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

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