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Supplementary Developing Core Regulatory Network Blood
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Flk1
Runx1
4SG
4SFG-
a
b
PS NP H
F 4S
0
20
40
60
80
Numberofcellsperembryo(x10)
3.94% 3.34% 5.30% 10.20%
4.27%
HFNPPS
3
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.
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PC2scores
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PC 1 scores
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PC 1 scores
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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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PC2
s
cores
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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
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40
50
60
0
10
20
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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.
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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)
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PC2
scores
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PC 1 scores
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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 ( )
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.
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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.
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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.
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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).
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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
HoxB3 HoxB3 Mm00650701 m1
HoxB4 HoxB4 Mm00657964 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
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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
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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
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
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Supplementary Table 3b
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
Ets1 Sox7 95Notch1 97
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
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continued from previous page
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
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Supplementary Table 3c
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
Scl Hhex 97Notch1 97
Sox7 98Etv2 98Fli1 99Ets1 100Scl 100
Etv2 No solution
Fli1 Notch1 97Etv2 97Sox7 98
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
Continued on next page
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continued from previous page
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
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Supplementary Table 3d
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
Scl Etv2 96Notch1 96
Ets1 96Hhex 96Fli1 97Sox7 97
Etv2 No solution
Fli1 Notch1 96Etv2 96Sox7 98
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
Ets1 Sox7 94Notch1 97
Gfi1 Gata1 88Gata1 Sox17 89
Gfi1b Gata1 85Nfe2 84
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Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
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Supplementary Table 3e
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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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Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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).
Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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
Ets1 Sox7 99Notch1 94
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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Gene Synthesised update functions % Non-observed transitions disallowed (Ni)
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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