SUPPLEMENTARY INFORMATION · KLS Colonies 0 5 10 15 20 25 30 %/MNC 0.0005 0.001 0.0015 ......

SUPPLEMENTARY INFORMATION

1www.nature.com/nature

doi: 10.1038/nature08851

Supplementary figure 1: The effect of Dicer1 deletion on osteoblasts and bone in vivo. a,b frequency of lineage-CD45-CD31-

GFP+ osteolineage cells in bone marrow (a) or collagenased bone fragments (b) (n=4) c-e impaired osteogenic differentiation

capacity of OCD fl/fl bone marrow stromal cells as shown by reduced CFU-Alk/CFU-F ratio (c) (n=2, performed in quadruplicate),

impaired in vitro osteogenic differentiation upon osteogenic induction (d) and decreased osteocalcin gene expression in primary

osteolineage cells as shown by RT-PCR on CD45-Lineage-CD31-GFP+ cells (n=5) (e) f, altered texture of the bone matrix g, normal

trabecular bone volume (BV) and h, increased cortical bone volume as assessed by histomorphometric analysis (n=8)(TV=total

volume; BA=bone area, TA total area). i,j reduced osteoblasts (Ob) number per bone surface (Bs) in OCD fl/fl mice as indicated by

histomorphometric analysis (n=8) k, the number of osteoclasts (OC) was unaltered in OCD fl/fl mice (n=8). Data are mean ± s.e.m. *

p≤0.05, **p≤0.01. CFU-F=colony-forming assay –fibroblast, CFU-ALK=colony forming unit alkaline phosphatase, AP=alkaline

phosphatas

i

00.10.20.30.40.50.60.70.80.9

* *

OCD fl/flOCD fl/+

CFU

Alk

/CFU

-F

c

g

0

5

10

15

20BV/TV

%

OCD fl/flOCD fl/+0

0.2

0.4

0.6

0.8

1Osteocalcin

Cop

ies

OC

N/c

opy

GA

PD

H

*

e

OCD fl/flOCD fl/+

i

0

5

10

15

20

25

30

35Ob/Bs

#/m

m

**

OCD fl/flOCD fl/+

h

0

10

20

30

BA/TA

%

**

OCD fl/flOCD fl/+

0

0.01

0.02

0.03

0.04

% o

f CD

45- c

ells

OCD fl/flOCD fl/+0

0.05

0.1

0.15

0.2%

of C

D45

-ce

lls

OCD fl/flOCD fl/+

0

0.5

1

1.5

2

2.5Oc/Bs

#/m

m

k

OCD fl/flOCD fl/+

f

j

OCD fl/+

OCD fl/fl

d APDay 0 Day 7

OCD fl/fl

OCD fl/+

AlizarinRedba

20 um

10 um

10 um

CFU

-Alk

/CFU

-F


doi: 10.1038/nature08851 SUPPLEMENTARY INFORMATION

Supplementary figure 2: Ineffective hematopoiesis and myelodysplasia in OCD fl/fl mice. a, Leukopenia comprised all leukocyte subsets (n=10). Cytopenias were present despite normal or increased cellularity of the bone marrow (b,c) and normal hematopoietic stem and progenitor cell numbers and function. d,e, frequency of immunophenotypically defined stem (LKS CD150+CD48-) and progenitor (LKS) cells (n=4). f, hematopoietic stem cell function as determined by competitive reconstitution capacity (n=5) g, hematopoietic progenitor cell function as assessed by CFU-C (n=10). h, increased frequency of (CD11b+Gr1+) myeloid cells (n=6) and i, decreased frequency of (CD43+B220+CD19+/-) B-cell progenitors in the bone marrow (n=5) j, increased bone marrow vascularity confirmed by the endothelial cell marker CD31 (PECAM). Inset: CD31 staining of micro-megakaryocytes. Data are mean ± s.e.m. * p≤0.05, **p≤0.01. LY=lymphocytes, BASO=basophils, NE=neuthrophils, MO=monocytes, EO=eosinophils, CFU-C=colony forming unit in culture. LKS= lineage –C-kit+ Sca1+ cells LKS-SLAM= lineage –C-kit+ Sca1+ CD150+ CD48- cells, H&E=hematoxylin/eosin.

05

101520253035404550

CD43+B220+

OCD fl/+

OCD fl/fl

%/M

NC

CD43 B220+-B220+

**

**

**

** * H&E

CD31

100 um

OCD fl/fl

OCD fl/+

H&E

fl/fl

fl/+

0

20

40

60

80

Col

onie

s

CFU-C

OCDfl/+ OCDfl/fl

g

i

010

20

30

40

50

60

70

% C

D11

b+G

r1+

cells

/MN

C

**

OCDfl/+ OCDfl/fl

h

0

0.00005

0.0001

0.00015 LKS/CD150+/CD48-

%/M

NC

OCDfl/flOCDfl/+

d

F

0

0.0005

0.001 LKS

%/M

NC

OCDfl/flOCDfl/+

e

0

2

4

6

8

OCD fl/fl

OCD fl/+

Dic

er (C

D45

.2)/C

ompe

titor

(C

D45

.1)

2 weeks 24 weeks16 weeks8 weeks4 weeks

*

*

****

Competitive reconstitutionf

j

bc

50 um

OCD fl/fl

OCD fl/+10 um

50 um

a

0

1

2

3

4

5

6

7

Leukocytes

NELYMOEOBASO

**

**

**

103 /µ

l OCD fl/+

OCD fl/fl

CD43+B220+

CD19+ CD19

Cellularity

0

0.5

1

OCDfl/flOCDfl/+C

ells

(10

6 )/fe

mur

/gr

(total)


SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature08851

Supplementary figure 3: Extramedullary hematopoiesis rescues peripheral cytopenia in a subfraction of OCD fl/fl mice. Extramedullary hematopoiesis (EMH) was observed in a subset of 3/10 animals investigated for its presence as indicated by a,

histology showing increased hematopoiesis with increased numbers of magakaryocytes. Megakaryocytes display normal morphology b,increased CFU-C spleen, spleen cellularity , relative frequency of erythroid progenitor (CD45+ter119+) cells and

primitive hematopoietic (LKS) progenitors (n=3). c,d, significant anemia and thrombocytopenia in OCD fl/fl mice lacking EMH (n=7) in

comparison to Dicer fl/+ littermates. Conversely, the occurrence of EMH (n=3) rescued anemia and thrombocytopenia in these mice.

Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

b

d

0

100

200

300

400

500

600

700 Platelets

EMHNO EMH

OCDfl/+

*

103 /

µl

OCDfl/fl

0

2

4

6

8

10

12

14 RBC

NO EMH EMH

*

106 /

µl

OCDfl/fl

OCDfl/+

a

0

10

20

CFU-C Spleen

30

40

50Spleen Cellularity

CD45+/Ter119+

0

KLS

Col

onie

s

0

5

1015

20

2530

%/M

NC

0.0005

0.001

0.0015

0.002

0.0025

%/M

NC

OCDfl/+

OCDfl/fl

00.20.40.60.81

1.21.4

Cel

ls(

106 )

/gr

b.w

.

OCD fl/+ OCD fl/fl

OCD fl/+ OCD fl/fl c

25 um

100 um 5 um



Supplementary figure 4: Representative example of flow-cytometric assessment of primitive hematopoietic LKS/CD150/CD48 subpopulations

CD150

0

102

103

104

105

CD

48

38.125.3

30.4

6.09

Sca-1

0102

103

104

105

c-Ki

t

1.45

Lineage

0

5000

10K

15K

20K

# C

ells

2.31

0 50K 100K 150K 200K 250KFSC

0

50K

100K

150K

200K

250K

SSC

46.5

0

102

103

104

10564.8

10.1

20.34.810

10K

20K

30K

3.68

0

102

103

104

105

1.16

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

68.2

OCD fl/+

OCD fl/fl0 101 102 103 104

0 101 102 103 1040 101 102 103 104

0 101 102 103 104 0 101 102 103 1040 101 102 103 104



Supplementary figure 5: Myelodysplastic features of OCD fl/fl mice. a, neutrophilic dysplasia in peripheral blood. Arrow indicating giant platelet. b, micro-megakaryocytes with hyperchromatic nuclei. c, frequency of dysplastic cells (n=12 mice, ≥ 20 cells counted/sample; average ± sem, range 15.0%-89.5% and 6.5%-92.0% for neutrophils and megakaryocytes respectively)

a b

OCD fl/+

OCD fl/+

OCD fl/fl

OCD fl/fl

c

0

25

50

75

100

%

dysp

last

ic

Neutrophils Megakaryocytes

5 um

5 um



Supplementary figure 6: Apoptosis in hematopoietic progenitor cells in OCD fl/fl mice: representative FACS plots.

