Reduced bone formation in <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A) Skeletons of 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (right) mice stained with alcian blue (cartilage) and alizarin red (bone). The skeleton of the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mouse is remarkably smaller. (B) Alcian blue- and alizarin red-stained skull from 6-day-old <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. (D) Plain X-radiography of the tibiae from 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice (right). The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (right) mice. The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice showed reduced trabecular (arrowheads) and cortical bones (arrows). (F–H) Quantitative μ-CT data showing that the 6-day-old <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice also presented reduced material density although no statistically significant difference was observed (H).</p

Cell proliferation was reduced in the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A–B) BrdU immunohistochemical staining of the femur sections of 7-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice, compared to the control mice (<i>n</i> = 4, *<i>P</i><0.05). The data were expressed as the percentage of BrdU-positive nuclei versus total nuclei. Scale bars: 100 µm.</p

Quantitative real-time PCR analyses of osteoblast differentiation markers.

<p>Real-time PCR was performed with total RNA isolated from the long bones of the one-week-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. The expressions of key transcription factors associated with osteoblast differentiation (Runx2 and osterix), osteoblast markers (<i>Alp</i>, <i>Ocn</i> and <i>Bsp</i>) and osteocyte marker (<i>Dmp1</i>) were reduced in <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. The mRNA levels in the control mice were set as one, and the mRNA levels of <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice were expressed as folds of those in the control mice. The data represented three analyses (<i>n</i> = 3) for each group.</p

Reduced FGF signaling in <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A) Quantitative real-time PCR was performed to analyze the mRNA levels of <i>Fgf2</i>, <i>Fgfr1</i>, <i>Fgfr2</i>, <i>Fgfr3</i> and <i>Fgfr4</i>, as well as two effector molecules, <i>Erm</i> and <i>Pea3</i>, using total RNA isolated from the long bones of 8-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. The mRNA levels in the control mice were set as one, and the mRNA levels of <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice were expressed as folds of those in the control mice. The data represented three analyses (<i>n</i> = 3) for each group. (B) Immunohistochemistry showed that the level of the Fgfr2 protein (signal in brown) was reduced in the femurs of the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice, compared to the control mice. Scale bars: 100 µm.</p

Histological examination of <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A–B) Femur sections of 6-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice were stained with H&E. The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. Note that the osteoclasts (red arrows) appeared to be similar in size and distribution in the control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. The osteoclast densities were 0.55±0.06/0.01 mm² in the controls (<i>n</i> = 5) and 0.60±0.02/0.01 mm² in the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice (<i>n</i> = 5, <i>P</i>>0.05). (D–F) <i>In situ</i> hybridization analyses (signal in blue) of the transcripts of <i>Alp</i> (D), <i>Ocn</i> (E) and <i>Dmp1</i> (F) in the femurs of one-week-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> 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 <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. Scale bar = 100 µm.</p

Effects of Twist1, Twist2 and E12 on the activity of a 4.9<i>Fgfr2</i> promoter fragment.

<p>C3H10T1/2 (A) and MC3T3-E1 cells (B) were transiently co-transfected with a 4.9 kb <i>Fgfr2</i> 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).</p

The effect of deletion of <i>Klotho</i> on histological features of the <i>Dmp1^−/−</i> growth plate and metaphysis area.

<p>(<b>A</b>) Growth plates and metaphysis of proximal tibias obtained from WT, <i>Dmp1^−/−</i>, <i>kl/kl</i>, and <i>Dmp1^−/kl/kl</i> (Safranin O staining x200). Note that there is an expansion of metaphysis in the <i>kl/kl</i>, which is similar to that of the <i>Dmp1^−/−</i> and <i>Dmp1^−/−kl/kl</i> mice. (<b>B</b>) growth plate width comparison between all four groups of the mice shows increase of phosphate in both <i>kl/kl</i> and <i>Dmp1^−/−kl/kl</i> mice resulted in decreased growth plate width compared to WT (P = 0.009 and 0.024 respectively). Values reported in mean ± SE from more than 3 mice in each group at 6 weeks of age. *P<0.05 compared to WT; § P<0.05 compared to <i>Dmp1^−/−</i> group.</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p

Sharply increased ectopic calcification and apoptosis in the compound deficient (<i>Dmp1^−/− kl/kl</i>) mice.

<p>(<b>A</b>) Representative X-ray images of kidney and aorta in four groups show moderate ectopic calcifications in the <i>kl/kl</i> kidney and aorta, and massive ectopic calcification in the <i>Dmp1^−/−kl/kl</i> kidney and aorta. (<b>B</b>) von-Kossa stain (left panel) and TUNEL assay (right panel) of the <i>Dmp1^−/−kl/kl</i> kidney showing a close correlation between the calcified- and apoptotic cells. (<b>C</b>) A similar linkage between the ectopic calcification (von-Kossa stain; right panel) and the apoptosis (TUNEL assay; right panel) was observed in the adjacent <i>Dmp1^−/−kl/kl</i> aorta. (<b>D</b>) The quantitative mineral content is sharply increased in both the <i>kl/kl</i> and the <i>Dmp1^−/−kl/kl</i> kidney by µCT analysis with significantly higher in the <i>Dmp1^−/−kl/kl</i> kidney than that of the single kl/kl kidney (P = 0.041). Values are means ± SEM of 3–5 kidneys. Note that there was no calcification in the WT and the <i>Dmp1^−/−</i> mice. (<b>E</b>) Representative DMP1 Western blots with WT at left panel and <i>kl/kl</i> at right panel shows higher density of DMP1 in <i>kl/kl.</i> Osteocalcin (OCN) is the matrix protein which does not show difference between these two groups. (<b>F</b>) Quantitative kidney RT-PCR data showing over two fold-increases of <i>Dmp1</i> mRANA in <i>kl/kl</i> compared to the WT control. n = 3, *P<0.05.</p

The changes of bone volume/mineral content (reflected by BV/TV) and femur length in <i>Dmp1^−/−</i>, <i>Klotho</i> deficient (<i>kl/kl</i>) and the compound deficient (<i>Dmp1^−/− kl/kl</i>) at age of 6-weeks.

<p>(<b>A</b>) Representative X-ray (upper panel) and backscattered SEM (lower panel) images of the hind limbs obtained from wild type (WT), <i>Dmp1^−/−</i>, <i>kl/kl</i> and <i>Dmp1^−/− kl/kl</i> mice. (<b>B</b>) Representative µCT images of the above four group femurs at the longitudinal front view (upper panel), and the midshaft cross section view (lower pane). (<b>C</b>) Quantitative µCT data shows moderate changes of femur length in <i>Dmp1^−/−</i>, <i>kl/kl</i> and <i>Dmp1^−/−kl/kl</i> mice compared to the age-matched WT control (P<0.001, P<0.001, and P = 0.03 respectively; n = 10). Note that the femur length difference between the <i>Dmp1^−/−kl/kl</i> mice and <i>kl/kl</i> (P<0.001) or <i>Dmp1^−/−</i> (*P<0.05) are significant. (<b>D</b>) The Quantitative µCT data show a sharp reduction of BV/TV in the <i>Dmp1^−/−</i> cortical bone (P<0.01), and there is no significant change between WT and <i>kl/kl</i> or the <i>Dmp1^−/−kl/kl</i> (left panel). In contrast, there is a significant increase in metaphysis BV/TV in <i>Dmp1^−/−</i> (>80%, P<0.01) or in the <i>kl/kl</i> (>50%, P<0.01) or in the <i>Dmp1^−/−kl/kl</i> mice (>60%, P<0.01) separately (right panel).</p