The type I BMP receptor ACVR1/ALK2 is required for chondrogenesis during development

Abstract

Bone morphogenetic proteins (BMPs) are crucial regulators of chondrogenesis. BMPs transduce their signals through three type I receptors: BMPR1A, BMPR1B, and ACVR1/ALK2. Fibrodysplasia ossificans progressiva (FOP), a rare disorder characterized by progressive ossification of connective tissue, is caused by an activating mutation in Acvr1 (the gene that encodes ACVR1/ALK2). However, there are few developmental defects associated with FOP. Thus, the role of ACVR1 in chondrogenesis during development is unknown. Here we report the phenotype of mice lacking ACVR1 in cartilage. Acvr1(CKO) mice are viable but exhibit defects in the development of cranial and axial structures. Mutants exhibit a shortened cranial base, and cervical vertebrae are hypoplastic. Acvr1(CKO) adult mice develop progressive kyphosis. These morphological defects were associated with decreased levels of Smad1/5 and p38 activation, and with reduced rates of chondrocyte proliferation in vertebral cartilage. We also tested whether ACVR1 exerts coordinated functions with BMPR1A and BMPR1B through analysis of double mutants. Acvr1/Bmpr1a and Acvr1/Bmpr1b mutant mice exhibited generalized perinatal lethal chondrodysplasia that was much more severe than in any of the corresponding mutant strains. These findings demonstrate that ACVR1 is required for chondrocyte proliferation and differentiation, particularly in craniofacial and axial elements, but exerts coordinated functions with both BMPR1A and BMPR1B throughout the developing endochondral skeleton.

Keywords: ACVR1; ALK2; BMP; CHONDROGENESIS; MOUSE.

Figure 1. ACVR1/ALK2 protein expression in axial elements

A, C. and E are stained with alcian blue-nuclear fast red. B, D, F, G, H are immunofluorescent images counterstained with DAPI. A,B, E11.5 cervical region showing ACVR1/ALK2 protein is expressed in sclerotome (asterisk). C,D. E11.5 cervical region showing expression in precartilaginous mesenchyme (detected by alcian blue staining in C), and notochord (arrows). E,F, Sections through E13.5 cervical spine showing expression in nucleus pulposus and chondrocytes in the vertebrae. G, E15.5 cervical spine showing expression in nucleus pulposus and proliferating chondrocytes in vertebral bodies. H, P0 cervical spine showing expression in endplate cartilage and nucleus pulposus. Low levels of expression are present in the periosteum of the vertebrae. Af, annulus fibrosus; ep, endplate; np, nucleus pulposus; po, periosteum; vb, vertebral body.

Figure 2. Axial defects in Acvr1^fx/fx;Col2a1-Cre(Acvr1^CKO) mutants

All images are whole-mount skeletal preparations of neonatal (P0) littermates stained with alcian blue/alizarin red. A, B, Cervical and thoracic spines of P0 WT (A) and Acvr1^CKO (B) littermates. C-E, transverse views of isolated WT cervical (C1-C3) vertebrae. C’-E’, C1, C2, and C3 vertebrae, respectively, from Acvr1^CKO P0 littermate. ap, anterior process; c, centrum; tp, transverse process; va, vertebral arch.

Figure 3. Impaired BMP signaling in Acvr1^fx/fx;Col2a1-Cre(Acvr1^CKO) mutant axial elements

All images are sagittal sections through the vertebral column of E17.5 mice counterstained with DAPI. A,A’, Immunofluorescence staining for pSmad1/5 in sections through E17.5 cervical vertebrae from WT and Acvr1^CKO littermate. B, Quantitation revealed a reduction in Acvr1^CKO mice in the percentages of cells positive for pSmad1/5 compared to WT littermates (n = 16, p < 0.0001). C,C’, Immunofluorescence staining for p-p38 in sections through E17.5 cervical vertebrae from WT and Acvr1^CKO littermate. D, Quantitation revealed a reduction in Acvr1^CKO mice in the percentages of cells positive for p-p38 compared to WT littermates (n = 9, asterisk, p < 0.0001). E,E’, PCNA immunofluorescence on E17.5 cervical vertebrae from WT and Acvr1^CKO littermate. F, Quantitation of percentage of PCNA positive cells reveals a significant decrease in mutants. (n = 6, Asterisk, p < 0.005). Size differences in the nucleus pulposus for WT vs. Acvr1^CKO mice in panels A and C are not real and are a result of plane of section.

Figure 4. ACVR1/ALK2 exhibits overlapping functions with BMPR1A and BMPR1B in axial elements

All images are cleared skeletal preparations of P0 spines (disarticulated from ribs) stained with alcian blue/alizarin red. A-F, dorsal views. A’-F’, lateral views. Arrow in B highlights reduced ossification of the centra in Acvr1^CKO mice. Arrow in D highlights lack of segmentation, evidenced by gaps in staining in regions where ossified centra should be located. Arrow in F highlights vertebral fusion. Arrow in F’ highlights discontinuous vertebral arch. C, cervical; T, thoracic; va, vertebral arch. Asterisks in A’-F’ demarcate the axis (C1). Arrowheads in A’-F’ demarcate the atlas (C2).

Figure 5. Appendicular defects in Acvr1^fx/fx;Bmpr1a^fx/fx;Col2a1-Cre(Acvr1/Bmpr1a^CKO) mice

A-D, skeletal preparations from E17.5 forearms stained with alcian blue/alizarin red. Double-headed arrows in C and D highlight shortening of radius and ulna in Acvr1/Bmpr1a^CKO mice compared to Bmpr1a^CKO mice. Arrows in B and D highlight delayed ossification of digits in Acvr1^CKO mice, which is exacerbated in Acvr1/Bmpr1a^CKO mice. E-H, sagittal sections through proximal femurs of E17.5 mice of the indicated genotypes stained with alcian blue/nuclear fast red.

Figure 6. Appendicular defects in Acvr1^fx/fx;Bmpr1b−/−;Col2a1-Cre (Acvr1^CKO/Bmpr1b−/−) mice

Cleared skeletal preparations from E17.5 autopods of forelimbs (A-D) and hindlimbs (E-H) stained with alcian blue/nuclear fast red. A blue filter was applied in Photoshop to the images to enhance visualization of alcian blue-stained regions. Arrows in B and F point to reduced or absent ossification in phalangeal elements in Acvr1^CKO mice. Arrow in H highlights absence of ossification in the Acvr1^CKO/Bmprb−/− hindlimb compared to the Acvr1^CKO and Bmpr1b−/− single mutant strains.