LKS= lineage –C-kit+ Sca1+ cells LKS-SLAM= lineage –C-kit+ Sca1+ CD150+ CD48- cells L-K+= lineage-c-kit+ cells L-K-int=lineage-

Ckit intermediate

OCD fl/+

OCD fl/fl

OCD fl/+

OCD fl/fl

0 102 103 104 105

0102

103

104

105

0 50K 100K150K 200K 250K0

50K

100K

150K

200K

250K

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

FSC

SSC Lineage

Lin -

Sca 1

C-

kit

CD150

CD

48

LKSL - k+

L - kit - int

LKS - SLAMC-

kit

0 50K 100K150K 200K 250K0

50K

100K

150K

200K

250K

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

2 3 4 5

0102

103

104

105

15.8

0 102 103 104 105

0102

103

104

105

13.113.1

0 102 103 104 105

0102

103

104

105

11.711.7

0 102 103 104 105

0102

103

104

105

14.814.8

Annexin V

7AAD

LKS - SLAM LKS L - k+ L - kit - int

0 102 103 104 105

010

2

103

104

105

34.034.0

0 102 103 104 105

010

2

103

104

105

25.725.7

0 102 103 104 105

0102

103

104

5

24.7

2 3 4 5

2

3

4

10 5

24.7

0 102 103 104 105

0102

103

104

105

25.1

2 3 4 5

2

3

4

5

25.1

10

0 10 10 10 10



Supplementary figure 7: Apoptosis in hematopoietic progenitor subsets in OCD fl/fl mice.

a. Representative FACS plots of progenitor identification and apoptosis rates, b. overall data (n=4). Data are mean ± s.e.m. * p≤0.05,

**p≤0.01.

OCD fl/+

b

a

0

10

20

30

40

50

Anne

xin

V+ 7

AAD

CMP GMP MEP

OCD fl/flOCD OCD fl/+

**

0 102 103 104 105

0102

103

104

105

MEP CMP

GMP

0 10 10 10 10

0102

103

104

105

MEP CMP

GMP

Lin- ckit+CD127- cells

CD34

CD

16/3

2

OCD fl/fl

0 10 2 10 3 10 4 10 5

0102

103

104

105

MEP CMP

GMP

CD34

CD

16/3

2

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0

102

103

104

105CMP GMPMEP

Annexin V

7AAD

9.19 26.1 9.24

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0

102

103

104

105CMP GMPMEP

Annexin V

7AAD

11.4 12.942.4

Lin- ckit+CD127- cells



Supplementary figure 8: Increased proliferation of hematopoietic stem and progenitor cells at the endosteal surface in OCD fl/fl mice. a, representative FACS histogram of BRDU+ cells by in vivo BRDU labeling b, confocal microscopy of transplanted DiD-

labeled LKS cells. (osteoblasts green, vasculature red, LKS cells (arrows) white) c, distance to endosteal surface (n=2, 20±3.6

cells/animal) and d, frequency of cell duplets. Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

OCD fl/+OCD fl/+

OCD fl/flOCD fl/fl

0

10

20

30

40

0/32

17/75

10

20

30

40

0/32

% o

f eve

nts

OCDfl /flOCDfl /+

17/75

0

10

20

30

**

µm

OCD fl/+

OCD fl/fl

b c

d

aLKS/CD150+CD48-

# C

ells

# C

ells

# C

ells

# C

ells

# C

ells

# C

ells

OCD fl/+

OCD fl/fl

2.42.21

21.27.37

BRDU

Cel

ls

BRDU

BRDU

Cel

ls

OCD fl/+

OCD fl/fl

LKS#

Cel

ls#

Cel

ls#

Cel

ls#

Cel

ls#

Cel

ls#

Cel

ls

OCD fl/+

OCD fl/fl

2.42.21

21.27.37

BRDU

Cel

ls

BRDU

BRDU

Cel

ls

OCD fl/+

OCD fl/fl

0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5

0 10 2 10 3 10 4 10 50 10 2 10 3 10 4 10 5

Distance to Endosteal Surface



Supplementary figure 9: Representative example of flow-cytometric assessment of B-cell subpopulations

0 101 102 103 104B220

0

101

102

103

104

CD

43

76.9 4.01

11.3

0 200 400 600 800 1000FSC

0

200

400

600

800

1000

SSC

37.8

0 101 102 103 104CD19

0

101

102

103

104

CD

43

61.1

0 101 102 103 1040

101

102

103

10439.9

0 101 102 103 1040

101

102

103

10463.9 6.63

23.5

0 200 400 600 800 10000

200

400

600

800

1000

65.9

OCD fl/fl

OCD fl/+



Supplementary figure 10: Hematological abnormalities in OCD fl/fl mice cannot be propagated in a hematopoietic cells autonomous manner. Bone marrow mononuclear cells of OCD fl/fl or littermate OCD fl/+ mice (n=2) were transplanted into lethally

irradiated WT (B6.SJL) mice (n=4 per OCD mouse). a, near complete donor chimerism 16 weeks post- transplant with normalization

of b, peripheral blood numbers, and c, apoptosis of primitive progenitors. Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

% a

nnex

inV+

/ 7

AAD

-

% a

nnex

inV+

/ 7

AAD

-

c

OCD fl/+ OCD fl/fl0

10

20

30

40

50

60

0

10

20

30

40

50

60

OCD fl/+ OCD fl/fl

OCD fl/fl

a

0

20

40

60

80

100

% C

D45

.2 d

onor

cel

ls in

PB

OCD fl/+

OCD fl/+024681012

106 /

µl

Platelet

010203040506070

RBC

024681012

106 /µ

l10

4 /µl

Platelet

010203040506070

104 /µ

l

Platelets

RBC

OCD fl/+OCD fl/fl

b Platelets

RBC

OCD fl/fl

OCD fl/flOCD fl/flOCD fl/+ OCD fl/+

OCD fl/fl



Supplementary figure 11: Hematopoietic cells from OCD fl/fl mice do not confer hematological abnormalities: Data of mutant

into WT transplantation at 12 months post-transplant. a, bone marrow mononuclear cells of OCD fl/fl (n=4) or littermate OCD fl/+ mice

(n=6) were transplanted into lethally irradiated WT (B6.SJL) mice. b, near complete donor chimerism c, peripheral blood numbers, d, granulocyte and megakaryocyte morphology, e, frequency of B cells and B cell progenitors. f, frequency of myeloid cells g,

apoptosis of primitive progenitors. Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

a b

cd

e f

g

OCD fl/flOCD fl/+

0

20

40

60

%An

nexi

nV+

7AA

D - Lineage- c-kit+

0

20

40

60

% G

r1+C

D11

b+ c

ells

/MN

C

OCD fl/+ OCD fl/fl

0

2

4

6

8

10

X106

/ul

OCD fl/+ OCD fl/fl

RBC

0

2

4

6

8 WBC

OCD fl/+ OCD fl/fl

X103

/ul

OCD fl/fl

OCD fl/+

B220 total

CD43+B220+ CD43+B220+ CD19 -

0

5

10

15

20

25

30

%/M

NC

0

20

40

60

80

100

OCD fl/flOCD fl/+

%C

D45

.2 O

CD

OCD fl/+ OCD fl/fl0

200

400

600

800

1000

X103

/ul

platelets

DONORS

B6.SJL (45.1)

OCD fl/+

OCD fl/fl (45.2)

(45.2)

DONORS

OCD fl/fl

f

5 um

5 um



Supplementary figure 12: Myelodysplasia in OCD fl/fl mice is induced by the bone marrow microenvironment. Wildtype

(CD45.1) B6.SJL cells were transplanted into OCD fl/+ (n=8) or OCD fl/fl (n=8) mice. a,b, hematopoiesis at 8 weeks was predominantly

of WT origin but c, dysplayed prominent dysplastic features (n=4; 40 neutrophils or megakaryocytes were scored per sample) d,e, anemia and thrombopenia f, increased frequency of myeloid cells in the bone marrow with g, reduced frequency of B-cells and B-cell

progenitors and h, increased apoptosis of hematopoietic progenitors. Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

