- Email: [email protected]
The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification
Developmental Cell
Erratum
The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification Annie Moisan, Miguel N. Rivera, Sutada Lotinun, Sara Akhavanfard, Erik J. Coffman, Edward B. Cook, Svetlana Stoykova, Siddhartha Mukherjee, Jesse A. Schoonmaker, Alexa Burger, Woo Jae Kim, Henry M. Kronenberg, Roland Baron, Daniel A. Haber,* and Nabeel Bardeesy* *Correspondence: [email protected] (D.A.H.), [email protected] (N.B.) DOI 10.1016/j.devcel.2012.04.017
Developmental Cell 20, 583–596; May 17, 2011 In the course of compiling all the data for this manuscript and preparing composite figures, some confusion arose in labeling and presenting some of the data. In some cases, the similarities between controls and wild-type (e.g., Figures 2A and S3B) made it difficult to distinguish the correct image. In other cases, images were inadvertently swapped or inverted between analogous experiments that showed very similar results (e.g., Figures 4B and 5E). In total, eight subpanels of the main figures were affected, as well as five subpanels of the supplemental material. None of these changes alter the conclusions of the manuscript in any way. A joint review of these errors, conducted by Harvard Medical School and Massachusetts General Hospital, did not find any evidence of research misconduct to warrant further investigation. A complete listing of the affected panels is therefore included below, along with the corrected panels (shown in the context of the entire figures to which they belong). We most sincerely apologize for the confusion that our errors have caused. The corrected panels in the document are: From the main text: Figures 2A (the two leftmost panels for Col2a-Cre mice), 4A (labeling of the nonspecific band), 4B, 5A, 5C, 5E, 5F, and 8A. From the supplement: Figures S3B, S6A, S6C, S7B, and S8C (bottom panel for Ocn-Cre). For clarification, we also include a listing of panels that are unchanged but in which a common control is present in two figures; these reflect cases in which the primary data from the same experiment are divided into two figures. Figure 2A, second panel for Prx-Cre ribs, also in Figure 6C. Figure 4B, bottom-left panels for ‘‘shControl adipo,’’ also in Figure 5E. Figure 5A, panels for ‘‘osteo day 4,’’ also in Figure 8A. Figure 7A, third panels from left for P0 calvaria, also in Figure S8C. Figure S6C, actin panels at bottom, also in Figure S7B.
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1109
Developmental Cell Erratum
Wtx+
Wtx Lox
Wtx+
Wtx Lox
Wtx Lox
Wtx+
Prx1+ multipotent progenitor
Prx1-Cre
Col2a+ chondrocyte precursor
Col2a-Cre
A
*
Ocn-Cre
Osx+ osteoblast precursor Ocn+ differentiated osteoblast
B
Wtx Lox
Wtx+
Wtx+
D
Prx1-Cre
Prx1-Cre
*
25 20 15 10 5 0
500 400 300 200
40 30 20 10
100
40 20
60
30 20
100
10
0
0
**
*
60
0
40
192.2 ± 8.7
80
3.5
50
83.1 ± 9.8
120
0
**
MS/BS (%)
BFR/TV (%/year)
700 600
***
50
Tb.Th (μm)
BV/TV (%)
***
*
3 2.5 2 1.5 1
E
Wtx+
Wtx Lox
N.S.
Prx1-Cre
60
35
*
*
Prx1-Cre: Wtx Lox
N.Ob/T.Ar (/mm2)
Prx1-Cre: Wtx+
MAR (μm/day)
C
30
Wtx Lox
0.5 0
276.3 ± 21.4
103.8 ± 21.7
Figure 2. Targeted Inactivation of Wtx in Mesenchymal Progenitors Causes Osteosclerosis and Impaired Adipogenesis
1110 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Developmental Cell
GFP
C
Runx2 n.s.
