supplementary materials for€¦ · 03/08/2016 · specification of tissue-resident macrophages...
TRANSCRIPT
www.sciencemag.org/cgi/content/full/science.aaf4238/DC1
Supplementary Materials for
Specification of tissue-resident macrophages during organogenesis
Elvira Mass, Ivan Ballesteros, Matthias Farlik, Florian Halbritter, Patrick Günther,
Lucile Crozet, Christian E. Jacome-Galarza, Kristian Händler, Johanna Klughammer,
Yasuhiro Kobayashi, Elisa Gomez-Perdiguero, Joachim L. Schultze, Marc Beyer,
Christoph Bock, Frederic Geissmann*
*Corresponding author. Email: [email protected]
Published 4 August 2016 on Science First Release
DOI: 10.1126/science.aaf4238
This PDF file includes:
Supplementary Text
Figs. S1 to S13
Captions for Tables S1 to S6
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencemag.org/cgi/content/full/science.aaf4238/DC1)
Tables S1 ro S6 (Excel files)
Mass et al., Fig. S1 C
Developmental stage (time in days)
EMP à MΦ 954 genes 1396 transcripts
YS FL YS FL
head caudal
YS liver
head brain limb
skin/epi. kidney
lung
EM
P pM
ac
MΦ
birth
A
B
● ●
●
●
●
●
●
●
−100 0 100 200 300
−150
−100
−50
0
50
100
150
200
Principal component 1 (18.9%)
Prin
cipa
l com
pone
nt 2
(11.
1%)
●
●
●
EMPpMacMac
● headcaudalFLYS
D EMP pMac MΦ
head caudal FL YS
0 1 2 3 4
05
1015
Average expression
Dis
pers
ion
4632428N05RikAdam15
Adgrg1
AdslAgpsAnxa3
Apbb1ip
Apoe
Aur kaBclaf1Brd4
C1qa C1qbC1qc
Caprin1
Cbfa2t3Ccnb1
Ccnd2Cd34
Cd47Cd53Cdk6Cdt1CebpbChchd4Cited2
Clint1CluhCnn2CopaCreg1 Csf1r
CtsbCtsc
CtslCx3cr1
Dab2
Ddx17
Def6Dnajc2Dpysl2 Efhd2Ehd4
Eif5aEmbEmilin2Evl
Fcgr1
Fcgr3Fmnl1Fth1
Ftl1Fzr1
Galk1GlulGrnGrpel1
H19 Hba−a1
Hba−xHbb−bh1
Hmga2
Hnr npa2b1Hnr nph1Hp
Hsp90aa1
Ifi30
Ifngr1Igfbp4
IghmIl17r a
Il4raImp3Iqgap1
Irf8Itga4 Lgals1
LgmnLmo2LplLsp1Lyz2Maea
Maf
Malat1
MarcksMef2c
MpoMrc1
Mrto4
Mt1
Ncf1Nedd4Nhp2l1Nop10
Nop56NptnNudt4Ogfr l1 Pabpc1Pfdn6
Plac8Plk1
Pou2f2Prdx2Prpf40a
Pr tn3
Pycard Rap1bRhoaRpa2Runx1Scd2
Sepp1SetSlc25a12
Smc4
Snx2
Spns2
SrmSrrm2
Stab1
Thg1lTimm9Tm6sf1 Top2aTpm4
Tuba4a
Tubb2aTub b4b
Tubb6Ube2c
Ube2q1Ugcg VimYbx1
Zfpm1
Zmiz1
B
A
B C
D
E F
Single-cell RNA-seq of 576
CD45low/+ cells
576 cells - 17766 genes
QA/ QC
removal of low quality cells
408 cells - 8657 genes
Dimensionality reduction and computational
clustering
Mapping of gene signature
expression scores to tSNE clusters
Pseudotemporal ordering of single
cells
ATCA
TCTA CTAC
-20
-10
0
10
20
-20 0 20 PC1
PC
2
0 5
10 15
0 1 2 3 5 9 10 Number of clusters
F r e q u e
n c y a m
o n g a l l
i n d i c e s
Cluster 1
Cluster 3 Cluster 2
-20
-10
0
10
20
-20 0 20 PC1
PC
2
-3
-2
-1
0
1
-2 0 2 Component 2
Cluster 1
Cluster 3 Cluster 2
-1 1 z-score expression
Com
pone
nt 1
G
Mass et al., Fig. S2
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
CD16.2 CD64 Tim4 CD206
AYS
FL
head
limbs
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
1000 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
Il4ra Il13ra1 Tnfr2 Ifngr
Kit+
pMacs
MΦ
E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5, gated on YFP+ cells
Il4ra Il13ra1 Tnfr2 Ifngr
brain
liver
lung
skin
Dectin-1 CD64 Tim4 CD206
E14.5 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5 B
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
1000 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
1000 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
1000 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
FMO, YFP+
FMO, all
all YFP+
Gated on MΦ
Mass et al., Fig. S3
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
YFP Dectin-1 Iba1 merge
E14.5 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @8.5
lung
YFP Iba1 F4/80
kidn
ey
head
merge
liver
brai
n liv
er
YFP Iba1 F4/80
kidn
ey
lung
E18.5 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5
merge
YFP Grn F4/80 merge
lung
he
ad
liver
E14.5 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5
E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5 C
D
E
F
YFP Kit F4/80 merge
E9.5 Csf1riCre; Rosa26LSL-YFP
head
ca
udal
YFP F4/80 merge Iba1 B
Mass et al., Fig. S4 E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP+ OH-TAM @E8.5
merge YFP CD206 Iba1
YFP Iba1 Ifngr
YFP Iba1 Tnfr2
merge
merge
YFP Dectin-1 Iba1
merge YFP Iba1 F4/80
merge
YFP Iba1 Trem2 merge
YFP Iba1 CD16/32 merge
YFP Iba1 Grn merge
caud
al
head
ca
udal
he
ad
caud
al
head
ca
udal
he
ad
caud
al
head
ca
udal
ca
udal
he
ad
caud
al
head
he
ad
Aliv
er
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CD45
Kit
F4/8
0
CD11b YFP
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
Tnfrsf11aCre; Rosa26LSL-YFP E10.25 (YS)
0 102 103 104 105
0
102
103
104
105
0 102 103 104 1050
20
40
60
80
100
CD48
CD
150
0 102 103 104 1050
20
40
60
80
100
YFP 0 102 103 104 105
0
20
40
60
80
100
LSK LT-HSCs
ST-HSCs MPPs
E14.5 Tnfrsf11aCre; Rosa26LSL-YFP
brai
n ki
dney
merge YFP Iba1 F4/80
A
B
Mass et al., Fig. S5
liver kidneybrain epidermis0
10
20
30
40
50
0
6
12
0
50
100
150
200
250
0.00
0.01
0.02
0.03 Cx3cr1+/-Cx3cr1-/-
0
5
10
15
lung
p < 0.001
merge
head
ca
udal
GFP Iba1 F4/80
E10.25 Cx3cr1gfp/+
F4/8
0+ /
g of
org
an (x
106)
cells
/ ea
r (x1
06)
E14.5
adult (3-4 months)
F4/8
0+ /
orga
n (x
104 )
F4/8
0+ /
org
an (%
)
E12.5
brain limbsliver0.0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
5
0.0
0.1
0.2
0.3
0.4
brain kidneyliver0.0
0.5
1.0
1.5
0.00
0.03
0.06
0.09
0.12
0
1
2
3
4
5
cells
/org
an (1
04 )
0.0
0.2
0.4
0.6
lung
p = 0.015
A
B C
D
Mass et al., Fig. S6
A
pMacMac
Mac
vs.
