Supplemental Figures - Development · Supplemental Figures Supplemental Figure 1. Phenotypes in...
15
Development • Supplementary information Development 143: doi:10.1242/dev.128793: Supplementary information Supplemental Figures Supplemental Figure 1. Phenotypes in Runx2prx1 −/− mice. (A) The sections of calvarial sagittal suture of Runx2 flox/flox and Runx2prx1 −/− mice at E18.5 were stained with anti-Runx2, anti-Osx, anti-OPN, anti-FABP4 and anti-type II collagen. (B) Total RNA was isolated from the calvaria of Runx2 flox/flox and Runx2prx1 −/− mice at E18.5, followed by determination of Runx2 and Prx1 mRNA expression by qPCR (n = 4). **P < 0.01; Student’s t-test. Error bars indicate standard error of the mean (SEM). (C) Primary osteoblasts from Runx2 flox/flox and Runx2prx1 −/− mice at E18.5 were cultured, followed by Alizarin Red or Van Gieson staining.
Transcript of Supplemental Figures - Development · Supplemental Figures Supplemental Figure 1. Phenotypes in...
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figures
Supplemental Figure 1. Phenotypes in Runx2prx1−/− mice. (A) The sections of calvarial
sagittal suture of Runx2flox/flox and Runx2prx1−/− mice at E18.5 were stained with anti-Runx2,
anti-Osx, anti-OPN, anti-FABP4 and anti-type II collagen. (B) Total RNA was isolated from
the calvaria of Runx2flox/flox and Runx2prx1−/− mice at E18.5, followed by determination of
Runx2 and Prx1 mRNA expression by qPCR (n = 4). **P < 0.01; Student’s t-test. Error bars
indicate standard error of the mean (SEM). (C) Primary osteoblasts from Runx2flox/flox and
Runx2prx1−/− mice at E18.5 were cultured, followed by Alizarin Red or Van Gieson staining.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 2. Tracking the cell fate of Prx1- or Nestin-Cre derived cells.
(A-B) A confocal image of the sagittal suture of Prx1-Cre;Rosa26-tdTomato mice at P1. The
sections were stained with an anti-Osx antibody (A) and anti-OPN antibody (B). (C-D) A
confocal image of the sagittal suture of Nestin-Cre;Rosa26-tdTomato mice at P1. The sections
were stained with an anti-Osx antibody (C) and anti-OPN antibody (D).
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 3. Quantification in the images of the sagittal suture of
Prx1-Cre;Rosa26-tdTomato, Nestin-Cre;Rosa26-tdTomato, Prx1-GFP and Nestin-GFP
mice. (A) The percentage of tdTomato-positive cells among Runx2, Osx or OPN-positive
cells in a confocal image of the sagittal suture of Prx1-Cre;Rosa26-tdTomato and
Nestin-Cre;Rosa26-tdTomato mice at P1 (n = 3 – 4, 2-3 slices per independent mouse). (B)
The percentage of Runx2-positive cells among Prx1-GFP+ cells in a confocal image of the
sagittal suture of Prx1-GFP and Nestin-GFP mice at P1 (n = 3, 2-3 slices per independent
mouse). Error bars indicate SEM. The numbers of cells were individually counted in the
calvarial region 500 μm away from the center of the suture.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 4. Targeting validation of Nestin-Cre in mouse calvaria at various
postnatal stages. (A) A confocal image of the sagittal suture of Nestin-Cre;Rosa26-tdTomato
mice at P10 or 12 week-old. The sections were stained with an anti-Runx2 antibody, anti-Osx
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
antibody and anti-OPN antibody. (B) Total RNA was isolated from the calvaria of Runx2flox/flox
and Runx2nestin−/− mice at 6 month-old, followed by determination of Runx2 mRNA
expression by qPCR (n = 3). N.S.; not significant. Error bars indicate SEM. (C) Calvariae
from Runx2flox/flox and Runx2nestin−/− mice at 6 month-old were stained with anti-Runx2. (D)
X-ray CT analyses of calvaria of Runx2nestin−/− mice at 6 month-old.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 5. Targeting validation of Prx1-Cre in mouse frontal bone. (A)
tdTomato fluorescence in frontal bone and parietal bone in Prx1-Cre;Rosa26-tdTomato mice
at P1. (B) The tdTomato images of the frontal and sagittal suture of
Prx1-Cre;Rosa26-tdTomato mice at P1. (C) The sections of frontal suture of
Prx1-Cre;Rosa26-tdTomato mice at P1 were stained with an anti-Osx antibody and anti-OPN
antibody.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 6. Non-colored confocal images of Fig. 2B and Fig. 2C. (A)
Magnified confocal images of the area defined by the square in Fig. 2B. (B) Magnified
confocal images of the area defined by the square in Fig. 2C.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 7. The presence of Runx2hiPrx1+ and Runx2lowPrx1+ cells in the
sagittal suture of Prx1-GFP mice. (A) Magnified images of Runx2hiPrx1+ cells (arrow) and
Runx2lowPrx1+ cells (arrowhead) in Fig. 2B. (B) Intensity profile of Figure 2B. The upper
right panel and lower three panels show the signal intensity of Prx1-GFP, Runx2/Alexa 633
and Hoechst33342 at the arrow in the upper left panel. The arrow was drawn according to the
Hoechst 33342 staining. The signal intensities of Runx2hiPrx1+ and Runx2lowPrx1+ cells are
indicated.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 8. Targetting validation of Prx1-Cre in calvaria of Prx1-GFP mice.