Relative expression
adipo Wtx
osteo Wtx
Wtx
shRNA:
GFP
ni
A
GFP
Erratum
adipogenic
250
shGFP shWtx
200 150 100 50
*
0
Klf5
shControl
B
shWtx
shGFP
Cebpb Cebpd
eGFP
D
shGFP
shWtx
*
*
Pparg Cebpa Fabp4
PPAR-γ
shWtx
shGFP
shWtx
day 6
day 12
Oil Red-O
ni
adipo
shGFP
shRunx2
ST2 cells
day 6
adipo
osteo
ALP
day 6
ALP
WT
PC
osteo
adipo
Oil Red-O
E
KO
F
P0 femur WT
PC
KO
Wtx HC
HC
Runx2
Runx2
Osterix Sox9 Ppar-γ Actin
600
Wtx+ Wtx -
*
400
*
*
*
200
*
*
C
N
P O
sx
AL
O
2
un
1a ol
ol
1a C
C
x2
0 1
HC
neonates Relative expresison
Osterix
HC
G
R
PC
PC
Figure 4. Wtx Acts Upstream of Runx2 and Pparg to Control Osteogenic and Adipogenic Fate Determination
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1111
Developmental Cell Erratum
A
B
C
E
D
F
G
Figure 5. Wtx-Mediated Control of b-Catenin Levels in BMPCs Determines Osteoblast and Adipocyte Specification
1112 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Developmental Cell Erratum
day 8 + Lox
MC3T3-E1 day 10 + Lox
ABC β-cat
Wtx+ Wtx Lox
2500
Relative Expression
Wtx
day 4 + Lox
Prx1-Cre BMPC
B
Prx1-Cre BMPC
sh G FP sh W tx
A
Actin
2000 1500 1000 500 0
Axin2
Tcf1
Axin2
ni shβ-catenin
shControl
C ni
osteo
ni
MeBIO
D ni
osteo
Tcf1
osteo day 10 BIO
osteo
ni
osteo
E Wtx
Wtx promotes
Wtx
Wtx inhibits
Associated with destabilization of β-catenin
adipocyte cchondrocyte precursor (Col2a+)
Wtx
mesenchymal progenitor (Prx1+)
bipotential progenitor Wtx
chondrocyte
osteoblast bl
osteoblast precursor (Osx+) Wtx
mature osteoblast (Ocn+)
Wtx
Figure 8. Wtx Inactivation Impairs Osteoblast Maturation Independent of b-Catenin Deregulation
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1113
Developmental Cell Erratum
A
Gata1-Cre Wtx +
Prx1-Cre Wtx +
Wtx Lox
Col2a1-Cre
Wtx Lox
CB
Wtx Lox
CB
C
Osx-Cre
D
Osx-Cre
Osx-Cre
Wtx Lox
Wtx+
Wtx +
Ocn-Cre Wtx +
Wtx Lox
Wtx+
B
Wtx +
Wtx Lox
Wtx Lox
CB
Osx-Cre: Wtx+
E 14
40
**
12
70
*
35 30
10
40 30
150
20
10
100
5
10
50
0
0
0
0
Y
2
Wtx Lox/Y
/Y tx -
tx W
W
Lo
x/
F
**
200
15
4
350
250
20
6
400
300
50
25
8
*
60
Osx-Cre: Wtx Lox
Gata1
Prx1
Osx
Col2a
Ocn
Lox band null band
1
2
3
4
5
6
7
8
9
10
11 12
Figure S3. Skeletal Phenotypes in Wtx-Deficient Mice
1114 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
50 45 40 35 30 25 20 15 10 5 0
N.S.
45 4 35 3 25 2 5 1 05 0
N.S.
