Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP)

Abstract

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1^R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1^R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1^R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

Keywords: ACVR1; Bone morphogenetic protein; Chondrogenesis; Digit; Fibrodysplasia ossificans progressiva; Joint.

Figure 1.. Acvr1 R206H delays and disrupts digit development in a mouse model of FOP.

Digits of forelimbs and hindlimbs of Acvr1^R206H/+;Prrx1-Cre and control littermate mice were stained to detect cartilage (Alcian blue) and mineralized bone (Alizarin red) over time from E14.5 to P28. Numbers in (A) indicate digit number from anterior to posterior; all panels, same arrangement. (A-D) At E14.5, mineralization has not yet occurred, and only cartilage is detected. Cartilaginous digit rays are segmented at presumptive joints in controls but not mutants. (E-H) In P0 mutants, Alizarin red staining is limited to digit tips, metacarpals/tarsals of digits 2–5, and first phalanges of digits 3 and 4 of hindlimbs. In addition, starting at age P0 and continuing through development, phalangeal joints in mutants fail to fully cleave in digits 1 and 5 (insets G’ and H’, with arrowheads showing expected sites of cleavage). (I-L) In P14 mutants, this failure to cleave in hindlimb digit 1 and 5 (asterisks) and all forelimb digits (J) leads to highly dysmorphic joints (insets I’ and J’, with arrowheads as in G’ and H’). At this stage, medial phalanges of hindlimb digits 2 and 5 and forelimb digits 2 through 5 have failed to form. (M-P) By P28, Alcian blue staining appears reduced in mutants compared to controls in all digit joints, particularly in the area of the growth plates and joints (insets O’ and P’, with arrowheads as in G’ and H’). Asterisk in N denotes example of cartilaginous bridge between phalanges that have failed to separate in forelimb digit 3. Arrows in P indicates the stunted metatarsal of digit 1. Controls were identified by phenotype. All experiments, n≥3.

Figure 2.. Fused joints and skeletal malformation in Acvr1^R206H/+;Prrx1-Cre P14 hindlimb digits.

Sagittal sections of the metatarsophalangeal joints of digits 1 (A, B), 3 (C, D), and 5 (E, F) of P14 mutants and control littermates were stained to detect cartilaginous matrix (Alcian blue) and collagens (picrosirius red). While only minimal defects could be detected in digit 3 in mutants, (C, D), digits 1 and 5 of Acvr1^R206H/+ mice exhibited several anomalous features including dorsal-ventral orientation of apparent endochondral ossification (asterisk, B; B’), ectopic cleavage events (arrow, B), incomplete joint cleavage (arrowheads, B, F, and F”) and expanded regions of chondrogenesis (asterisks, B, B’, F, and F’). Ventral, left (with digit sesamoids); dorsal, right. mt, metatarsal; p1, phalanx 1; p2, phalanx 2; ss, sesamoid. n≥7, all groups. Controls were identified by phenotype. Scale bars, 250μm.

Figure 3.. Malformed joints fail to fully cleave in Acvr1^R206H/+;Prrx1-Cre P28 hindlimb digits.

Histological sections of the metatarsophalangeal (MTP) joints of digits 1 (A, B), 3 (C, D), and 5 (E, F) of P28 mutants and control littermates were stained to detect cartilaginous matrix (Alcian blue) and collagens (picrosirius red). (A,B) In digit 1 of mutant mice, endochondral ossification has progressed despite failure to fully cleave at the presumptive MTP joint (arrow, B) and nascent osseous tissue has begun replacing the aberrant growth plate cartilage present at P14 (Fig. 2B), leading to dysmorphic skeletal elements. The metatarsal is also significantly smaller than in controls (mt, B). (C, D) Joints of digit 3 (and other medial digits) remain minimally affected. (E, F) In digit 5 of mutants, 3/7 mice showed cleavage of the MTP joint (arrow, F; compare with arrow and bracket in E, indicating normal separation between bones) and tissue is disorganized throughout the joint. The morphology of the sesamoid is also altered in mutants. mt, metatarsal; p1, phalanx 1; p2, phalanx 2; ss, sesamoid. n≥7, all groups. Controls were identified by phenotype. Scale bars, 250μm.

Figure 4.. Dysregulated BMP-pSmad1/5 pathway activity throughout the developing Acvr1^R206H/+ digit rays.

Whole-mount immunohistochemistry and confocal imaging detected pSMAD1/5 at E12.0 (A-D), E12.5 (E-H), E13.0 (I-L), and E13.5 (M-P) in forelimbs and hindlimbs of control and Acvr1^R206H/+;Prrx1-Cre mice. As indicated in panel M, all panels show digit 1 at left; pSMAD1/5 (blue) is detected in the interdigital tissue (indicated by asterisks) and in the developing skeletal elements (example indicated by arrow) as well as the digit crescent (arrowhead). Arrows in P highlight delayed emergence of discrete zones of BMP signaling in digit rays 3 and 4. All experiments, n≥3. Controls were pooled wild type and single heterozygotes; no differences were observed among control genotypes. Scale bar panel A, 500 μm.

Figure 5.. Gdf5 expression is not appropriately localized to the presumptive joints.

(A) Whole mount in situ hybridization of control E14.5 hindlimb digits showed the expected bands of Gdf5-expressing cells (dark purple) in the developing digit (n=3). Digits are numbered 1 through 5 with developing phalanges numbered 1, 2, or 3 (proximal to distal; bolded numbers). (B) In contrast, Gdf5 expression in mutant littermates is at the digit periphery (arrowheads, B; n=2; also see Supplementary Figure S6); each digit shows loss of at least one band of Gdf5 expression with corresponding reduction in nascent skeletal elements (numbers). (C) Sense probe, negative control. Control mice were pooled from 2 Acvr1^fl/+ heterozygotes and 1 Prrx1-Cre hemizygote; Acvr1^fl/+ is shown). Scale bar is shown in panel A, 500 μm.