GFP and tdTomato fluorescence in the calvaria of Prx1-GFP;Prx1-Cre;Rosa26-tdTomato
mice at P1 (n = 3, 2-3 slices per independent mouse).
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 9. Targetting validation of Nestin-Cre in calvaria of Nestin-GFP
mice. (A) Prx1-GFP+ cells and Nestin-GFP+ cells were sorted from the calvaria of Prx1-GFP
or Nestin-GFP mice at P1, respectively, followed by determination of Runx2 mRNA
expression by qPCR (n = 3 - 4). The values were compared with those obtained from cultured
osteoblasts. **P < 0.01; Student’s t-test. Error bars indicate SEM. (B) GFP and tdTomato
fluorescence in the calvaria of Nestin-GFP;Nestin-Cre;Rosa26-tdTomato mice at P1. (C) A
confocal image of the sagittal suture of Nestin-GFP;Nestin-Cre;Rosa26-tdTomato mice at P1
(n = 3, 2-3 slices per independent mouse). The sections were stained with an anti-Runx2
antibody.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 10. The fraction size of differentiated osteoblasts and Prx1-GFP+
cells among total calvarial cells in flow cytometric analysis. Flow cytometric analysis of
calvarial cells isolated from (A) α1(I)-collagen-Cre;Rosa26-tdTomato mice and (B)
Prx1-GFP mice.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 11. The original data of flow cytometric analysis in Fig. 3. (A) The
original data of the flow cytometric analysis in Fig. 3A. (B) The original data of the flow
cytometric analysis in Fig. 3B.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 12. Localization of Prx1+Sca1+ cells in calvaria. (A) A confocal
image of the sagittal suture of Prx1-GFP mice at P1. The sections were stained with anti-Sca1
antibody (green) and anti-Runx2 antibody (red). (B) Magnified confocal images of the area
defined by the square in Fig. 11A. Arrows indicate Prx1-GFP and Sca1 double positive cells.
(C) Magnified images of Runx2hiPrx1+Sca1- cells (arrow) and Runx2lowPrx1+Sca1+ cells
(arrowhead) in a confocal image of the sagittal suture of Prx1-GFP mice at P1.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 13. mRNA expression of MSC surface markers in sorted
Prx1+Sca1+ and Prx1+Sca1- cells. Stromal Prx1+Sca1+ or Prx1+Sca1− cells were sorted from
the calvaria of Prx1-GFP mice at P1, followed by determination of Itgb3 (CD61) and Thy1
(CD90) mRNA expression by qPCR (n = 3). **P < 0.01; Student’s t-test. Error bars indicate
SEM.
Dev
elo
pmen
t • S
uppl
emen
tary
info
rmat
ion
Development 143: doi:10.1242/dev.128793: Supplementary information
Table S1. Antibodies used in the analysis
For immunohistochemistry
antibody origin cat.# company Diluted
in TBST
anti-Runx2 rabbit #12556 Cell Signaling Technology 400：1
anti-Osterix rabbit ab22552 Abcam 800：1
anti-Osteopontin rabbit #18621 Immuno-Biological
Technology 400：1
anti-FABP4 rabbit #3544 Cell Signaling Technology 400：1
anti-type II
collagen rabbit LB-1297 LSL 400：1
anti-Sca1 rat 553333 BD Biosciences 100：1
TBST; Tris-buffered saline with 0.1% Tween 20
For flow cytometry and cell sorting
antibody fluorochrome clone company diluted in
2%FBS/PBS
anti-CD29 APC HMb1-1 eBioscience 100:1
anti-CD31 APC MEC 13.3 BD Biosciences 100:1
anti-CD45 APC 30-F11 BD Biosciences 100:1
anti-CD49e PE HMa5-1 eBioscience 100:1
anti-CD51 PE RMV-7 Biolegend 100:1
anti-CD61 Alexa Fluor 647 2C9.G2 Biolegend 100:1
anti-CD90.2 APC 53-2.1 eBioscience 100:1
anti-CD105 PE MJ7/18 eBioscience 100:1
anti-PDGFRα BV421 APA5 BD Biosciences 100:1
anti-PDGFRβ APC APB5 eBioscience 100:1
anti-Sca1 PE/Cy7 D7 BD Biosciences 100:1
anti-Ter-119 APC TER-119 BD Biosciences 100:1
rat IgG2b, Isotype Ctrl APC RTK4530 Biolegend 100:1
rat IgG2a, Isotype Ctrl PE RTK2758 Biolegend 100:1
rat IgG2a, Isotype Ctrl PE/Cy7 RTK2758 Biolegend 100:1
rat IgG2a, Isotype Ctrl BV421 R35-95 BD Biosciences 100:1