pM
acsi
gnat
ure
Mac
vs.
EM
Psi
gnat
ure
pMac
vs.
EM
Psi
gnat
ure
EMPMac EMPpMacRanked by expression (higher expression in ...)
B
Nor
mal
ized
enr
ichm
ent s
core
D
C
EMPpMacMac
Cell type:
****
***
*
***
***
***
***
***
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Cell typescompared
Cell typescompared
Nor
mal
ized
enr
ichm
ent s
core
Gau
tier
et a
l. (2
012)
Cor
e MΦ
sig
natu
re
**
***
***
0
1
2
3
4
5
...
# % #%
GSEA result summary (panels B, D):
MΦ MΦ
MΦ
MΦ
MΦ MΦ
MΦ
(2012)
B
D
C A
Mass et al., Fig. S7
A
Thbs1
C
Pu.1 Cebpα Cebpβ
Maf Stat1 Stat6
Irf8 JunB Rela Atf3
35
100
100
300
300
200
250
70
100
300 50 kb
Mrc1
50 kb
Pu.1 Cebpα Cebpβ
Maf Stat1 Stat6
Irf8 JunB Rela Atf3 35
100
100
300
300
200
250 70
100
300 50 kb 50 kb
Emr1 Cx3cr1
Pu.1 Cebpα Cebpβ
Maf Stat1 Stat6
Irf8 JunB Rela Atf3 35
100
100
300
300
200
250
70
100
300 50 kb 50 kb
Myod1 Gata1
B
E2f1
Spi1
Suz12 Irf1 Hif1a Ring1 Rela Atf3 Batf3 Cebpa Irf8 Egr1 JunB Maf Cebpb Stat1 Ezh2 Stat6 Relb Rel Stat3 Stat5b Gfi1b
6 -6
Rel
ativ
e ex
pres
sion
(Log
2)
E9.
5 ca
udal
E
9.5
head
E
9.5
YS
E10
.5 h
ead
E10
.25
FL
E10
.25
YS
E10
.5 li
mbs
E
10.2
5 lim
bs
E10
.25
YS
E
10.2
5 FL
E10
.25
FL
E10
.25
limbs
EMP pMac
E10
.25
head
E10
.5 F
L
E10
.25
head
E10
.5 Y
S
E9
YS
E10
.25
YS
MΦ
Myb
Mass et al., Fig. S8
Cell type:EMPpMacMac
Time:E9E9.5E10.25E10.5E12.5E14.5E18.5P2P8P21
Tissue:Limbs / skinHead / brainKidneyLiverLungYolksac
1.00.50.0-0.5-1.0
Row
z-s
core
Samples (n = 91)
Diff
eren
tially
exp
ress
ed g
enes
(n =
13,
717)
CA
������
Tissue:Limbs / skinHead / brainKidneyLiverLungYolksac
Cell type:EMPpMacMac
�200 �100 0 100 200
�200
�100
0
100
200
Principal component 1
Prin
cipa
l com
pone
nt 2
Head / brainLimbs / skin Kidney
Liver YolksacLung
E10.25
P21
P21 E10.25E14.5
P21
E10.25
E10.5
E14.5
P21P21
E10.25
Time (days)
�� � �
�
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��
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Gen
e ex
pres
sion
rela
tive
to m
axim
um (m
ean
+/- s
.d.)
Limbs / skin signature
Head / brain signature
Kidney signature
Liver signature
Lung signature100%
50%
0%
100%
50%
100%
50%
100%
50%
100%
50%
E10.25, 10.5
E12.5E14.5
E18.5P2 P8 P21
B�
�
Limbs / skinHead / brain
�
�
KidneyLiver
�
�
LungYolksac
B A C
Mass et al., Fig. S9
limbs/skin brain liver lung kidney
6 -6
Relative expression (Log2)
P8
P21
P21
P21
P8
P2
E12
.5
E14
.5
E18
.5
P8
P2
E12
.5
E14
.5
E18
.5
P8
P2
E12
.5
E14
.5
E18
.5
P8
P2
E14
.5
E18
.5
P2
E14
.5
E18
.5
P21
P21
D
E
Id1 Id3 Sall1 Sall3
6 -6
Relative expression (Log2) E9.
