discarding10% of the state space data and rerunning the analysis.

Table 2: Synthesised Boolean functions

Gene Synthesised update functions Comments

Gata2 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 (Gata1 Cebpa) Pu.1(Gata2 Fog1) Pu.1(Gata1 Gata2) Pu.1(Gata1 Gata2 Fli1) Pu.1Other functions of the form(X Y Z) Pu.1

Fog1 Gata1 Unique

EKLF Gata1 Fli1 Unique

Fli1 Gata1 EKLF Unique

Scl Gata1Gata1 Pu.1

Cebpa Cebpa (Fog1 Scl)Cebpa (Cebpa (Scl Fog1))Cebpa (Fog1 (SclCebpa))Cebpa (Fog1 (SclGata1))Cebpa (Fog1 (SclGata2))Cebpa (Gata1 (Fog1 Scl)Cebpa (Scl (Fog1 Cebpa)Cebpa (Scl (Fog1 Gata1)

Pu.1 Pu.1 Gata2(Pu.1 Cebpa) Gata2Pu.1 (Gata1 Gata2)Other functions of the formPu.1 (Gata2X)Pu.1 (Gata2Cepba)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(CebpaX) (Gata2 Y)Other functions of the form(Pu.1 X) (Gata2 Y)Other functions of the form(Cebpa Pu.1) (Gata2X)

cJun Pu.1 Gfi1 Unique

EgrNab (cJunGata1) Gfi1 IncorrectGfi1 Cebpa EgrNab Unique


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REFERENCES

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