Table 1.
Primers used for real-time PCR.
Figure 1.
Reduced bone formation in Twist1flox/+; Twist2Cre/+ mice.
(A) Skeletons of 6-day-old control (left) and Twist1flox/+; Twist2Cre/+ (right) mice stained with alcian blue (cartilage) and alizarin red (bone). The skeleton of the Twist1flox/+; Twist2Cre/+ mouse is remarkably smaller. (B) Alcian blue- and alizarin red-stained skull from 6-day-old Twist1flox/+; Twist2Cre/+ mice (right) showed delayed fusion of interfrontal suture and open posterior fontanel (arrows), compared with the control mice (left). (C) Alcian blue- and alizarin red-stained hind foot of 6-day-old control (left) and Twist1flox/+; Twist2Cre/+ (right) mice. Note the delayed ossification in metatarsals (mt) and phalanges (pl), and an additional toe (arrow) originating from the same (or duplicated) metatarsal as the hallux in Twist1flox/+; Twist2Cre/+ mice. (D) Plain X-radiography of the tibiae from 6-day-old control (left) and Twist1flox/+; Twist2Cre/+ mice (right). The Twist1flox/+; Twist2Cre/+ mice had shorter tibiae and reduced radiopacity, compared to the control mice. (E) Representative three-dimensional μ-CT images of tibiae from 6-day-old control (left) and Twist1flox/+; Twist2Cre/+ (right) mice. The Twist1flox/+; Twist2Cre/+ mice showed reduced trabecular (arrowheads) and cortical bones (arrows). (F–H) Quantitative μ-CT data showing that the 6-day-old Twist1flox/+; Twist2Cre/+ mice had a significant decrease in the ratio of bone volume (BV)/total volume (TV) (F) and in apparent bone density (G), compared to the control mice (n = 6, P<0.001). The Twist1flox/+; Twist2Cre/+ mice also presented reduced material density although no statistically significant difference was observed (H).
Figure 2.
Histological examination of Twist1flox/+; Twist2Cre/+ mice.
(A–B) Femur sections of 6-day-old control and Twist1flox/+; Twist2Cre/+ mice were stained with H&E. The Twist1flox/+; Twist2Cre/+ mice displayed reduced metaphyseal trabecular bone (A, red arrows) and a decreased thickness of the periosteum (B, blue arrows) and cortical bone (B, red arrows). (C) TRAP staining of femur sections of 6-day-old control and Twist1flox/+; Twist2Cre/+ mice. Note that the osteoclasts (red arrows) appeared to be similar in size and distribution in the control and Twist1flox/+; Twist2Cre/+ mice. The osteoclast densities were 0.55±0.06/0.01 mm2 in the controls (n = 5) and 0.60±0.02/0.01 mm2 in the Twist1flox/+; Twist2Cre/+ mice (n = 5, P>0.05). (D–F) In situ hybridization analyses (signal in blue) of the transcripts of Alp (D), Ocn (E) and Dmp1 (F) in the femurs of one-week-old control and Twist1flox/+; Twist2Cre/+ mice. (G, H) Immunohistochemical analyses (signal in brown) of the osterix (G) and biglycan (H) protein levels in the femurs of the 6-day-old control and Twist1flox/+; Twist2Cre/+ mice. Scale bar = 100 µm.
Figure 3.
Quantitative real-time PCR analyses of osteoblast differentiation markers.
Real-time PCR was performed with total RNA isolated from the long bones of the one-week-old control and Twist1flox/+; Twist2Cre/+ mice. The expressions of key transcription factors associated with osteoblast differentiation (Runx2 and osterix), osteoblast markers (Alp, Ocn and Bsp) and osteocyte marker (Dmp1) were reduced in Twist1flox/+; Twist2Cre/+ mice. The mRNA levels in the control mice were set as one, and the mRNA levels of Twist1flox/+; Twist2Cre/+ mice were expressed as folds of those in the control mice. The data represented three analyses (n = 3) for each group.
Figure 4.
Cell proliferation was reduced in the Twist1flox/+; Twist2Cre/+ mice.
(A–B) BrdU immunohistochemical staining of the femur sections of 7-day-old control and Twist1flox/+; Twist2Cre/+ mice. The BrdU-positive cells (signal in brown) were counted in a 100-µm zone of the metaphysis, demarcated by the chondro-osseous junction and the marked line (A), and in the femoral diaphysis (B). The osteoblast/osteoprogenitor proliferation was reduced in both the metaphysis (C) and diaphysis (D) in the Twist1flox/+; Twist2Cre/+ mice, compared to the control mice (n = 4, *P<0.05). The data were expressed as the percentage of BrdU-positive nuclei versus total nuclei. Scale bars: 100 µm.
Figure 5.
Reduced FGF signaling in Twist1flox/+; Twist2Cre/+ mice.
(A) Quantitative real-time PCR was performed to analyze the mRNA levels of Fgf2, Fgfr1, Fgfr2, Fgfr3 and Fgfr4, as well as two effector molecules, Erm and Pea3, using total RNA isolated from the long bones of 8-day-old control and Twist1flox/+; Twist2Cre/+ mice. The mRNA levels in the control mice were set as one, and the mRNA levels of Twist1flox/+; Twist2Cre/+ mice were expressed as folds of those in the control mice. The data represented three analyses (n = 3) for each group. (B) Immunohistochemistry showed that the level of the Fgfr2 protein (signal in brown) was reduced in the femurs of the Twist1flox/+; Twist2Cre/+ mice (right), compared to the control mice (left). (C) Immunohistochemistry showed that there was less phospho-Erk1/2 (signal in brown) than in the femurs of Twist1flox/+; Twist2Cre/+ mice, compared to the control mice. Scale bars: 100 µm.
Figure 6.
Effects of Twist1, Twist2 and E12 on the activity of a 4.9Fgfr2 promoter fragment.
C3H10T1/2 (A) and MC3T3-E1 cells (B) were transiently co-transfected with a 4.9 kb Fgfr2 promoter luciferase construct and the indicated expression constructs, along with a pRL-TK construct as an internal control. The luciferase activities were determined by a dual luciferase assay system, and the promoter activities were expressed as luciferase activities relative to that of the control. The values represented mean ± SD. n = 3 for each group. “a” indicates significant difference from the control (p<0.05); “b” denotes a significant difference from all other groups (p<0.05).