5 ca
udal
E
9.5
head
E
9.5
YS
E10
.25
FL
E10
.25
YS
E10
.25
YS
E
10.2
5 FL
E10
.25
limbs
EMP pMac
E10
.25
head
E9
YS
B
A
C
Mass et al., Fig. S10
Mass et al., Fig. S11
Dimensionality reduction and computational
clustering
Mapping of gene (co-)expression to
tSNE plot (Fig. 6B)
Calculation of macrophage tissue signatures from bulk RNA-seq data
all MΦ 10.25/10.5 all MΦ 10.25/10.5 all MΦ 10.25/10.5
brain MΦ 14.5/18.5 skin MΦ
14.5/18.5 liver MΦ 14.5/18.5
Common signature Tissue signatures
|FC| > 1.5, FDR < 0.05 Mapping of tissue
signatures to PCA of pMacs (Fig. 6C)
A
Kit
0.0
0.5
1.0
1.5
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
Stab1
0.0
0.5
1.0
1.5
2.0
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
Maf
0.0
0.5 1.0 1.5 2.0 2.5
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
Cx3cr1
0.0 0.5 1.0 1.5 2.0 2.5
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
B
Id1
0.0
0.5
1.0
1.5
2.0
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
Id3
0.0
0.5
1.0
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
Sall3
0.0
0.5
1.0
1.5
2.0
EMP pMA CS MA CS
n o r m a l i z e
d e x p r e s
s i o n
C
D Id1 Id3 Sall3 relative expression
0.00 0.05 0.10
0.00 0.02 0.04 0.06
0.00 0.01 0.02 0.03 0.04 0.05
relative expression
-20
-10
0
10
20
-20 0 20 tSNE_1
tSN
E_2
-20
-10
0
10
20
-20 0 20 tSNE_1
tSN
E_2
-20
-10
0
10
20
-20 0 20 tSNE_1
tSN
E_2
relative expression
Skin
Liver
Brain
EMP
pMac
Mac
Common
Cluster 6Cluster 5Cluster 4
Cluster 3Cluster 2Cluster 1
0 0.05 1
E
E
14 0
Expression (Log2)
Id3+/- Id3-/-
Kupffe
r cell
s
hepa
tocyte
s0
1
2
3
4
5
rela
tive
expr
essi
on
(nor
mal
ized
to G
AP
DH
) Id3
Kupffe
r cell
s
hepa
tocyte
s0.0
0.5
1.0
1.5Nr1h3A YFP ID3 DAPI F4/80
Id3+/-
Id3-/- C
D31
+ ar
ea (u
m2 /
mm
2 )
Id3-/-
Id3+/-
Id3+/- Id3-/- F4/80 CD31
B
C D
Cre+
Cre-
0
200
400
600Id3-/-Id3+/-
0
200
400
600RankCre+ Id3f/fRankCre- Id3+/f
CD
31+
area
(um
2 / m
m2 )
merge
Cre+; Id3f/f Cre-; Id3f/+
Mass et al., Fig. S12
liver
br
ain
GO Term (273 genes) corrected p-value
negative regulation of apoptotic process 0.0420 negative regulation of metabolic process 0.0420 response to cytokine 0.0420 negative regulation of programmed cell death 0.0420 negative regulation of macromolecule metabolic process 0.0420 signal transduction 0.0420 negative regulation of macromolecule biosynthetic process 0.0420 negative regulation of biosynthetic process 0.0420 negative regulation of protein kinase activity 0.0495
GO Term (1320 genes) corrected p-value
carboxylic acid metabolic process 3.36E-25 monocarboxylic acid metabolic process 2.44E-24 organic acid metabolic process 1.06E-23 oxoacid metabolic process 8.79E-23 arachidonic acid monooxygenase activity 2.09E-17 blood microparticle 3.04E-17 small molecule metabolic process 3.70E-17 fatty acid metabolic process 2.88E-16 xenobiotic metabolic process 4.40E-16 drug catabolic process 6.32E-16
Id3-/- > Id3+/-
genes 2-fold enriched with t-test <0.05, FDR <0.05
Id3+/- > Id3-/-
genes 2-fold enriched with t-test <0.05, FDR <0.05; (Top 10 GO-terms)
F
Expression (Log2)