OCD fl/flOCD fl/+

% C

D45

.1 W

T do

nor

cells

in B

M

0

20

40

60

80

100a

d

e

OCD fl/+ OCD fl/fl

1

6

8

0

12

X106 /

ul

P=0.07

P=0.06

0

200

400

600

800

1000

X103 /

ul

OCD fl/+ OCD fl/fl

RBC

Platelets

%C

D11

b+G

r1+

cells

/MN

C

0

20

40

60

80

100 **f

OCD fl/flOCD fl/+

Anne

xin

V /

7AAD

-

0

10

20

30

40**

Lineage-,c-kit+ cells

h0

20

40

% M

NC

* *

* *

B 220 total BCD43+/B220+ BCD43+/B220+CD19-

gOCD fl/flOCD fl/+

b

0

20

40

60

80

100

Megakaryocytes(CD41+)

Neutrophils

CD

45.1

WT

(%/to

tal) c

0Megakaryocytes

(CD41+)Neutrophils

20

40

60

80

100

Dys

plas

ia (

%/to

tal)

OCD fl/flOCD fl/+



Supplementary figure 13: Co-culture assays reveal direct effects of OCD fl/fl osteolineage cells on hematopoietic progenitor cell proliferation and megakaryocytic differentiation

Two hundred DS-red LKS or MEP were co-cultured on bone marrow derived stromal cells for 7 days as described in the section

„methods‟ a, osterix and osteocalcin gene expression in stromal cells in A-MEM indicating osteolineage commitment in the absence

of terminally differentiated (osteocalcin+) osteoblasts b, The total number of hematopoietic cells was significantly increased in LKS

co-cultured with OCD fl/fl stroma with c, preferential expansion of ckit+lin- cells in the absence of decreased apoptosis in this fraction

(d) consistent with increased proliferation of primitive hematopoietic subsets on OCD fl/fl stroma. Plating of MEPs on OCD fl/fl stromal

cells resulted in increased numbers of hematopoietic cells (e) but significantly impaired differentiation towards (CD41+)

megakaryocytes (f,g). Apoptosis was not increased in hematopoietic subsets under these conditions (h) i, representative picture of

morphological abnormalities of CD41+ megakaryocytic cells showing small megakaryocytes with condensed, hypolobular nuclei on

OCD fl/fl stroma. (Data show representative experiments with n=2 mice, experiments performed at least in triplicate; data are mean ±

s.e.m. * p≤0.05, **p≤0.01). ND=not detectable, AMEM=minimal essential medium alpha

0

500

1000

1500

2000

2500

3000

Num

ber

of

DSr

edce

lls LKS **

0

0.005

Osx Ocn

Cop

ies/

copy

GAP

DH

ND

Osx Ocn

ND

a b c

02468

1012141618

cKit+

lin-(

% d

sRed

Cel

ls)

*d

0

400

800

1200

1600

Nr.

Of D

S-r

ed c

ells

MEP **e

29.6

3.53

DS -red

CD

41

29.6

3.53

f

0

5

10

15

20

25 LKS

ckit+lin - ckit+lin + ckit - lin +

% A

nnex

inV

+

h

0

10

20

30

40

50

60 MEP

ckit+CD41- ckit+CD41+ ckit -CD41+

OCD fl/+

OCD fl/fl

OCD fl/+

OCD fl/fl

**

%

Anne

xin

V +

dsRed CD41-FITC DAPI

OCD fl/fl

OCD fl/+i

-

g

0

5

10

15

20

25

30

cKit+CD41 -%

of D

Sre

d ce

llscKit+CD41+ cKit -CD41+

**

**

g

OCD fl/flOCD fl/+

OCD fl/flOCD fl/+

OCD fl/flOCD fl/+

25 um

OCD fl/fl

OCD fl/fl

OCD fl/fl

OCD fl/+

OCD fl/+

OCD fl/+



Supplementary figure 14: Deletion of Dicer1 in terminally differentiated osteoblasts does not confer myelodysplasia. a, deletion of Dicer1 in long bones and calvarium of ocn-cre+Dicer fl/+ and fl/fl animals. b, alteration in texture of bone matrix and, c, increased cortical bone volume (n=2) . Effects on hematopoiesis were examined in 4-6 weeks old animals showing d, no effect on

peripheral blood cell numbers, e, peripheral blood cells morphology, f, bone marrow vasculature or megakaryocyte morphology g, frequency of myeloid cells and h, B-cell progenitors. n=5. Data are mean ± s.e.m.

a

c

b

d

e

f

0

5

10Leukocytes

Ocn-Dfl/+ 0

5

10

15RBC

0

500

1000Platelets

103 /

µl

Ocn-Dfl/+Ocn-Dfl/+ Ocn-Dfl/fl

0

20

40

60

% G

r1+ C

D11

b+ce

lls/M

NC

Ocn-Dfl/+ Ocn-Dfl/fl

Ocn-Dfl/fl

103 /

µl

Ocn-Dfl/fl

0

20

40

B220+ CD43+B220+ CD43+B220+CD19-

%/M

NC

Ocn-Dfl/fl

Ocn-Dfl/+

106 /

µl

OCD fl/+Ocn-Dfl/+

Ocn-Dfl/fl

Ocn-Dfl/+

Ocn-Dfl/+ Ocn-Dfl/+

Ocn-Dfl/flOcn-Dfl/fl

Osteocalcin-cre:Dicer1

Δ Dicer1309 bp

LiverFemur CalvariumOcn-Dfl/+ Ocn-Dfl/+Ocn-Dfl/+ Ocn-Dfl/flOcn-Dfl/flOcn-Dfl/fl

Ocn-Cre-Dicer fl/+Ocn-Cre-Dicer fl/fl

e

f

g h

0

0.1

0.2

0.3

0

0.1

0.2

0.3

Ocn-Cre-Dicer fl/+ Ocn-Cre-Dicer fl/fl

BA

/TA

25 um

25 um

5 um

100 um



Supplementary figure 15: Hematopoietic phenotype of OCD fl/fl mice at the age of three weeks. a, anemia, b, dysplasia of

neutrophils and megakaryocytes, c, decreased frequency of B-cell progenitors, d, increased frequency of myeloid cells, e, Increased

apoptosis of hematopoietic progenitor cells. (n=5 for all analyses). Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

OCD fl/fl OCD fl/+a b

c d

e

5

5.5

6

6.5

7

7.5

8 RBC

OCD fl/+ OCD fl/fl

*

x106

/ ul

0

10

20

30

40

50

60

OCD fl/fl

OCD fl/+

B220 total

CD43+B220+ CD43+B220+ CD19 -

**

**

%/M

NC

0

10

20

30

40

50

60

OCD fl/flOCD fl/+

**

% G

r1+C

D11

b+ c

ells

/MN

C

0

24

68

1012

14

OCD fl/flOCD fl/+

*

%An

nexi

nV+

7AA

D-

Lineage- ckit+

5 um

20 um



a

b

c

Supplementary figure 16: Genomic location and probe information on cytogenetic abnormalities in myeloid sarcomas as detected by CGH a, tumor1 b, tumor2 c, tumor3



Supplementary figure 17: Infiltrative tumor behavior a, infiltration of tumor into muscle (a1) and glandular tissue (a2).

Tumor

25 um

100 um



Supplementary figure 18: Targeted deletion of the Sbds gene from osteoprogenitor cells

a, altered texture of the cortical bone similar to OCD fl/fl mice, b, reduced frequency of B cells and pre-B cells in the bone marrow

(n=8) c, increased frequency of myeloid cells in the bone marrow (n=8). Data are mean ± s.e.m. * p≤0.05, **p≤0.01.

c

0

20

40

60

**

OCS fl /+ OCS fl /fl

%C

D11

b+G

r1+

cells

/MN

C

0

20

40

60

80

% M

NC

B220 (total) CD43+B220+ CD43+B220+/CD19-

**

**

OCS fl/+

OCS fl/fl

bOCS fl /fl

aOCS fl /+



Supplementary tables

Supplementary table 1: Significantly (p<0.05 (t-test) up (> 2-fold) - and down regulated (>2-fold) genes in OCD fl/fl in comparison to OCD fl/+ osteolineage cells

Gene name Description T-test p-value

Fold Change Status

ESF1 2610101J03RIK 0.036 5.42682 up

PRG2 PRG2:proteoglycan 2, bone marrow (natural killer cell activator, eosinophil granule major basic protein)

0.04697 4.69371 up

FABP6 FABP6:fatty acid binding protein 6, ileal (gastrotropin) 0.02693 4.16036 up