Developmental Cell Erratum
A
B
osteogenic
Relative expression
8000
Wtx
GFP
Wtx
shRNA:
GFP
shControl shRunx2
Wtx Runx2
200
*
120 4000 80 2000
40
0
shControl shRunx2
shControl shRunx2
Alp
Pparg
D
*
Relative expression
1600
Wtx
GFP
Wtx
GFP
shRNA:
PPARγ
Wtx n.s. PPARγ
osteogenic
shGFP shWtx
1400 1200 1000
*
800 600 400
*
200 0
Runx2 Alp Ocn
Actin
F
E17.5
KO
WT
PC
Sox9
Runx2 Osterix Sox9
HC
HC
TB
TB
H
E15.5 limb WT
HC
trabeculae
TB
G
PC
trabeculae
TB
HC
Ocn
E15.5
KO
WT
Runx2 Alp
PPARγ
eGFP
ni
E
*
*
0
ni
eGFP
shGFP shWtx
6000
Actin
C
adipogenic
160
Wtx
1600
Runx2
1200
Osterix
Wtx+ Wtx +/-
adults 2000
KO
* * *
*
800 400
*
*
Sox9
N O C
sx
AL P
O
x2
2
un R
1a
1a
ol C
ol C
Actin
1
0
Figure S6. Wtx Acts Upstream of Runx2 and Pparg
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1115
Developmental Cell Erratum
β-cat
β-cat
Actin
Actin
eGFP
ni
C 2000
1200
800
*
GF P
Wt x
shGFP shWtx
shGFP shWtx
1600
400
*
300
200
100
*
Tcf1
E
F
β-catenin +/+ Wtx Lox
HC
trabeculae
Prx1-Cre
SC HE
CB
SC
β-catenin L/+ Wtx Lox Wtx +
CB
SC
CB SC
HE
Figure S7. Role of Wtx in Regulation of b-Catenin Stability in Mesenchymal Progenitors
1116 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Fa
bp
4
pa
ar g
shaft
β-catenin +/+ Wtx + Wtx Lox
G
eb
pb
KO
B
C
Pp
β-catenin L/+ Wtx +
Prx1-Cre
WT
shβ-catenin
Wtx +
B
eb
1
shControl
C
2 in Ax
Tc f
4 bp
pa eb
ar g
C
Pp
1 Tc f
in Ax
pb
shβ-catenin
eb
shControl
C
Axin2 ALP
0 2
Axin2 ALP
*
*
0
Fa
400
ST2-adipogenic
500
Relative Expression
Relative Expression
*
ni
*
D
ST2-osteogenic
PPARγ
Wt x
GF P
GF P Wt x
Wt x
shRNA:
GF P
shControl shRunx2
B
Wt x
GF
P
Wt x
GF
shRNA:
P
MG132
A
HE
CB
Wtx Lox
Developmental Cell Erratum
A
P14
HET
WT
OF
OF
E15.5
B
P0 CB
TB
WT
CB
MC
TB
KO
CB
CB
Gata1-Cre
Osx-Cre
Ocn-Cre
D 0.4 0.35
OV/TV (%)
Wtx Lox
Wtx +
C
0.3 0.25
*
Prx1-Cre: Wtx+ Prx1-Cre: Wtx Lox
0.2 0.15 0.1 0.05 0
Figure S8. Mineralization Defects in Wtx-Deficient Bone
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1117
Erratum
The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification Annie Moisan, Miguel N. Rivera, Sutada Lotinun, Sara Akhavanfard, Erik J. Coffman, Edward B. Cook, Svetlana Stoykova, Siddhartha Mukherjee, Jesse A. Schoonmaker, Alexa Burger, Woo Jae Kim, Henry M. Kronenberg, Roland Baron, Daniel A. Haber,* and Nabeel Bardeesy* *Correspondence: [email protected] (D.A.H.), [email protected] (N.B.) DOI 10.1016/j.devcel.2012.04.017
Developmental Cell 20, 583–596; May 17, 2011 In the course of compiling all the data for this manuscript and preparing composite figures, some confusion arose in labeling and presenting some of the data. In some cases, the similarities between controls and wild-type (e.g., Figures 2A and S3B) made it difficult to distinguish the correct image. In other cases, images were inadvertently swapped or inverted between analogous experiments that showed very similar results (e.g., Figures 4B and 5E). In total, eight subpanels of the main figures were affected, as well as five subpanels of the supplemental material. None of these changes alter the conclusions of the manuscript in any way. A joint review of these errors, conducted by Harvard Medical School and Massachusetts General Hospital, did not find any evidence of research misconduct to warrant further investigation. A complete listing of the affected panels is therefore included below, along with the corrected panels (shown in the context of the entire figures to which they belong). We most sincerely apologize for the confusion that our errors have caused. The corrected panels in the document are: From the main text: Figures 2A (the two leftmost panels for Col2a-Cre mice), 4A (labeling of the nonspecific band), 4B, 5A, 5C, 5E, 5F, and 8A. From the supplement: Figures S3B, S6A, S6C, S7B, and S8C (bottom panel for Ocn-Cre). For clarification, we also include a listing of panels that are unchanged but in which a common control is present in two figures; these reflect cases in which the primary data from the same experiment are divided into two figures. Figure 2A, second panel for Prx-Cre ribs, also in Figure 6C. Figure 4B, bottom-left panels for ‘‘shControl adipo,’’ also in Figure 5E. Figure 5A, panels for ‘‘osteo day 4,’’ also in Figure 8A. Figure 7A, third panels from left for P0 calvaria, also in Figure S8C. Figure S6C, actin panels at bottom, also in Figure S7B.