Batf3 Pparg Erg1 Atf3 Relb Ring1 Lmo2 Phf19 Jun Stat5a Gata3 Rel Cebpa Stat5b Maf E2f1 Stat3 Tcf3 Hif1a Irf1 Cebpb Ezh2 Irf8 Rela Suz12 Gfi1b Runx1 Lyl1 Spi1
3.5 3.2 3.0 7.8 7.7 8.3 7.9 8.4 8.0 8.0 8.3 6.8 7.1 8.3 7.8 7.8 7.4 7.4 6.2 6.7 7.7 7.8 6.2 4.5 5.5 7.5 2.6 2.6 3.1 5.1 5.7 5.4 5.2 6.3 6.2 5.4 5.6 7.4 5.5 3.5 5.0 6.7 3.6 5.1 3.2 4.2 4.8 4.5 5.2 6.8 5.0 5.8 3.2 4.6 4.4 7.1 6.7 6.4 4.8 6.7 8.0 3.3 4.9 4.6 4.4 3.8 5.2 5.4 5.1 3.5 3.9 7.7 4.6 7.2 5.5 5.4 5.2 4.9 3.4 3.1 4.0 6.8 6.1 6.7 3.6 3.7 8.1 6.6 7.9 8.2 8.7 6.5 8.4 6.8 7.8 8.2 6.8 9.6 9.3 7.9 8.8 9.9 6.1 8.8 3.4 5.5 4.5 5.4 4.5 4.8 4.7 5.5 3.0 4.0 3.7 4.6 5.9 3.6 4.3 5.4 6.5 7.1 4.4 4.5 5.6 6.5 5.8 7.5 3.8 4.7 4.4 4.1 5.3 3.8 4.6 5.0 3.6 3.8 3.9 3.2 4.6 3.8 4.5 3.8 3.7 5.0 5.4 4.1 4.0 5.7 3.3 3.9 3.9 5.5 4.0 5.4 5.0 4.5 5.3 5.2 6.0 5.9 5.6 6.4 5.8 5.0 6.0 5.9 6.1 7.3 6.1 7.4 5.6 5.7 4.5 7.8 4.8 8.1 8.3 7.9 8.0 6.3 5.0 4.7 7.1 4.4 4.0 3.7 3.6 3.3 4.1 4.9 4.3 3.1 4.9 5.2 5.8 5.0 4.5 5.7 4.9 4.5 5.9 4.1 5.8 4.1 7.4 5.7 6.2 6.2 6.2 8.2 8.6 8.5 7.9 6.0 8.7 6.9 8.8 9.8 7.5 7.7 8.2 7.8 8.9 9.2 7.8 9.7 7.9 10 10 8.7 9.6 7.3 7.1 5.5 6.3 7.9 6.1 6.4 7.1 7.7 7.4 7.1 6.7 6.2 6.4 7.4 6.1 6.7 5.6 5.6 5.2 6.3 5.5 5.8 5.5 5.8 7.0 5.8 6.3 7.4 6.2 5.2 5.3 4.2 3.9 5.3 4.8 4.0 4.8 4.5 5.6 5.0 5.6 7.6 5.1 4.4 7.9 5.4 4.1 5.2 5.4 5.7 5.7 5.2 5.8 7.3 5.1 6.9 7.8 7.9 8.0 7.6 6.8 8.0 7.7 7.9 6.8 8.8 6.5 8.3 7.8 7.5 5.7 8.4 6.7 8.9 8.2 7.6 8.3 7.4 7.5 5.8 6.5 7.2 7.8 7.8 7.4 7.8 7.7 7.3 7.0 7.0 7.0 7.1 6.8 7.2 6.4 7.3 6.5 8.6 7.1 8.4 7.7 6.7 7.1 6.6 7.2 8.2 7.3 7.2 6.9 8.0 7.1 7.5 7.5 7.7 8.2 9.5 8.2 8.3 7.0 7.5 7.7 7.3 6.8 5.7 7.1 8.8 9.6 8.0 5.4 6.2 7.4 6.4 4.9 9.9 8.9 7.9 9.3 9.5 9.0 9.8 10 9.4 10 10 9.6 9.7 7.4 11 7.5 10 9.4 9.7 9.4 9.6 11 10 8.0 5.5 7.4 8.5 8.4 6.9 8.1 9.1 8.4 8.3 8.4 7.7 8.5 8.5 7.6 5.4 8.7 9.0 6.5 6.8 7.0 6.4 8.3 9.2 6.5 6.8 7.5 9.0 7.8 7.4 7.5 8.2 6.8 6.2 7.6 7.3 6.4 8.9 7.1 6.0 5.6 3.6 6.7 6.7 7.0 8.2 6.1 5.8 7.3 8.5 6.9 6.2 6.9 5.7 7.4 5.8 5.7 6.4 6.1 6.6 5.9 5.7 5.5 6.5 6.3 5.2 7.2 5.3 7.4 7.0 7.3 5.5 6.7 5.6 5.1 6.3 6.5 7.3 8.0 9.0 9.3 7.7 8.1 7.5 6.9 7.6 7.1 7.5 7.9 7.9 8.3 8.3 9.6 7.6 8.4 7.7 8.2 8.2 7.2 4.8 9.8 6.3 6.6 6.8 7.6 8.9 7.0 6.7 7.3 7.1 7.2 6.5 6.2 6.5 6.7 7.8 6.2 6.4 6.5 6.9 7.0 7.2 6.6 9.6 9.7 10 8.6 9.5 9.6 8.6 8.6 7.0 7.9 7.9 8.4 7.3 8.0 7.6 7.9 7.6 7.9 9.1 7.6 8.2 8.3 8.2 7.7 9.1 5.4 7.6 8.4 8.1 7.4 8.5 7.5 9.1 9.1 9.3 8.8 9.3 9.1 8.8 9.1 9.4 9.1 9.8 8.7 6.5 8.0 8.4 7.9 8.8 8.9 7.1 9.2 7.5 7.7 8.5 7.0 8.8 8.8 8.2 9.4 8.7 8.2 11 11 12 11 11 12 12 11 11 11 11 12 11 10 11.1 8.7 13 10 12 12 12 11 12 8.7 9.7 7.4 8.0 8.5 8.0 8.1 7.5 8.7 8.1 8.9 11 7.9 7.0 8.0 8.5 9.0 10 8.2 7.6 9.0 8.9 10 9.5 8.5 7.3 9.3 8.7 9.0 10 10 10 9.9 10 9.9 10 9.5 8.8 9.8 9.0 8.6 10 9.8 9.0 8.9 9.9 9.4 10 8.3 9.8 9.6 7.6 9.2 11 10 6.1 7.2 4.4 7.2 4.3 7.8 5.3 5.0 8.3 3.7 3.7 5.2 7.7 3.2 3.6 2.9 3.7 9.1 7.1 4.3 3.6 3.7 5.9 10 10 10 9.4 9.8 10.0 9.3 9.4 9.9 8.8 8.9 8.7 9.5 8.6 9.2 8.9 9.2 9.2 7.1 9.4 7.8 4.9 10 9.6 9.3 8.9 11 11 11 10 11 10 10 9.1 9.9 9.5 10 12 10 11 9.6 8.9 10 11.6 7.9 9.9 10 11 12 11 11 12 11 12 11 12 12 12 12 11 12 12 12 13 11 12 11 10 11 12 9.8 11 12 12 12 11 10 12
Ccr1 Cx3cr1 Ccr5 Cxcr2 Cxcr3 Ccr3 Cxcr4 Cxcr6 Ccr9 Ccr7 Cxcr5 Xcr1 Ccr2
E10
.25
head
E9.
