Fig 1.
Defective patterning and ossification in Hand1 mutant long bones.
(A) Wild-type (a) and Hand1Tg/+; Twist2-Cre mutants (b,c) at P1. (B) Hand1Tg/+; Twist2-Cre mutant (Tg) and wild-type (Wt) littermate at P21. (C) Bone staining of P1 Wt and Hand1 mutant forelimbs (top panels) and hindlimbs (bottom panels), as indicated. Scale Bars: 1mm. (D) Bone staining of Wt and Hand1 mutants at E16.5, P1, and P10, as indicated. Scale Bars: 1 mm (a,b,d,e), 2 mm (c,f). fl, forelimb; hl, hindlimb; r, radius; u, ulna; hu, humerus; s, scapula; fe, femur; t, tibia; fi, fibula.
Fig 2.
Hand1 mutants present hypoplastic primary and secondary ossifications.
(A) Hypoplastic primary ossification in Hand1 mutant femurs. (a,b) von Kossa staining of Wt (a) and Hand1 mutant (b) femurs at E16.5. Brackets indicate the osoteogenesis domain. Scale bars: 200 μm. (B) Delayed secondary ossification in Hand1 mutant femurs (b,d,f,h) compared to Wt (a,c,e,g) at P7 and P21. (c,e) and (d,f) show enlargement of the boxed regions of the adjacent panels (a) and (b), respectively. Immunohistochemistry for Spp1 (e,f) indicates the presence of osteoblasts. ch, chondrocytes; po, periosteum; o, ossified tissue. Scale bars: 200 μm (a,b), 50 μm (c-f), 400 μm (g,h).
Fig 3.
Hand1 downregulates Ihh and Runx2 expression in vivo.
(A) Endogenous expression of Hand1, Ihh and Runx2 in E12.5 cartilage primordium. Alcian blue staining (a-c) and immunohistochemical analysis for Hand1 (d,e), Ihh (f,g), and Runx2 (h,i) in the distal (b,d,f,h) and central (c,e,g,i) part of a wild-type forelimb cartilage primordium at E12.5. (b,c) Magnified images of the distal and central regions inside of the boxes in (a). Hand1 is strongly expressed in the distal part of the cartilage primordium (arrows in d). The expression pattern of Ihh (f,g) is opposite to Hand1 (d,e). Runx2 is expressed both in the distal (h) and the central part (i) of the immature chondrocytes. ch, chondrocytes. Scale bars: 100 μm (a), 25 μm (b-i). (B, C) qPCR analysis of Hand1 (a), Ihh (b) and Runx2 (c) transcripts in femoral epiphyseal cartilage at E16.5 (B) and P1 (C). All data were normalized to Ppia expression. (D) Expression of Ihh (a,b) and Runx2 (c,d) was detected by immunohistochemistry of E16.5 Wt (a) and Hand1 mutant (b) femurs. The zone of Ihh-positive chondrocytes (brackets in a,b) and the region of Runx2-positive preosteoblasts in the periosteum (brackets in c,d) is decreased in Hand1 mutants compared to Wt. p, proliferating chondrocytes; hy, hypertrophic chondrocytes; po, periosteum; o, ossified tissue. Scale bars: 50 μm.
Fig 4.
Hand1 inhibits the Ihh promoter through Runx2 transactivation.
(A) qPCR analysis of Hand1 (a), Ihh (b), and Runx2 (c) transcripts in ATDC5 cells stably transfected with an empty vector (Control) or Hand1 expression vector. (B) Luciferase assays. COS1 cells were transiently cotransfected with pIhh-luc reporter and the indicated expression vectors. Luciferase data (b) is shown as a percentage of Runx2 activation (normalized to 1.0). The data represent the mean ± SD. (C) Model for transcriptional regulation of endochondral ossification by Hand1. Hand1 inhibits Runx2-dependent Ihh expression, which normally promotes Runx2 expression in the perichondrium and the periosteum, which, in turn, is required for osteoblast differentiation.