SLC26A6 SLC26A6:solute carrier family 26, member 6 0.02134 3.83104 up

AA591059 na 0.04541 3.76227 up

TUBA4 TUBA4:tubulin, alpha 4 0.03639 3.27486 up

C430042M11RIK na 0.02185 3.23017 up

CRTAC1 CRTAC1:cartilage acidic protein 1 0.0473 3.09731 up

GP49A /// LILRB4 na 0.04749 2.75924 up

D3ERTD108E na 0.03166 2.70639 up

ZFP397 na 0.0182 2.68709 up

COL4A4 COL4A4:collagen, type IV, alpha 4 0.05033 2.60923 up

KIF13B KIF13B:kinesin family member 13B 0.0291 2.60515 up

SLC7A2 SLC7A2:solute carrier family 7 (cationic amino acid transporter, y+ system), member 2

0.04061 2.5453 up

2310007H09RIK na 0.00718 2.49706 up

4930441O14RIK na 0.05163 2.49114 up

CML3 na 0.0004 2.4063 up

ITSN2 ITSN2:intersectin 2 0.05006 2.3858 up

4930579C12RIK na 0.05379 2.35791 up

WRN WRN:Werner syndrome 0.03028 2.30526 up

TEX19 na 0.05461 2.30136 up

ARL6IP2 ARL6IP2:ADP-ribosylation factor-like 6 interacting protein 2 0.04964 2.29179 up

LOC548102 na 0.05069 2.27919 up

CUZD1 CUZD1:CUB and zona pellucida-like domains 1 0.02772 2.24676 up

RABGAP1 RABGAP1:RAB GTPase activating protein 1 0.04486 2.21999 up

SMEK1 1110034C04RIK 0.03913 2.19501 up

MLSTD1 MLSTD1:male sterility domain containing 1 0.01118 2.18289 up

SYT1 SYT1:synaptotagmin I 0.03581 2.1705 up

2700022O18RIK na 0.03128 2.15625 up

LSAMP LSAMP:limbic system-associated membrane protein 0.0478 2.14451 up

IL2RB IL2RB:interleukin 2 receptor, beta 0.01355 2.13979 up

AI844685 na 0.01838 2.13573 up



RAD23B RAD23B:RAD23 homolog B (S. cerevisiae) 0.02611 2.1244 up

STX19 A030009B12RIK 0.01625 2.12178 up

9430081G06RIK na 0.0154 2.12048 up

1810033B17RIK na 0.004 2.1112 up

DHPS DHPS:deoxyhypusine synthase 0.03132 2.10313 up

1700034F02RIK na 0.01501 2.05275 up

DMT2 na 0.01204 2.05086 up

PCDHB16 PCDHB16:protocadherin beta 16 0.01963 2.04084 up

PMPCB PMPCB:peptidase (mitochondrial processing) beta 0.05089 2.04056 up

RETNLA na 0.003 2.03683 up

SCN1A SCN1A:sodium channel, voltage-gated, type I, alpha 0.01654 2.03638 up

BC035295 na 0.003 10.8597 down

8430408J09RIK na 0.00004 7.43661 down

WDR32 WDR32:WD repeat domain 32 0.05195 6.5184 down

LEO1 LEO1:Leo1, Paf1/RNA polymerase II complex component, homolog (S. cerevisiae)

0.02355 6.06723 down

MICAL2 MICAL2:microtubule associated monoxygenase, calponin and LIM domain containing 2

0.00827 5.95856 down

AI413194 na 0.00001 5.94452 down

AW742931 na 0.03429 5.34601 down

B3GALTL B3GALTL:beta 1,3-galactosyltransferase-like 0.01187 5.23733 down

ZFP408 na 0.04506 5.1633 down

2410018G20RIK na 0.01427 5.11795 down

DFNA5H na 0.05083 5.09683 down

AA408296 na 0.04759 5.07906 down

UACA UACA:uveal autoantigen with coiled-coil domains and ankyrin repeats

0.02384 4.96892 down

IRAK1BP1 IRAK1BP1:interleukin-1 receptor-associated kinase 1 binding protein 1

0.005 4.87327 down

0610037P05RIK na 0.00738 4.86756 down

BC054438 na 0.001 4.82693 down

MGA MGA:MAX gene associated 0.02833 4.66628 down

CYGB CYGB:cytoglobin 0.03967 4.59107 down

JAG1 JAG1:jagged 1 (Alagille syndrome) 0.03387 4.58492 down

ADM ADM:adrenomedullin 0.05096 4.57084 down

TASP1 TASP1:taspase, threonine aspartase, 1 0.04421 4.49225 down

D15WSU126E na 0.04732 4.49113 down

ICA1 ICA1:islet cell autoantigen 1, 69kDa 0.00802 4.39416 down

MRPS35 MRPS35:mitochondrial ribosomal protein S35 0.02985 4.34184 down

GCC1 GCC1:GRIP and coiled-coil domain containing 1 0.01746 4.30058 down

AI450540 na 0.0093 4.24837 down

HS2ST1 HS2ST1:heparan sulfate 2-O-sulfotransferase 1 0.02254 4.24774 down



SHF SHF:Src homology 2 domain containing F 0.004 4.09982 down

GLOD4 2700085E05RIK 0.04616 4.06067 down

NR2F6 NR2F6:nuclear receptor subfamily 2, group F, member 6 0.0285 4.03937 down

RUSC2 RUSC2:RUN and SH3 domain containing 2 0.01736 4.03404 down

TIPRL TIPRL:TIP41, TOR signalling pathway regulator-like (S. cerevisiae)

0.0255 4.03344 down

BTBD5 BTBD5:BTB (POZ) domain containing 5 0.0146 4.02652 down

EIF2C2 EIF2C2:eukaryotic translation initiation factor 2C, 2 0.0503 3.89805 down

NFIA NFIA:nuclear factor I/A 0.0445 3.89446 down

PROS1 PROS1:protein S (alpha) 0.02681 3.83777 down

MAP4K3 /// LOC675560

na 0.0438 3.82749 down

LDB2 LDB2:LIM domain binding 2 0.002 3.79608 down

RBM19 RBM19:RNA binding motif protein 19 0.0466 3.78786 down

MCMDC1 MCMDC1:minichromosome maintenance deficient domain containing 1

0.01879 3.74637 down

SMO SMO:smoothened homolog (Drosophila) 0.001 3.72639 down

DDX24 DDX24:DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 0.02098 3.69234 down

PLAC9 PLAC9:placenta-specific 9 0.01527 3.65417 down

2610014I16RIK na 0.04731 3.64898 down

GLT28D2 na 0.04701 3.61421 down

NAGLU NAGLU:N-acetylglucosaminidase, alpha- (Sanfilippo disease IIIB) 0.01324 3.60552 down

SLC10A3 SLC10A3:solute carrier family 10 (sodium/bile acid cotransporter family), member 3

0.02873 3.59452 down

NFATC2IP NFATC2IP:nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 interacting protein

0.05147 3.59288 down

MCOLN1 MCOLN1:mucolipin 1 0.02559 3.59093 down

FBXO18 FBXO18:F-box protein, helicase, 18 0.05395 3.57977 down

HOXA10 HOXA10:homeobox A10 0.01077 3.57055 down

DNAJA4 DNAJA4:DnaJ (Hsp40) homolog, subfamily A, member 4 0.05295 3.56585 down

9230104K21RIK na 0.04956 3.56259 down

TMEM16K TMEM16K:transmembrane protein 16K 0.04528 3.55635 down

ENOPH1 2310057D15RIK 0.04873 3.54497 down

ABHD14B ABHD14B:abhydrolase domain containing 14B 0.03567 3.52646 down

NINJ1 NINJ1:ninjurin 1 0.004 3.5251 down

SMAD3 SMAD3:SMAD, mothers against DPP homolog 3 (Drosophila) 0.0405 3.50195 down

TRABD 5730502D15RIK 0.03846 3.4988 down

YIPF2 YIPF2:Yip1 domain family, member 2 0.0275 3.48257 down

MTIF2 MTIF2:mitochondrial translational initiation factor 2 0.0486 3.46648 down

METTL7A /// UBIE /// na 0.05451 3.46179 down

ISLR ISLR:immunoglobulin superfamily containing leucine-rich repeat 0.001 3.45555 down