5 ca
udal
E
9.5
head
E
9.5
YS
E10
.5 h
ead
E10
.25
FL
E10
.25
YS
E10
.5 li
mb
E10
.25
limb
E10
.5 Y
S
E10
.5 l
iver
E10
.25
head
E9
YS
E
10.2
5 Y
S
E10
.25
FL
E12
.5 b
rain
E
14.5
bra
in
E10
.25
liver
E10
.25
limbs
E12
.5 li
ver
E14
.5 li
ver
E12
.5 li
mbs
E
14.5
ski
n
E14
.5 k
idne
y E
14.5
lung
6.0 6.6 6.4 9.2 8.7 10.0 10.3 9.4 11.1 10.5 11.1 11.2 10.9 11.0 11.2 10.7 10.1 11.4 9.1 9.6 8.3 5.5 11.0 12.5 11.3 10.1 4.3 4.3 4.6 7.9 7.8 7.4 7.9 8.8 8.1 8.5 8.1 7.0 9.3 8.7 8.8 8.2 7.9 8.4 4.9 6.5 2.8 3.9 3.7 3.6 3.7 3.5 5.4 4.8 5.0 7.9 7.0 8.5 8.5 8.1 8.6 9.0 9.5 8.5 7.9 8.8 7.8 8.6 7.6 8.4 3.1 7.2 3.2 7.5 3.8 4.8 6.2 4.6 6.6 8.0 5.2 6.5 7.2 7.8 6.5 8.2 6.0 7.4 5.6 5.0 4.1 3.6 3.7 3.8 4.0 3.5 3.2 4.2 3.1 4.1 4.1 3.8 3.9 3.7 6.1 8.7 4.9 4.7 4.8 4.5 2.8 3.8 3.0 6.3 4.5 3.6 5.7 4.9 4.7 4.6 4.8 5.5 6.2 4.0 2.7 4.6 3.7 4.1 4.0 3.6 2.6 3.3 3.2 4.6 2.9 3.2 2.4 3.5 5.7 2.5 4.2 3.1 4.2 3.7 3.9 3.1 3.1 3.4 5.0 4.1 4.4 5.8 3.8 4.1 6.7 4.0 2.6 4.3 3.1 3.6 3.9 3.7 3.2 3.7 3.0 2.8 3.5 4.6 4.6 3.5 4.1 3.7 3.3 3.5 3.5 4.3 3.2 5.7 6.9 8.1 5.4 6.8 3.6 3.0 3.2 2.5 3.4 4.8 2.6 3.3 2.8 2.7 3.7 4.8 5.3 4.0 3.9 3.5 5.0 5.5 4.8 3.8 3.2 4.0 4.1 5.8 6.1 3.5 4.7 2.9 4.2 2.7 3.9 3.8 2.9 3.9 3.2 3.4 5.0 4.6 3.6 3.8 3.7 3.8 2.9 3.5 7.4 9.9 6.2 6.4 9.8 9.8 7.8 7.4 5.6 7.4 3.0 7.8 3.2 5.0 5.5 3.7 3.3 3.1 3.4 3.4 3.5 3.6 3.8 3.6 2.9 3.5 2.7 3.8 2.9 3.9 3.9 3.7 3.7 3.5 2.8 2.6 3.0 6.4 4.5 3.4 2.8 3.7 3.0 2.8 4.0 3.3 3.7 3.6 3.8 3.7 4.8 4.1 7.8 11 9.7 8.0 10.0 5.4 6.6 6.3 3.5 2.7 5.3 4.8 5.7 5.2 5.4 6.4 3.3 5.3 4.7 4.0 4.9 4.2 4.2 4.6 3.7 4.1 2.6 3.8 2.8 3.9 3.7 3.6 4.0 6.1 4.4 7.3 6.0 8.1 6.2 7.1 7.5 7.6 7.8 6.9 5.7 5.4 5.6 8.8 7.2 8.1 4.9 5.8 3.5 4.7 3.5 4.0 4.9 4.6 4.6 4.0
EMP pMac MΦ
E10
.25
YS
S100a4
S100a8
S100a1
S100a14
S100a6
S100a10 S100a11
S100a9
S100a13
S100a16 4.1 6.4 4.6 2.4 3.0 3.2 3.0 3.6 2.5 3.7 3.9 3.1 4.2 3.4 3.9 4.0 3.6 3.3 6.5 3.9 8.9 9.2 3.7 3.9 3.9 4.2 2.6 2.7 3.1 2.5 3.0 3.2 2.7 3.5 2.9 2.6 3.2 3.2 3.5 3.5 3.8 3.5 3.0 3.4 2.4 3.9 2.7 4.0 3.6 3.8 3.8 3.6 2.7 2.5 3.0 2.4 3.5 3.5 3.7 4.0 7.0 4.0 4.5 4.9 3.7 3.6 5.0 7.9 3.4 3.5 3.5 3.8 3.0 4.4 3.9 3.6 3.7 4.0 2.8 2.5 2.9 2.7 4.0 3.7 2.9 4.2 4.4 3.2 5.2 5.2 3.7 3.7 3.7 7.2 2.8 3.6 2.8 3.7 2.8 3.9 3.8 3.6 3.6 3.8 3.5 5.2 6.1 6.8 4.8 5.4 5.2 3.1 4.7 5.6 6.2 6.2 5.8 5.0 7.6 5.7 4.8 5.8 3.8 5.5 3.7 4.2 3.8 4.2 3.9 3.8 5.6 6.4 6.2 7.0 6.8 6.9 6.5 7.2 6.9 7.0 7.7 9.1 6.5 7.0 8.0 7.0 6.4 7.2 3.4 4.4 3.9 4.4 4.9 4.4 4.7 4.5 6.6 8.0 8.0 6.0 7.0 6.9 6.5 7.6 7.2 6.8 7.3 8.0 5.8 8.2 6.8 6.7 7.1 8.1 6.8 5.8 6.7 7.1 6.6 6.5 7.0 6.0 8.0 9.7 9.5 7.7 7.7 7.6 7.9 9.0 8.0 8.6 9.2 10.5 8.5 9.0 9.7 8.1 8.2 8.5 7.2 4.8 8.6 6.0 8.4 8.3 7.0 8.8 7.0 7.1 7.1 7.5 7.2 7.5 7.9 8.9 8.2 8.2 8.4 8.7 7.9 8.6 7.6 7.7 7.4 7.7 6.3 8.0 7.6 7.7 7.9 7.9 7.2 8.4 6.3 7.0 7.0 7.1 4.9 5.9 6.9 8.1 7.1 7.2 7.1 8.0 8.4 7.3 7.9 7.9 6.8 7.0 6.3 5.8 7.9 5.3 8.6 7.4 7.5 6.5
Mass et al., Fig. S13
Supplementary figure legends Fig. S1: Quality control and analysis of bulk RNA-seq. (A) Number of reliably detected transcripts (covered with a least 25 reads) in each library. The colored circles underneath indicate the batch (sequencing flow cell), time, cell type, and tissue of each sample with the color code as given below panel B. (B) Hierarchical clustering with Euclidean distance and complete linkage of all samples based on their RNA-seq gene expression profiles. The colored circles underneath the cluster dendrogram indicate the batch (sequencing flow cell), time, cell type, and tissue of each sample with the color code as given below. (C) Scorecard analysis of differentially up-regulated genes (DESeq2 Wald test, adjusted p-value<0.05, BH-correction) in early macrophages (E10.25, E10.5) in comparison to EMPs. The table shows the relative enrichment of differentially upregulated genes in macrophages across cell types and tissues (y-axis) and developmental time points (x-axis, from E9 to P21). See Methods for details of the scorecard. (D) Principal component analysis (PCA) plot of EMPs (red, E9-E10.25), pMacs (yellow, E9.5-E10.25) and macrophages (purple, E10.25-E10.5) from the head, caudal, fetal liver (FL) and yolk sac (YS). The shape of each dot indicates the tissue the sample was taken from. The first and second principal component explain 18.9% and 11.1% of the entire variation in the data, respectively. Fig. S2: Quality control and analysis of single-cell RNA-seq. (A) Workflow of the MARS-seq single cell data analysis. (B) Mean-variability plot shows average expression and dispersion for each gene. This analysis was used to determine highly variable genes (labeled by gene symbol). These 138 highly variable genes were used to perform a dimensionality reduction of the single-cell data by a principal component analysis. (C) The highest gene loadings in the first and second principal component from the PCA of 408 high quality cells, colored by batch association, showed even distribution of cells among the PCA plot based on the 138 most highly variable genes. (D) Heatmap of 138 highly variable genes among single-cell clusters as defined by DBScan clustering. (E) Optimal cluster number was identified by calculation of diverse indices for determining