Brachy-syndactyly caused by loss of Sfrp2 function

Abstract

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.

Figure 1

Sfrp2 expression pattern by mRNA in situ hybridization. Panel A shows Sfrp2 expression in a full embryo section at E15.5 (composite image). Panels B,C,D,E,F,G,H,I show hematoxylin and eosin stained serial sections of the corresponding panels B′,C′,D′,E′,F′,G′,H′,I′ which were hybridized with the Sfrp2 antisense probe. Sfrp2 specific signal was pseudo-colored in red. The hybridizations with the sense probe were negative and are not shown. (B′,C′,D′) E13.5 distal forelimb sections showing Sfrp2 expression in perichondrium surrounding the digits, in mesenchyme around the carpal bones and in forming joints. (E′,F′) At E15.5 Sfrp2 expression strongly localizes into joints as well as mesenchymal cells around them. Note expression flanking distal and proximal epiphysis of metacarpal and phalangeal elements (arrows). (G′,H′,I′) At E17.5 Sfrp2 expression is still present in joints, although weaker, and becomes more strongly localized in tendons and ligaments, including the achille's and patellar tendons (arrow in G′).

Figure 2

(A) Schematic diagram showing the strategy used to obtain Sfrp2-/- mice. (B) Southern blot genotyping using the 5′ flanking probe and BamHI digestion. (C) Northern blot showing decreased or absent Sfrp2 mRNA transcript in heterozygous or homozygous null mice, respectively, normalized with Gapdh (bottom).

Figure 3

Brachydactyly observed in the Sfrp2-/- mice (in each panels A,B,C,D WT limbs are to the left and Sfrp2-/- to the right). (A) Skeletal preparation of distal limbs of newborn mice stained with Alcian blue (cartilage) and Alizarin red (calcified bone matrix). Note decreased length of all skeletal elements, most evident by looking at their calcified portions (arrowheads point to fifth metacarpal element). Also note consistent lack of ossification centers in the second phalangeal elements of Sfrp2-/- compared with WT littermate mice (arrows). (B) Forelimb skeletal preparations of four days old pups (P4). The overall length of autopod is consistently decreased in the mutant mice. (C) The same is observed in P4 hindlimb skeletal preparations (the length measurement of the third metatarsal is shown). (D) Newborn forelimb skeletal preparations showing mild mesomelic shortening in the mutant mice. (E) Statistical analysis of forelimb autopod bone lengths showing significant shortening of each skeletal elements in the Sfrp2-/- mice versus controls (n=8, p < 10e-15).

Figure 4

Syndactyly observed in the Sfrp2-/- mice. (A) E15.5 distal hind limbs showing unilateral syndactyly between the third and fourth digit. (B) Newborn hind limb section stained with hematoxylin and eosin. The arrow points to failed inter-digital mesenchyme regression with absence of cleavage between third and fourth digit. (C) Incomplete hindlimb syndactyly in an adult Sfrp2 null mouse.

Figure 5

mRNA in situ hybridization analysis of mesenchymal, cartilage and bone differentiation markers at E13.5, E15.5 and newborn (P1) stage (HL= hindlimb). (A) Sox9 and (B) Indian hedgehog antisense probes show comparable pattern of expression between Sfrp2-/- and WT controls. (C) Type X collagen is normally expressed, however the probe detected a delay in chondrocyte differentiation of Sfrp2-/- mice demonstrated by the smaller distance between the hypertrophic zones in the metaphyses of the first phalanx at P1 (arrows). (D) The antisense probe specific for Osteocalcin shows no overt difference at P1 between the two genotypes.

Figure 6

Chondrocyte proliferation defect in Sfrp2-/- distal limb elements. (A) BrdU labeling shows decreased number of BrdU positive proliferating chondrocytes in E17.5 first phalanx sections of Sfrp2-/- versus controls. Also note the consistent hypertrophy delay in mutant phalanges. (B) Percentage of chondrocytes positively labeled with BrdU staining. The difference between WT and Sfrp2-/- mice (n=3) was statistically significant (p<0.05).

Figure 7

Sfrp2 modulation of beta-catenin in vitro and in vivo. (A) Transfection assay using the Top/Fop-flash luciferase reporter system. Wnt1and to a lesser extent, Wnt4 and Wnt9a activated the Top-flash reporter gene, demonstrating that they can signal via the canonical pathway and co-transfection with Sfrp2 inhibited such activation. Wnt5a and Wnt5b had no effect on the reporter gene, suggesting they are not signaling through the canonical pathway in our experimental system. (B) Sections of interphalangeal joint at E15.5 stained with a beta-catenin specific antibody. Note subtle increased accumulation of beta-catenin in the epiphyseal region (arrow) of the Sfrp2-/- mouse (right panel). (C) Sections of first phalanx at E17.5 stained with the same antibody. Notice the increase beta-catenin localization in the epiphyseal area (arrow) in the mutant mouse versus the WT. (D) Sections of first phalangeal elements of TOPGAL/Sfrp2 trangenic mice, showing increased β-galactosidase staining in the articular chondrocytes of some interphalangeal joints of the Sfrp2 null mice (arrow).

Figure 7

Sfrp2 modulation of beta-catenin in vitro and in vivo. (A) Transfection assay using the Top/Fop-flash luciferase reporter system. Wnt1and to a lesser extent, Wnt4 and Wnt9a activated the Top-flash reporter gene, demonstrating that they can signal via the canonical pathway and co-transfection with Sfrp2 inhibited such activation. Wnt5a and Wnt5b had no effect on the reporter gene, suggesting they are not signaling through the canonical pathway in our experimental system. (B) Sections of interphalangeal joint at E15.5 stained with a beta-catenin specific antibody. Note subtle increased accumulation of beta-catenin in the epiphyseal region (arrow) of the Sfrp2-/- mouse (right panel). (C) Sections of first phalanx at E17.5 stained with the same antibody. Notice the increase beta-catenin localization in the epiphyseal area (arrow) in the mutant mouse versus the WT. (D) Sections of first phalangeal elements of TOPGAL/Sfrp2 trangenic mice, showing increased β-galactosidase staining in the articular chondrocytes of some interphalangeal joints of the Sfrp2 null mice (arrow).