ACYP1 ACYP1:acylphosphatase 1, erythrocyte (common) type 0.03783 3.43518 down

FBXL3 FBXL3:F-box and leucine-rich repeat protein 3 0.05226 3.40165 down

HNRPH3 /// LOC669773

na 0.02436 3.39531 down

PQLC3 PQLC3:PQ loop repeat containing 3 0.0431 3.3691 down

PALLD PALLD:palladin, cytoskeletal associated protein 0.04649 3.36097 down

5330431N19RIK na 0.03947 3.33596 down

ALCAM ALCAM:activated leukocyte cell adhesion molecule 0.01269 3.33139 down

RG9MTD2 RG9MTD2:RNA (guanine-9-) methyltransferase domain containing 2

0.04216 3.32609 down

LRRC17 LRRC17:leucine rich repeat containing 17 0.02977 3.24375 down

EIF2AK1 EIF2AK1:eukaryotic translation initiation factor 2-alpha kinase 1 0.01791 3.23972 down

EXTL2 EXTL2:exostoses (multiple)-like 2 0.03894 3.22632 down

CRTC3 CRTC3:CREB regulated transcription coactivator 3 0.0159 3.21783 down

PAOX PAOX:polyamine oxidase (exo-N4-amino) 0.00757 3.20999 down

HMBOX1 HMBOX1:homeobox containing 1 0.046 3.19186 down

GTF3C2 GTF3C2:general transcription factor IIIC, polypeptide 2, beta 110kDa

0.02639 3.19022 down

NEK8 NEK8:NIMA (never in mitosis gene a)- related kinase 8 0.02814 3.17498 down

BNIP1 BNIP1:BCL2/adenovirus E1B 19kDa interacting protein 1 0.04001 3.13605 down

RFX5 RFX5:regulatory factor X, 5 (influences HLA class II expression) 0.005 3.13276 down

KNS2 KNS2:kinesin 2 0.01367 3.12315 down

SORBS2 SORBS2:sorbin and SH3 domain containing 2 0.01624 3.12002 down

COL5A3 COL5A3:collagen, type V, alpha 3 0.03875 3.11783 down

EXT1 EXT1:exostoses (multiple) 1 0.04278 3.07762 down

1600002K03RIK na 0.02141 3.0713 down

CLIP3 1500005P14RIK /// AL 0.05159 3.06071 down

POLR3C POLR3C:polymerase (RNA) III (DNA directed) polypeptide C (62kD)

0.0366 3.05909 down

TBCC TBCC:tubulin-specific chaperone c 0.03781 3.04434 down

ERC1 ERC1:ELKS/RAB6-interacting/CAST family member 1 0.04342 3.04195 down

TRIP6 TRIP6:thyroid hormone receptor interactor 6 0.04633 3.04116 down

HSBP1 HSBP1:heat shock factor binding protein 1 0.04248 3.01409 down

PPP2CB PPP2CB:protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform

0.0088 2.98921 down

PANK3 PANK3:pantothenate kinase 3 0.01014 2.97531 down

TMEM50A TMEM50A:transmembrane protein 50A 0.03696 2.95722 down

AV025504 na 0.00837 2.95663 down

WDFY2 WDFY2:WD repeat and FYVE domain containing 2 0.03629 2.9552 down

DDR2 DDR2:discoidin domain receptor family, member 2 0.03946 2.95262 down

ZFP618 /// LOC667396 na 0.00761 2.94734 down



4632411B12RIK na 0.0169 2.94262 down

PTX3 PTX3:pentraxin-related gene, rapidly induced by IL-1 beta 0.03079 2.92064 down

BC017612 na 0.05424 2.89922 down

ATG4C ATG4C:ATG4 autophagy related 4 homolog C (S. cerevisiae) 0.00837 2.89476 down

RBM43 0610033I05RIK 0.04051 2.87449 down

RBM42 3100004P22RIK /// 23 0.03254 2.87222 down

ALOX12 ALOX12:arachidonate 12-lipoxygenase 0.01951 2.86592 down

NCOA5 NCOA5:nuclear receptor coactivator 5 0.01723 2.86006 down

WNK4 WNK4:WNK lysine deficient protein kinase 4 0.04324 2.85777 down

PGP 1700012G19RIK 0.02712 2.84566 down

MYO18A MYO18A:myosin XVIIIA 0.02934 2.84086 down

CYB5R3 CYB5R3:cytochrome b5 reductase 3 0.02474 2.83949 down

AA536749 na 0.03247 2.8289 down

BC005624 na 0.03948 2.82785 down

IPO9 IPO9:importin 9 0.03469 2.81871 down

EXOSC7 EXOSC7:exosome component 7 0.03156 2.81313 down

AP3S1 AP3S1:adaptor-related protein complex 3, sigma 1 subunit 0.05259 2.8084 down

OXNAD1 OXNAD1:oxidoreductase NAD-binding domain containing 1 0.05392 2.80498 down

RRAS RRAS:related RAS viral (r-ras) oncogene homolog 0.03705 2.79071 down

2610002F03RIK na 0.04775 2.78836 down

ZCCHC14 ZCCHC14:zinc finger, CCHC domain containing 14 0.0329 2.77777 down

STS STS:steroid sulfatase (microsomal), arylsulfatase C, isozyme S 0.00883 2.76881 down

RCOR3 RCOR3:REST corepressor 3 0.02713 2.74565 down

ST3GAL2 ST3GAL2:ST3 beta-galactoside alpha-2,3-sialyltransferase 2 0.01937 2.7313 down

HPS4 HPS4:Hermansky-Pudlak syndrome 4 0.04004 2.68525 down

1110005A03RIK na 0.01849 2.68281 down

D930050J11 na 0.01927 2.67308 down

SLC45A4 SLC45A4:solute carrier family 45, member 4 0.04853 2.67167 down

PANK1 PANK1:pantothenate kinase 1 0.04199 2.67069 down

CCDC84 CCDC84:coiled-coil domain containing 84 0.03089 2.66777 down

SAPS1 SAPS1:SAPS domain family, member 1 0.04673 2.64874 down

POMT1 POMT1:protein-O-mannosyltransferase 1 0.04783 2.64598 down

PSD3 4931420C21RIK 0.03173 2.64312 down

REXO1 REXO1:REX1, RNA exonuclease 1 homolog (S. cerevisiae) 0.02204 2.63142 down

MLLT1 MLLT1:myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 1

0.00544 2.62691 down

CARM1 CARM1:coactivator-associated arginine methyltransferase 1 0.04902 2.60999 down

PBXIP1 PBXIP1:pre-B-cell leukemia transcription factor interacting protein 1

0.04893 2.60147 down



ANTXR2 ANTXR2:anthrax toxin receptor 2 0.04345 2.59669 down

PARG PARG:poly (ADP-ribose) glycohydrolase 0.004 2.59156 down

3110082I17RIK na 0.03931 2.5905 down

2700097O09RIK na 0.003 2.57518 down

2510003E04RIK na 0.04721 2.57024 down

MED25 MED25:mediator of RNA polymerase II transcription, subunit 25 homolog (S. cerevisiae)

0.0162 2.56425 down

FKBP1B FKBP1B:FK506 binding protein 1B, 12.6 kDa 0.04954 2.5559 down

C030013C21RIK na 0.01692 2.55466 down

METAPL1 na 0.04655 2.53018 down

PDPK1 PDPK1:3-phosphoinositide dependent protein kinase-1 0.05226 2.52208 down

A130038J17RIK na 0.04504 2.50464 down

PXMP3 PXMP3:peroxisomal membrane protein 3, 35kDa (Zellweger syndrome)

0.03348 2.496 down

ZFAND2B ZFAND2B:zinc finger, AN1-type domain 2B 0.045459 2.493982 down

5230400M03RIK na 0.034336 2.483538 down

2410006H16RIK na 0.034442 2.476582 down

B230216N24RIK na 0.008545 2.472954 down

ENO3 ENO3:enolase 3 (beta, muscle) 0.010466 2.472312 down

CNNM2 CNNM2:cyclin M2 0.023387 2.453917 down

WHSC2 WHSC2:Wolf-Hirschhorn syndrome candidate 2 0.052867 2.451304 down

2610507B11RIK na 0.042471 2.451252 down

4930471M23RIK na 0.045363 2.445177 down

PREPL PREPL:prolyl endopeptidase-like 0.040585 2.443074 down

COQ6 COQ6:coenzyme Q6 homolog, monooxygenase (S. cerevisiae) 0.036103 2.430283 down