the best clustering scheme using the NbClust R package. (F) PCA plot of 408 single cells colored by cluster association. Clusters were defined by PCA + DBScan clustering. (G) Kinetic diagram shows the pseudotemporal ordering of single cells as determined by Monocle 2. Dots indicate individual cells and are colored according to the cluster association as in (F). Black line indicates the progression of single cells over developmental pseudotime. Fig. S3 Expression of surface markers on EMP-derived cells during development. (A) Flow cytometry analysis of E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP (OH-TAM at E8.5) tissues showing expression of Il4ra, Il13ra1, Tnfr2, Ifngr, CD16.2, CD64, Tim4, and CD206 on YFP+ Kit+ progenitors (gray), pMacs (blue) and macrophages (orange). Histograms represent the fluorescence intensity for each antibody in each cell subset. Data are representative of n=4 independent experiments with 4-6 embryos per marker. (B,C) Flow cytometry analysis of Csf1rMeriCreMer; Rosa26LSL-YFP (OH-TAM at E8.5) liver, brain, lung, and skin F4/80+ cells from E14.5 embryos showing expression of Il4ra, Il13ra1, Tnfr2, Ifngr, Dectin-1, CD64, Tim4, and CD206. Black dotted line depicts expression by all macrophages (CD45+CD11blowF4/80high) and green tinted line shows expression by the subsets of YFP+ macrophages. Gray histograms show the fluorescence intensity of the FMO controls for all macrophages (grey line) and YFP+ macrophages (grey tinted line). Data are representative of n=2 independent experiments with 3-4 embryos per marker. Fig. S4 Expression of the core macrophage program on EMP-derived cells. (A) Immunostaining on cryosections from E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP embryos, pulse-labeled with OH-TAM at E8.5 with antibodies against YFP (green), Iba1
(red/cyan), and CD206 (red), Ifngr (red), Tnfr2 (red), Dectin-1 (red), Trem2 (red), CD16/32 (red), Granulin (Grn, red), or F4/80 (cyan). Scale bars represent 10 µm. Data are representative of n=3 embryos for each marker. (B) Whole mount immunostaining of E9.5 Csf1riCre; Rosa26LSL-YFP embryo labeled with antibodies against YFP (green), Iba1 (red), F4/80 (cyan) and DAPI (white). Scale bars represent 10 µm. Data are representative of n=3 embryos. (C) Immunostaining on cryosections from E10.25 Csf1rMeriCreMer; Rosa26LSL-YFP embryo liver, pulse-labeled with OH-TAM at E8.5 with antibodies against YFP (green), Dectin-1 (red) and Iba1 (cyan) (upper panel) or YFP (green), Kit (red) and F4/80 (cyan) (lower panel) Scale bars represent 15 µm. Data are representative of n=4 embryos. (D, E, F) Immunostaining on cryosections from E14.5 (D,F) and E18.5 (E) Csf1rMeriCreMer; Rosa26LSL-YFP mouse embryos stained with antibodies against YFP (green), Iba1 (red) or Grn (red), and F4/80 (cyan). Data are representative of n=3 embryos. Scale bars represent 10 µm. Fig. S5 Analysis of Tnfrsf11aCre; Rosa26LSL-YFP mice. (A) Gating strategy for Tnfrsf11aCre; Rosa26LSL-YFP embryos in E10.25 YS pMacs (Kit- CD45+ F4/80- CD11blow
Gr1- Ter119-; green) and macrophages (CD45+F4/80+CD11blo; blue) (upper panel), and in E14.5 fetal liver LT-HSCs (Lin-Kit+Sca1+CD150+CD48-; orange), ST-HSCs (Lin-
Kit+Sca1+CD150-CD48-; blue) and MPPs (Lin-Kit+Sca1+CD150-CD48+; purple) (lower panel). Histograms represent YFP expression in Tnfrsf11aCre-; Rosa26LSL-YFP (grey) and Tnfrsf11aCre+; Rosa26LSL-YFP (color for cell type indicated in gating strategy). Data are representative of n=2 independent experiments. (B) Immunostaining on cryosection from E14.5 Tnfrsf11aCre; Rosa26LSL-YFP embryo, with antibodies against YFP (green), Iba1 (red) and F4/80 (cyan). Data are representative of n=2 embryos. Scale bars represent 10 µm. Fig. S6 Analysis of Cx3cr1-deficient mice. (A) Immunostaining on cryosection from E10.25 Cx3cr1gfp/+ embryo with antibodies against GFP (green), Iba1 (red) and F4/80 (cyan). Data are representative of n=2 embryos. (B, C, D) Flow cytometry analysis in Cx3cr1+/- and Cx3cr1-/- of F4/80+ macrophages from liver, brain, and limbs at E12.5 (B), liver, brain, limbs, and lung from at E14.5 (C) and liver, brain, limbs, lung, and epidermis from 12 week-old mice (D). Circles represent individual mice from n=7 independent experiments. Fig. S7 Gene set enrichment analysis (GSEA) of cell type-specific expression patterns. (A) GSEA plots illustrating the relative enrichment of genes that we found statistically significantly upregulated (see Methods) in the comparison of macrophage vs. pMac (top row), macrophage vs. EMP (middle row), or pMac vs. EMP (bottom row). The black bars in the middle of each plot indicate the transcripts which are in each respective lists. The order in which transcripts were input into the analysis was defined by the relative expression change (logarithmic fold change) in macrophage vs. pMac (left), macrophage vs. EMP (center), or pMac vs. EMP (right), respectively. (B) The GSEA results from panel A are summarized here by the normalized enrichment score (NES) of each analysis. The colored squares indicate the two cell types compared with the color code as defined at