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1109
Developmental Cell Erratum
Wtx+
Wtx Lox
Wtx+
Wtx Lox
Wtx Lox
Wtx+
Prx1+ multipotent progenitor
Prx1-Cre
Col2a+ chondrocyte precursor
Col2a-Cre
A
*
Ocn-Cre
Osx+ osteoblast precursor Ocn+ differentiated osteoblast
B
Wtx Lox
Wtx+
Wtx+
D
Prx1-Cre
Prx1-Cre
*
25 20 15 10 5 0
500 400 300 200
40 30 20 10
100
40 20
60
30 20
100
10
0
0
**
*
60
0
40
192.2 ± 8.7
80
3.5
50
83.1 ± 9.8
120
0
**
MS/BS (%)
BFR/TV (%/year)
700 600
***
50
Tb.Th (μm)
BV/TV (%)
***
*
3 2.5 2 1.5 1
E
Wtx+
Wtx Lox
N.S.
Prx1-Cre
60
35
*
*
Prx1-Cre: Wtx Lox
N.Ob/T.Ar (/mm2)
Prx1-Cre: Wtx+
MAR (μm/day)
C
30
Wtx Lox
0.5 0
276.3 ± 21.4
103.8 ± 21.7
Figure 2. Targeted Inactivation of Wtx in Mesenchymal Progenitors Causes Osteosclerosis and Impaired Adipogenesis
1110 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Developmental Cell
GFP
C
Runx2 n.s.
Relative expression
adipo Wtx
osteo Wtx
Wtx
shRNA:
GFP
ni
A
GFP
Erratum
adipogenic
250
shGFP shWtx
200 150 100 50
*
0
Klf5
shControl
B
shWtx
shGFP
Cebpb Cebpd
eGFP
D
shGFP
shWtx
*
*
Pparg Cebpa Fabp4
PPAR-γ
shWtx
shGFP
shWtx
day 6
day 12
Oil Red-O
ni
adipo
shGFP
shRunx2
ST2 cells
day 6
adipo
osteo
ALP
day 6
ALP
WT
PC
osteo
adipo
Oil Red-O
E
KO
F
P0 femur WT
PC
KO
Wtx HC
HC
Runx2
Runx2
Osterix Sox9 Ppar-γ Actin
600
Wtx+ Wtx -
*
400
*
*
*
200
*
*
C
N
P O
sx
AL
O
2
un
1a ol
ol
1a C
C
x2
0 1
HC
neonates Relative expresison
Osterix
HC
G
R
PC
PC
Figure 4. Wtx Acts Upstream of Runx2 and Pparg to Control Osteogenic and Adipogenic Fate Determination
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1111
Developmental Cell Erratum
A
B
C
E
D
F
G
Figure 5. Wtx-Mediated Control of b-Catenin Levels in BMPCs Determines Osteoblast and Adipocyte Specification
1112 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Developmental Cell Erratum
day 8 + Lox
MC3T3-E1 day 10 + Lox
ABC β-cat
Wtx+ Wtx Lox
2500
Relative Expression
Wtx
day 4 + Lox
Prx1-Cre BMPC
B
Prx1-Cre BMPC
sh G FP sh W tx
A
Actin
2000 1500 1000 500 0
Axin2
Tcf1
Axin2
ni shβ-catenin
shControl
C ni
osteo
ni
MeBIO
D ni
osteo
Tcf1
osteo day 10 BIO
osteo
ni
osteo
E Wtx
Wtx promotes
Wtx
Wtx inhibits
Associated with destabilization of β-catenin
adipocyte cchondrocyte precursor (Col2a+)
Wtx
mesenchymal progenitor (Prx1+)
bipotential progenitor Wtx
chondrocyte
osteoblast bl
osteoblast precursor (Osx+) Wtx
mature osteoblast (Ocn+)
Wtx
Figure 8. Wtx Inactivation Impairs Osteoblast Maturation Independent of b-Catenin Deregulation
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1113
Developmental Cell Erratum
A
Gata1-Cre Wtx +
Prx1-Cre Wtx +
Wtx Lox
Col2a1-Cre
Wtx Lox
CB
Wtx Lox
CB
C
Osx-Cre
D
Osx-Cre
Osx-Cre
Wtx Lox
Wtx+
Wtx +
Ocn-Cre Wtx +
Wtx Lox
Wtx+
B
Wtx +
Wtx Lox
Wtx Lox
CB
Osx-Cre: Wtx+
E 14
40
**
12
70
*
35 30
10
40 30
150
20
10
100
5
10
50
0
0
0
0
Y
2
Wtx Lox/Y
/Y tx -
tx W
W
Lo
x/
F
**
200
15
4
350
250
20
6
400
300
50
25
8
*
60
Osx-Cre: Wtx Lox
Gata1
Prx1
Osx
Col2a
Ocn
Lox band null band
1
2
3
4
5
6
7
8
9
10
11 12
Figure S3. Skeletal Phenotypes in Wtx-Deficient Mice
1114 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
50 45 40 35 30 25 20 15 10 5 0
N.S.