SBDS SBDS:Shwachman-Bodian-Diamond syndrome 0.027655 2.430248 down

C920006C10RIK na 0.040843 2.429177 down

ZFAND3 ZFAND3:zinc finger, AN1-type domain 3 0.051068 2.411616 down

5830472M02RIK na 0.022833 2.407413 down

ACADVL ACADVL:acyl-Coenzyme A dehydrogenase, very long chain 0.051178 2.377887 down

5330421F07RIK na 0.031995 2.370213 down

TCF7L2 TCF7L2:transcription factor 7-like 2 (T-cell specific, HMG-box) 0.017742 2.366551 down

C1R C1R:complement component 1, r subcomponent 0.034414 2.365563 down

PVRL2 PVRL2:poliovirus receptor-related 2 (herpesvirus entry mediator B) 0.016279 2.364646 down

UBE2G1 UBE2G1:ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, yeast) 0.052156 2.364552 down

0610037M15RIK na 0.030055 2.359723 down

TOM1L2 TOM1L2:target of myb1-like 2 (chicken) 0.042417 2.357986 down



4930453N24RIK na 0.012838 2.333928 down

SKI SKI:v-ski sarcoma viral oncogene homolog (avian) 0.032083 2.333383 down

BC048355 na 0.021395 2.328441 down

PPCDC PPCDC:phosphopantothenoylcysteine decarboxylase 0.002518 2.31653 down

SKAP2 SKAP2:src kinase associated phosphoprotein 2 0.048548 2.316329 down

LOC619719 na 0.031426 2.310286 down

TPD52L1 TPD52L1:tumor protein D52-like 1 0.052966 2.303352 down

1810048P08RIK na 0.038413 2.295201 down

COMMD4 COMMD4:COMM domain containing 4 0.043682 2.292841 down

HEXA HEXA:hexosaminidase A (alpha polypeptide) 0.041359 2.285667 down

SLC44A1 SLC44A1:solute carrier family 44, member 1 0.020211 2.259007 down

3010001K23RIK na 0.038181 2.242436 down

FLI1 FLI1:Friend leukemia virus integration 1 0.006129 2.224242 down

DENND1A DENND1A:DENN/MADD domain containing 1A 0.010247 2.210609 down

TRIP10 TRIP10:thyroid hormone receptor interactor 10 0.022136 2.206315 down

RBL1 RBL1:retinoblastoma-like 1 (p107) 0.029564 2.190662 down

PHF2 PHF2:PHD finger protein 2 0.04758 2.187651 down

FZD1 FZD1:frizzled homolog 1 (Drosophila) 0.035345 2.181633 down

TMEM141 TMEM141:transmembrane protein 141 0.019349 2.166158 down

C030040A22RIK na 0.041693 2.147752 down

2810439F02RIK na 0.041809 2.136875 down

NME7 NME7:non-metastatic cells 7, protein expressed in (nucleoside-diphosphate kinase) 0.037948 2.135189 down

INPP5A INPP5A:inositol polyphosphate-5-phosphatase, 40kDa 0.050672 2.131062 down

IFT172 IFT172:intraflagellar transport 172 homolog (Chlamydomonas) 0.025341 2.111549 down

RNF19 RNF19:ring finger protein 19 0.042536 2.09939 down

E130303B06RIK na 0.034074 2.098219 down

SGTB SGTB:small glutamine-rich tetratricopeptide repeat (TPR)-containing, beta 0.040253 2.09804 down

MUM1 MUM1:melanoma associated antigen (mutated) 1 0.026382 2.092568 down

NCBP2 NCBP2:nuclear cap binding protein subunit 2, 20kDa 0.032454 2.089116 down

ARMCX1 ARMCX1:armadillo repeat containing, X-linked 1 0.037973 2.081989 down

SPHK1 SPHK1:sphingosine kinase 1 0.007448 2.076194 down

MTA3 MTA3:metastasis associated 1 family, member 3 0.005851 2.073348 down

C530028I08RIK na 0.015106 2.062652 down

6330403L08RIK na 0.047894 2.062485 down

IFI47 na 0.030068 2.056686 down

ARHGAP5 ARHGAP5:Rho GTPase activating protein 5 0.02651 2.05092 down



NUDCD3 NUDCD3:NudC domain containing 3 0.047587 2.048661 down

GAG na 0.040636 2.043669 down

D8BWG1414E na 0.04943 2.03895 down

PPP1R12B PPP1R12B:protein phosphatase 1, regulatory (inhibitor) subunit 12B 0.026326 2.020668 down

D15WSU169E na 0.028883 2.010003 down

ODZ4 ODZ4:odz, odd Oz/ten-m homolog 4 (Drosophila) 0.043972 2.004228 down



Supplementary table 2: GSEA: Gene sets significantly enriched in OCDfl/+ osteolineage cells

PATHWAY

NAME SIZE NES NOM p-value

FDR q-value

Osteogenic differentiation1

UP_GENES_OSTEOGENIC 43 1.60 0 0.000

TGF-β TGFBETA_EARLY_UP 45 2.578963 0 0.000

TGF_BETA_SIGNALING_PATHWAY 46 2.433784 0 0.008223

TGFBETA_ALL_UP 75 2.432784 0 0.006167

Wnt/βcatenin AMBROSETTI_UP-WNT ACTIVATION 54 1.797488 0 0.001945

KENNY_WNT_DN 39 1.757821 0 0.032086

ST_WNT_BETA_CATENIN_PATHWAY 30 1.699135 0 0.041116

UV UVC_LOW_ALL_DN 49 2.407943 0 0.004112

UVB_NHEK3_C5 33 2.405828 0 0.003524

UVB_NHEK1_DN 233 2.254912 0 0.005181

UVC_TTD_ALL_DN 319 2.227531 0 0.009852

Gene Sets from:

1 Schilling,T. et al., Microarray analyses of transdifferentiated mesenchymal stem cells, J. Cell Biochem. 103(2), 413 (2008).

2 Ambrosetti, D. et al., Fibroblast Growth Factor Signaling Uses Multiple Mechanisms To Inhibit Wnt-Induced Transcription in Osteoblasts. Molecular and Cellular Biology, 28 (15), 4759 (2008) Details of other gene sets can be found at http://www.broad.mit.edu/gsea/



Supplementary table 3: Cytokines, interleukins, growth factors and other secreted factors differentially expressed in OCD fl/fl osteolineage cells (>1.5-fold; t-test<0.05)

Symbol Name t-test Fold-difference

PF4 platelet factor 4 0.0006 1.58

NPTX2 neuronal pentraxin II 0.004 1.61

IL31 interleukin 31 0.005 1.78

CCK cholecystokinin 0.039 1.71

OLFM4 olfactomedin 4 0.039 1.94

PRG2 proteoglycan 2, bone marrow 0.046 4.69

CRTAC1 cartilage acidic protein 1 0.047 3.10

STC1 stanniocalcin 1 0.048 1.54

COL4A4 collagen, type IV, alpha 4 0.050 2.61

Symbol Name t-test Fold-difference

PLAC9 placenta-specific 9 0.015 3.65

PROS1 protein S (alpha) 0.027 3.84

JAG1 jagged 1 (Alagille syndrome)

0.034 4.58

COL5A3 collagen, type V, alpha 3 0.039 3.12

BAI1 brain-specific angiogenesis inhibitor 1

0.045 1.61

ADM adrenomedullin 0.050 4.57



Supplementary methods and materials

Mice and genotyping

Osx-Cre transgenic mice9, Ocn-Cre transgenic mice20 and floxed Dicer1 mice10,

have been described. B6.SJL-Ptprca Pep3b/BoyJ mice were purchased from the

Jackson Laboratory. Floxed Dicer1 mice were on a mixed C57/B6/J129

background. Other mice strains were on a C57/B6 background. Genotyping of

Cre transgenic mice was performed by PCR using primers detecting the Cre

sequence36. The floxed and wild-type Dicer1 alleles were detected by using

primers, P1: 5′-AGTGTAGCCTTAGCCATTTGC-3′ and P2: 5′-

CTGGTGGCTTGAGGACAAGAC-3′. These primers amplify the region spanning

the downstream loxP sequence. Deletion of the floxed sequence from the Dicer1

gene was demonstrated by using primers: P1: 28290: 5'-

AGTAATGTGAGCAATAGTCCCAG-3' and P2: 32050AS: 5'-

CTGGTGGCTTGAGGACAAGAC-3' OCD fl/fl animals were compared to OCD fl/+

littermates for the studies described in this paper. The Subcommittee on

Research Animal Care of the Massachusetts General Hospital approved all

animal work according to federal and institutional policies and regulations.