the bottom right of this figure. Asterisks adjacent to bars indicate significance (FDR-corrected p-value); *: q≤0.05, **: q≤0.01, ***: q≤0.001. (C) Comparison of transcripts upregulated in macrophage vs. pMac, macrophage vs. EMP, and pMac vs. EMP, and the core macrophage signature extracted from Gautier et al. (2012). After translating gene denominators to Ensembl transcript IDs, Gautier's list matches 179 transcripts, only 27 of which occur in one of the lists of upregulated genes in this study. (D) GSEA results of Gautier's core macrophage signature genes in the comparisons of macrophage vs. pMac, macrophage vs. EMP, or pMac vs. EMP from this study (same gene rankings used in panels A and B). In all three comparisons, the core macrophage signature is enriched significantly (q≤0.01).
Fig. S8 LOLA enrichment analysis and enhancer regions. (A) Results of a LOLA enrichment analysis of promoter-adjacent regions (TSS +/- 20kb) of upregulated genes during transition from EMPs to pMacs (upper panel) or EMPs to early macrophages (E10.25-E10.5; lower panel). Each dot represents one single ChIP-seq experiment with the size relative to the quantity of enrichment (log odds ratio) and colors indicating the cell type used in the respective experiment. The key below the plots denominates the color coding. The numbers (“x/y”) given behind the cell type specify the number of enriched (x) out of all available datasets (y) from the respective cell type. Shown are all transcription factors of the genomic binding locations, which are significantly enriched (adjusted p-value <0.001, Benjamini Yekutieli correction) in at least one dataset in either comparison. All transcription factors shown are expressed in at least one sample examined, with the exception of Gcgr and Maff (greyed out and marked with an asterisk). (B) Heat map representation of the expression of the transcription factors shown in (A) in EMPs, pMacs and early macrophages (E10.25-E10.5) (C) Genome browser tracks showing ChIP-seq signals for the indicated transcription factors at the Emr1, Cx3cr1, Mrc1, Thbs1, Gata1 and Myod1 loci. The tracks display ChIP-seq data from macrophage populations, except for Maf (T cells), and Irf8 (dendritic cells). Fig. S9 Coordinated changes of gene expression identified during the specification of tissue-resident macrophages. (A) Top panel shows an unsupervised, low-dimensional projection via principal component analysis of the RNA-seq data in this study. The color of each dot indicates the tissue the sample was taken from. Bottom panels show only the macrophage datasets from one tissue at a time (gray dots). The mean of the coordinates at each time point was calculated and indicated as a colored dot (with the same color code as above). Consecutive time points were then connected with arrows to visualize the differentiation trajectory described by the transcriptional profiles of these samples. (B) Line plots illustrating average expression of tissue-specific gene signatures in macrophages over time. Each panel corresponds to one list of differentially upregulated tissue-specific genes (Table S3) and each line to the average of all macrophage samples of one tissue at the given time. Shaded areas indicate +/- standard deviation. (C) Heatmap showing the expression levels of all RNA-seq datasets across all transcript differentially upregulated in any tissue in either stage (early embryonic, fetal, postnatal). Expression values have been scaled as z-scores per row, resulting in a color scheme in which red values represent highly expressed and blue values represent lowly expressed transcripts. Hierarchical Ward clustering with Euclidean distance was used to arrange the samples and genes and the resulting dendrogram was cut at equal height into seven (column-wise) or ten (row-wise) clusters to highlight the strongest clusters of genes and samples more clearly. The color bars on top of the heatmap indicate the time, cell type, and tissue of each sample. Fig. S10 Gene set enrichment analysis (GSEA) of tissue-specific expression patterns in adult macrophages. (A) GSEA plots illustrating the relative enrichment of genes that were found to be statistically significantly upregulated (see Methods) in the comparison to Langerhans cells, microglia, kidney macrophages, Kupffer cells, and alveolar macrophages (from left to right) in comparison to the respective other adult macrophage populations. The black bars in the middle of each plot indicate the transcripts, which are in each respective lists. The order in which transcripts were input into the analysis was defined by the relative expression change (logarithmic fold change) in macrophages in the corresponding tissue (e.g. head/brain for microglia) compared to other macrophages at the same developmental stage (from top to bottom: early, fetal, or postnatal). The colored hashes (#) beneath each plot link to the bars in panel B. (B) The GSEA results from (A) are summarized here by the normalized enrichment score (NES) of each analysis. The colored squares underneath the bar plots indicate the tissue, cell type, and developmental stage of the sample for which