45 4 35 3 25 2 5 1 05 0
N.S.
Developmental Cell Erratum
A
B
osteogenic
Relative expression
8000
Wtx
GFP
Wtx
shRNA:
GFP
shControl shRunx2
Wtx Runx2
200
*
120 4000 80 2000
40
0
shControl shRunx2
shControl shRunx2
Alp
Pparg
D
*
Relative expression
1600
Wtx
GFP
Wtx
GFP
shRNA:
PPARγ
Wtx n.s. PPARγ
osteogenic
shGFP shWtx
1400 1200 1000
*
800 600 400
*
200 0
Runx2 Alp Ocn
Actin
F
E17.5
KO
WT
PC
Sox9
Runx2 Osterix Sox9
HC
HC
TB
TB
H
E15.5 limb WT
HC
trabeculae
TB
G
PC
trabeculae
TB
HC
Ocn
E15.5
KO
WT
Runx2 Alp
PPARγ
eGFP
ni
E
*
*
0
ni
eGFP
shGFP shWtx
6000
Actin
C
adipogenic
160
Wtx
1600
Runx2
1200
Osterix
Wtx+ Wtx +/-
adults 2000
KO
* * *
*
800 400
*
*
Sox9
N O C
sx
AL P
O
x2
2
un R
1a
1a
ol C
ol C
Actin
1
0
Figure S6. Wtx Acts Upstream of Runx2 and Pparg
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1115
Developmental Cell Erratum
β-cat
β-cat
Actin
Actin
eGFP
ni
C 2000
1200
800
*
GF P
Wt x
shGFP shWtx
shGFP shWtx
1600
400
*
300
200
100
*
Tcf1
E
F
β-catenin +/+ Wtx Lox
HC
trabeculae
Prx1-Cre
SC HE
CB
SC
β-catenin L/+ Wtx Lox Wtx +
CB
SC
CB SC
HE
Figure S7. Role of Wtx in Regulation of b-Catenin Stability in Mesenchymal Progenitors
1116 Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc.
Fa
bp
4
pa
ar g
shaft
β-catenin +/+ Wtx + Wtx Lox
G
eb
pb
KO
B
C
Pp
β-catenin L/+ Wtx +
Prx1-Cre
WT
shβ-catenin
Wtx +
B
eb
1
shControl
C
2 in Ax
Tc f
4 bp
pa eb
ar g
C
Pp
1 Tc f
in Ax
pb
shβ-catenin
eb
shControl
C
Axin2 ALP
0 2
Axin2 ALP
*
*
0
Fa
400
ST2-adipogenic
500
Relative Expression
Relative Expression
*
ni
*
D
ST2-osteogenic
PPARγ
Wt x
GF P
GF P Wt x
Wt x
shRNA:
GF P
shControl shRunx2
B
Wt x
GF
P
Wt x
GF
shRNA:
P
MG132
A
HE
CB
Wtx Lox
Developmental Cell Erratum
A
P14
HET
WT
OF
OF
E15.5
B
P0 CB
TB
WT
CB
MC
TB
KO
CB
CB
Gata1-Cre
Osx-Cre
Ocn-Cre
D 0.4 0.35
OV/TV (%)
Wtx Lox
Wtx +
C
0.3 0.25
*
Prx1-Cre: Wtx+ Prx1-Cre: Wtx Lox
0.2 0.15 0.1 0.05 0
Figure S8. Mineralization Defects in Wtx-Deficient Bone
Developmental Cell 22, 1109–1117, May 15, 2012 ª2012 Elsevier Inc. 1117