RT-PCR

RNA extraction, real-time quantitative RT-PCR and relative gene expression

quantitation was performed on sorted cells (GFP+CD45-CD31-Lineage –) as

described previously37 using the following primers: Dicer1-F, 5′-

AATTGGCTTCCTCCTGGTTAT-3′ and Dicer1-R,

GTCAGGTCCTCCTCCTCCTC-3′; Osteocalcin-F, 5′-

CTGACCTCACAGATCCCAAGC -3′ and Osteocalcin-R, 5′-

TGGTCTGATAGCTCGTCACAAG -3′;. GAPDH-F, 5′-

AGGTCGGTGTGAACGGATTTG -3′ GAPDH-R, 5′-

TGTAGACCATGTAGTTGAGGTCA -3′



Isolation and osteogenic differentiation of bone marrow derived stromal cells

Mice were sacrificed; tibiae, femurs, and spine were removed and excess soft

tissue was eliminated. Using a pestle and mortar, the bones were crushed and

washed in PBS with 0.5% FBS and passed through a 40-μm filter into a

collection tube. Cells were spun at 1500rpm for 5 minutes; the supernatant was

removed, and cells were resuspended in a minimal volume of ACK lysing buffer

(Cambrex) for 4 minutes on ice and washed once with PBS. After pelleting once

again, the cells were resuspended and plated in αMEM, 20% fetal bovine serum

(HyClone), and penicillin and streptomycin solution (CellGro) - henceforth

referred to as αMEM20%- and incubated at 33°C with 5% CO2. After 3 weeks of

culture and expansion, plastic adherent cells were CD45 depleted by magnetic

isolation (Invitrogen; Dynabeads M-280 Streptavidin, 112-06D) using an anti-

mouse CD45 biotin antibody (BD Bioscience; 550539). The plastic adherent

CD45 negative cells were then maintained in αMEM20% as before. To assess

osteogenic differentiation, bone marrow derived stromal cells (passage 3) were

plated at 10 × 103 cells/well in a 96-well plate (BD Biosciences) at 33° in

osteogenic induction medium: α20% modified with glycerol 2-phosphate (2.16

mg/ml), 2-phospho-L-ascorbic acid (0.05 mg/ml), and dexamethasone (10 nM)

(Sigma-Aldrich, G6251, 49752 and D1756, respectively ). After 7 days of

differentiation, alkaline phosphatase staining was carried out with BCIP/NBT

solution (Sigma-Aldrich) per the manufacturer’s instructions . For the von Kossa

assay and staining, cells were fixed and washed in water, and a 5% silver nitrate

solution was added to the well under incandescent light for 20–45 minutes. After

granules developed, the silver nitrate was removed and wells were washed with

water to stop the reaction.

CFU-F and CFU-ALK

0.5 × 106 primary bone marrow cells were plated in 12-well plates in αMEM20%

for CFU-F assay or osteogenic medium for CFU-Alk assay. Medium was

changed at 24 hours to eliminate nonadherent cells. After 7 days, colonies were



assessed by methylene blue staining for the CFU-F assay or BCIP staining

(alkaline phosphatase) for CFU-Alk.

Histomorphometric analysis

Bones were fixed in 4% paraformaldehyde and undecalcified sections embedded

in methyl methacrylate resin. Five-micrometer sections were stained with Masson

Trichrome or coverslipped unstained, and histomorphometric analysis was

performed with the Osteomeasure system (Osteometrics Inc., Atlanta, GA) using

standard procedures. Tibial sections were measured in the proximal metaphysis

beginning 340 µm below the chondro-osseous junction. Osteoblasts were

identified as mononuclear cells directly abutting either mineralized bone or

osteoid and restricted to the endosteal surface.

In Situ Hybridization.

In situ hybridization was carried out as described36. Complementary 35S-labeled

riboprobes were transcribed from the plasmids encoding mouse osteocalcin (OC)

using Riboprobe systems from Promega (Madison, WI). Probes for Osteocalcin

were described36.

Hematological Measurements

Peripheral blood samples were obtained by lateral tail vein bleeding. Peripheral

blood cell counts were performed on a HEMAVET Multispecies Hematology

Analyzer (CDC Technologies).

Methylcellulose colony formation assay

Bone marrow or spleen cells (10X103) were plated into methylcellulose M3434

(StemCell Technologies) in a 6-well plate and grown for 10 days before being

scored.

FACS analysis



Hematopoietic progenitors were identified based on their expression of lineage

markers as well as c-Kit, Sca-1, CD48 and CD150 expression. Lineage staining

used a cocktail of biotinylated antimouse antibodies to Mac-1α (CD11b), Gr-1(Ly-

6G and Ly-6C), Ter119 (Ly-76), CD3, CD4, CD8a (Ly-2), and B220 (CD45R; BD

Biosciences). For detection we used lineage-streptavidin conjugated with

PERCP, c-Kit-APC (CD117), CD48-pacific blue (CD135), CD150-PE-Cy7 (all

from BD Biosciences) and Sca1-PE-Cy5.5 (Ly 6A/E; Caltag Laboratories). For

congenic strain discrimination, anti-CD45.1-PE and anti-CD45.2 FITC antibodies

(BD Biosciences) were used. For the apoptosis assay we used 7-AAD and

AnnexinV-APC (BD Biosciences) in combination with lineage-streptavidin-PE and

c-kit-FITC (both Biolegend). For the intracellular detection of BRDU-FITC, bone

marrow cells were fixed and permeabilized using BD Cytofix/Cytoperm

Fixation/Permeabilization Solution Kit (BD Biosciences) according to the

manufacturer's recommendations. Compensation and data analysis were

performed using Flowjo 8.5.3

FACS- sorting of osteolineage cells

Whole bone-marrow and bone cells were collected by crushing tibias and femurs

of mice, stained with biotin-conjugated lineage cocktail antibodies and subjected

to lineage depletion using magnetic isolation (Invitrogen; Dynabeads M-280

Streptavidin, 112-06D). The resulting lineage-depleted fraction was stained with

lineage and CD31-biotin-streptavidin APC-Cy7 (BD Biosciences) and CD45-APC

(eBioscience) and sorted using FACS DiVa or FACS ARIA (Becton Dickinson). A

small fraction of the collected cells was re-run through the sorter and over 95%

purity was consistently confirmed.

Collagenase treatment of bone for PCR

Collagenase digestion was preformed on the bone fragments left in the mortar

and 70-μm filter after crushing long bones. A solution of DMEM (Cellgro; 10-013-

CV), 0.2% collagenase (WAKO; 034-10533) and 10mM HEPES (Fisher; BP299-

100) was warmed to 37°C. In a centrifuge tube, bone fragments were added to



the collagenase solution and kept at 37°C for 90 minutes, vortexing every 15-30

minutes. Excess PBS was added to the slurry which was then filtered through a

40-μm filter. The flow-through was then pelleted.

Bone marrow histology and peripheral blood morphology

For histological analysis, long bones were dissected, fixed in paraformaldehyde

4%, decalcified in 10% EDTA, paraffin-processed, cut, and subjected to

hematoxylin/eosin staining. Peripheral blood smears were formalin fixed for 5

minutes, stained with May-Grunwald ( Sigma-Aldrich) for 5 minutes, rinsed in

distilled water with PBS and in Giemsa stain (Sigma-Aldrich) for another 30

minutes. Permount (Fisher Scientific) was used to mount the sections. Images

were acquired with a Nikon Eclipse 80i epifluorescence microscope equipped

with a Qimaging Micropublisher digital CCD colour camera. Bone and bone

marrow histology was assessed by two independent investigators blinded to mice

genotypes.