enrichment is shown, following the color code as used in panel C. The bars highlighted in color correspond to the plots shown in panel A. Additionally, these and other bars from samples from a tissue related to the signature at hand are indicated by shading. Asterisks adjacent to bars indicate significance (FDR-corrected p-value); *: q≤0.05, **: q≤0.01, ***: q≤0.001. (C) GSEA results against externally defined gene signatures extracted from Gautier et al. (2012), Gorgani et al. (2008), and Lavin et al. (2014). The same color code, shading, and annotations are used as in panel B. (D) Hierarchical clustering of differentially up-regulated genes (2-fold change, adj. P-value < 0.05, BH-correction) in post-natal (P2-P21) brain, liver, kidney, epidermis or lung macrophages comparing one population vs. the others (see also Table S3). Each sample represents the mean of at least two biological replicates and two technical replicates, except for E14.5 liver macrophages and P8 and P21 lung macrophages, which consist of one biological replicate and two technical replicates. (E) Heatmap representation of the expression of Id1, Id3, Sall1, and Sall3 in EMPs and pMacs from bulk RNA-seq data. Fig. S11 scRNA-seq analysis of specification of tissue-resident macrophages. (A) Workflow for overlaying transcription factor co-expression and tissue macrophage-specific signatures onto the tSNE plots. (B) Normalized expression of Kit, Stab1, Maf, and Cx3cr1 within EMPs, pMacs, and macrophages. (C) Normalized expression of Id1, Id3, and Sall3 within EMPs, pMacs, and macrophages. (D) tSNE plots showing expression of Id1, Id3, and Sall3. (E) Heatmap depicting enrichment of tissue macrophage-specific signatures as in Fig. 6C or differentiation signatures as in Fig. 1E in pMacs on gene clusters defined by multimodal gene expression with subsequent hierarchical clustering of scRNA-seq data. Fig. S12 Role of Id3 in Kupffer cell development (A) Relative expression of Id3 and Nr3h1 transcript by qRT-PCR (normalized to GAPDH) in sorted Kupffer cells and hepatocytes of C57BL/6 mice n=3. (B) Immunostaining with antibodies against YFP (green), ID3 (red) and F4/80 (cyan) on cryosection from adult (4 week-old) liver (upper panel) or brain (lower panel) from a pulse labeled Csf1rMeriCreMer; Rosa26LSL-YFP mouse (OH-TAM at E8.5). Nuclei were counterstained with DAPI (white). Scale bar represents 10 µm. Data are representative of n=3 mice. (C) Immunostaining with antibodies against CD31 and F4/80 on liver cryosections from 4 week-old Id3-/- and Id3+/- mice. The figure displays isovolume-rendered images. Scale bars represent 150 µm for the overview of the adult tissue and 50 µm for insets. Data are representative of n=5 mice. (D) CD31+ area quantification on liver sections from Id3+/- and Id3-/- 4 week-old mice (left panels) or from Tnfrsf11aCre+; Id3+/f and Tnfrsf11aCre+; Id3f/f 2 week-old mice (right panels). Data are representative of n=3 independent experiments. (E) Unsupervised hierarchical clustering on whole transcriptome from Id3+/- and Id3-/- Kupffer cells (Distance metric: Euclidian, linkage rule: Ward’s, number of genes: 18882) from three Id3+/- and two Id3-/- replicates. (F) Significantly enriched Gene Ontology (GO) terms identified for 2-fold up-regulated (left table) or down-regulated (right table) genes in Id3-
/- vs. Id3+/- Kupffer cells enriched by a t-test (P<0.05; FDR<0.05). GO analysis was performed using GeneSpring. GO terms are depicted and raked by corrected P-value (FDR false discovery rate corrected for multiple testing). Fig. S13 Heat map representation of the normalized counts (log2) reads of S100a mRNA found in our dataset.
Supplementary tables (Tables published online in Excel format) Supplementary Table S1: Genes differentially regulated in pMacs and macrophages (E10.25-E10.5) in bulk RNA-seq experiments Supplementary Table S2: Gene signatures derived from bulk RNA-seq experiments used for single cell RNA-seq analysis Supplementary Table S3: Genes differentially regulated in postnatal macrophages (P2-P21) in bulk RNA-seq experiments Supplementary Table S4: antibodies used for Flow cytometry Supplementary Table S5: complete bulk RNA-seq data http://www.biomedical-sequencing.at/bocklab/fhalbrit/macro/data/EC7D8Y7EL5QDMYIOL7GW2SECRWZUN1CWPQCUSJ47FFRO1WHD.zip Supplementary Table S6: RNA-seq analysis on Kupffer cells from Id3KO and control littermates Author contribution. FG, EM designed the study and wrote the manuscript. EM, IB performed cell sorting, flow cytometry, fate-mapping, immunostaining experiments and RNA-seq analysis on Id3-deficicient and control Kupffer cells. LC and CEJG helped with analysis of Tnfrsf11aCre+; Rosa26LSL-YFP mice. EGP assisted with the design of cell sorting experiments. MF designed and prepared bulk RNA-seq libraries. FH, JK, CB performed primary and differential analysis of the bulk RNA-seq data. KH and MB generated single-cell RNA-seq libraries and performed single-cell RNA-seq, PG, MB and JLS analyzed single cell RNA-seq data. All authors contributed to the manuscript.