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
Runx2prx1−/− mice at E18.5 were cultured, followed by Alizarin Red or Van Gieson staining.
Development • Supplementary information
indicate standard error of the mean (SEM). (C) Primary osteoblasts from Runx2flox/flox and
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).
Development • Supplementary information
Development 143: doi:10.1242/dev.128793: Supplementary information
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
Development • Supplementary information
calvarial region 500 μm away from the center of the suture.
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
Development • Supplementary information
Development 143: doi:10.1242/dev.128793: Supplementary information
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)
Development • Supplementary information
X-ray CT analyses of calvaria of Runx2nestin−/− mice at 6 month-old.
Development 143: doi:10.1242/dev.128793: Supplementary information
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.
Development • Supplementary information
Supplemental Figure 5. Targeting validation of Prx1-Cre in mouse frontal bone. (A)
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
Development • Supplementary information
confocal images of the area defined by the square in Fig. 2C.
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.
Development • Supplementary information
Development 143: doi:10.1242/dev.128793: Supplementary information
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
Development • Supplementary information
mice at P1 (n = 3, 2-3 slices per independent mouse).
Development 143: doi:10.1242/dev.128793: Supplementary information
Supplemental Figure 9. Targetting validation of Nestin-Cre in calvaria of Nestin-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.
Development • Supplementary information
mice. (A) Prx1-GFP+ cells and Nestin-GFP+ cells were sorted from the calvaria of Prx1-GFP
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)
Development • Supplementary information
Prx1-GFP mice.
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.
Development • Supplementary information
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.
Development • Supplementary information
Development 143: doi:10.1242/dev.128793: Supplementary information
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
Development • Supplementary information
SEM.
Development 143: doi:10.1242/dev.128793: Supplementary information
Table S1. Antibodies used in the analysis
For immunohistochemistry
Diluted
in TBST
antibody
origin
cat.#
company
anti-Runx2
anti-Osterix
rabbit
rabbit
#12556
ab22552
400：1
800：1
anti-Osteopontin
rabbit
#18621
Cell Signaling Technology
Abcam
Immuno-Biological
Technology
Cell Signaling Technology
LSL
400：1
BD Biosciences
100：1
anti-FABP4
rabbit
#3544
anti-type II
rabbit
LB-1297
collagen
anti-Sca1
rat
553333
TBST; Tris-buffered saline with 0.1% Tween 20
400：1
400：1
antibody
fluorochrome
clone
company
anti-CD29
anti-CD31
anti-CD45
anti-CD49e
anti-CD51
anti-CD61
anti-CD90.2
anti-CD105
anti-PDGFRα
anti-PDGFRβ
anti-Sca1
anti-Ter-119
rat IgG2b,  Isotype Ctrl
rat IgG2a,  Isotype Ctrl
rat IgG2a,  Isotype Ctrl
rat IgG2a,  Isotype Ctrl
APC
APC
APC
PE
PE
Alexa Fluor 647
APC
PE
BV421
APC
PE/Cy7
APC
APC
PE
PE/Cy7
BV421
HMb1-1
MEC 13.3
30-F11
HMa5-1
RMV-7
2C9.G2
53-2.1
MJ7/18
APA5
APB5
D7
TER-119
RTK4530
RTK2758
RTK2758
R35-95
eBioscience
BD Biosciences
BD Biosciences
eBioscience
Biolegend
Biolegend
eBioscience
eBioscience
BD Biosciences
eBioscience
BD Biosciences
BD Biosciences
Biolegend
Biolegend
Biolegend
BD Biosciences
diluted in
2%FBS/PBS
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
100:1
Development • Supplementary information
For flow cytometry and cell sorting