Bone marrow transplantation

All bone marrow transplantations were performed by retro-orbital venous plexus

injection. For competitive transplantation, 5 × 105 whole bone-marrow cells from

6-week-old OCD fl/+ or OCD fl/fl (CD45.2) littermates were mixed with 5 × 105

CD45.1+ (competitor) WT cells and injected into lethally irradiated (9 Gy, split

dose on the day of transplant) recipient BL6-SJL (CD45.1+) mice. Engraftment

efficiency in recipients was monitored by donor contribution of CD45.2+ cells

using FACS analysis. For limiting dilution assays, 2 × 105, 5 × 104 1 × 104 and

0.5 X 104 OCD fl/+ or OCD fl/fl mononuclear bone marrow cells were mixed with 2

× 105 wild type bone marrow and injected into lethally irradiated recipients (9

mice per cell dose per genotype). Engraftment efficiency in recipients was

monitored by donor contribution of cells using FACS analysis. The frequencies of

competitive repopulating units were calculated using the L-Calc software. Greater

than or equal to 1% donor cells in both myeloid and lymphoid lineages was used



to determine whether an animal had a positive engraftment. For “ wt into mutant”

experiments, wildtype congenic BL6/SJL (CD45.1+) bone marrow cells (1 x 106

cells/recipient) were transplanted into lethally irradiated 4 week old OCD fl/+ and

OCD fl/fl (CD45.2+) recipients. Complete donor cell engraftment by wildtype

CD45.1+ cells was confirmed by FACS. Conversely, for “mutant into wt”

experiments OCD fl/+ or OCD fl/fl (CD45.2+) were transplanted into lethally

irradiated 4 week old BL6/SJL (CD45.1+) animals. Complete donor cell

engraftment by CD45.1/CD45.2+ cells was confirmed by FACS.

Immunohistochemistry

For immunohistochemistry, antigen retrieval was carried out with proteinase K

(20 mg/ml, Roche), followed by 3% H2O2 treatment to block endogenous

peroxidase. The TSA Biotin system (PerkinElmer) was used according to the

manufacturer's instructions. Specimens were incubated with mouse anti-CD31

antibody (BD Biosciences) or anti-CD13 antibody (Santa Cruz Biotechnology) for

1 hr at room temperature.

BrdU Labeling and Detection.

Mice received 150 l BrdU solution (10 mg ml-1) via intraperitoneal injection. After

15 hrs bone marrow was harvested for flow cytometric detection of BrdU-FITC

uptake according to or the manufacture’s instructions (FITC-BrdU Flow Kit (BD

Biosciences).

In vivo imaging

In vivo imaging has been extensively described elsewhere14. Briefly, 1–5 105

wild-type (Bl6/SJL) LKS cells were stained with 5 M DiD in PBS without serum for

10 min at 37 °C, washed once in PBS and immediately injected into the tail vein

of recipient mice. Mice were anaesthetized and prepared for in vivo imaging as

described. Immediately before imaging 20 l of non-targeted Qdot 800 or 655

(Invitrogen) diluted in 130 l sterile PBS was injected retro-orbitally to allow

vasculature visualization. All mice were imaged with a custom-built confocal two-



photon hybrid microscope specifically designed for live animal imaging.

Microscopy and image processing have been described. Images were colored

and merged using Adobe Photoshop and LKS-microenvironment distance

measures were obtained using Adobe Illustrator and Microsoft Excel. A two-tailed

type 2 t-test was applied to all data. P values 0.05 were considered statistically

significant.

Co-culture studies

Bone marrow stromal cells were isolated and CD45 depleted by magnetic

isolation upon confluence and expanded for an additional week. The expanded

cells were then plated at 1750 cells/well in 384-well tissue culture plates coated

with fibronectin (Millipore; FC010) in either αMEM20% or osteogenic induction

media. After four days of culture, 200 LKS or MEP (megakaryocyte-erythroid

progenitor, lineage-, CD 127-, Sca-,kit+, CD34-,CD16/32- cells.) cells from 8-12

week old Actin-DsRed positive mice (Jackson Laboratory; 005441) were added

to each well. Co-culture was performed without any cytokines. After 7 days of co-

culture the number of DS-red cells was assessed by automated microscopy.

Megakaryocytes were quantified morphologically as large cells with prominent

multinucleated megakaryons (the identity of these cells was additionally

confirmed by CD41 staining).

Comparative genomic hybrydization

Direct amplification of DNA from paraformaldehyde fixed paraffin-embedded

(FFPE) tissue samples was performed using a REPLI-g FFPE kit (Qiagen)

following the manufactory instruction. Briefly, FFPE samples were incubated at

95C for 10 min followed by lysis at 60C for 60 min and ligation at 24C for 30 min.

Amplification took place at 30C for 2h. Agilent genomic DNA labeling kit was

used for the amplified FFPE DNA labeling and purification. For each 244K array,

2 ug of FFPE DNA and 2 ug of germline reference DNA were labeled with Cy5

and Cy3 respectively. Labeled FFPE DNA and reference DNA were combined

and mixed with Cot-1 DNA, blocking agent and hybridization buffer. After



denaturation at 95C for 3 min and incubation at 37C for 30 min, the hybridization

mix was loaded onto a gasket slide in a Agilent Surehyb chamber. Array slide

was placed on top the gasket slide. The SureHyb chamber was covered,

clamped and incubated in a rotator rack in 65C oven for 40 hour. In an ozone-

controlled environment, hybridized arrays were disassembled, and washed in

Agilent Oligo aCGH wash buffer 1 for 5 min, in wash buffer 2 for 1 min at 37C,

and immediately scanned using an Agilent DNA microarray scanner. Data

extraction was conducted using the feature extraction software. Finally FE data

files were analyzed using the Agilent DNA analytics software.

Oligonucleotide microarrays

RNA was isolated from sorted GFP+CD45-CD31-Lineage- cells by Trizol

extraction (Invitrogen) according to the manufacturer’s protocol. Up to 4 mice

were pooled per sample. Linear amplification of 20 ng of total RNA was

performed using the Ovation Biotin RNA Amplification and Labeling System

(Nugen). The biotinylated cRNA was hybridized to the Affymetrix Mouse430 v2

chip. Signal normalization was performed by RMA method. Data of three

samples of OCD fl/fl cells vs. three samples of OCD fl/+ cells was analyzed using

GEPAS package38. A t-test was carried to identify probes differentially expressed

between OCD fl/+ and OCD fl/fl samples. Gene set enrichment analysis was

performed using GSEA39. The signal-to-noise metric and permutation of gene

sets was used to rank the genes and calculate significance and false discovery

rate. Analysis was performed by collapsing probe sets to unique gene symbols

and used to interrogate an established collection of curated gene sets provided

by the Molecular Signatures Database (MsigDB,

http://broad.mit.edu/gsea/msigdb). The osteogenic gene expression signature

was collected from the literature21 and used to interrogate the gene expression

dataset comparing OCD fl/+ and OCD fl/fl samples for gene set enrichment.

Statistical analysis



In all cases, analysis was performed by a standard unpaired, 2-tailed Student’s t

test. All data have been plotted as average ± SEM. Statistical significance is

indicated by * (P≤0.05) or ** (p≤0.01). The number of experiments is indicated in

the figure legends.

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denaturation at 95C for 3 min and incubation at 37C for 30 min, the hybridization

mix was loaded onto a gasket slide in a Agilent Surehyb chamber. Array slide

was placed on top the gasket slide. The SureHyb chamber was covered,

clamped and incubated in a rotator rack in 65C oven for 40 hour. In an ozone-

controlled environment, hybridized arrays were disassembled, and washed in

Agilent Oligo aCGH wash buffer 1 for 5 min, in wash buffer 2 for 1 min at 37C,

and immediately scanned using an Agilent DNA microarray scanner. Data

extraction was conducted using the feature extraction software. Finally FE data

files were analyzed using the Agilent DNA analytics software.

Oligonucleotide microarrays

RNA was isolated from sorted GFP+CD45-CD31-Lineage- cells by Trizol

extraction (Invitrogen) according to the manufacturer’s protocol. Up to 4 mice

were pooled per sample. Linear amplification of 20 ng of total RNA was

performed using the Ovation Biotin RNA Amplification and Labeling System

(Nugen). The biotinylated cRNA was hybridized to the Affymetrix Mouse430 v2

chip. Signal normalization was performed by RMA method. Data of three

samples of OCD fl/fl cells vs. three samples of OCD fl/+ cells was analyzed using

GEPAS package38. A t-test was carried to identify probes differentially expressed

between OCD fl/+ and OCD fl/fl samples. Gene set enrichment analysis was

performed using GSEA39. The signal-to-noise metric and permutation of gene

sets was used to rank the genes and calculate significance and false discovery

rate. Analysis was performed by collapsing probe sets to unique gene symbols

and used to interrogate an established collection of curated gene sets provided

by the Molecular Signatures Database (MsigDB,

http://broad.mit.edu/gsea/msigdb). The osteogenic gene expression signature

was collected from the literature21 and used to interrogate the gene expression

dataset comparing OCD fl/+ and OCD fl/fl samples for gene set enrichment.

Statistical analysis

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