The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling
Yukio Nakamura, Gilbert Weidinger, Jennifer O. Liang, Allisan Aquilina-Beck, Keiko Tamai, Randall T. Moon, Matthew L. Warman
Yukio Nakamura, Gilbert Weidinger, Jennifer O. Liang, Allisan Aquilina-Beck, Keiko Tamai, Randall T. Moon, Matthew L. Warman
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The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling

Abstract

In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium–containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor–related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.

Authors

Yukio Nakamura, Gilbert Weidinger, Jennifer O. Liang, Allisan Aquilina-Beck, Keiko Tamai, Randall T. Moon, Matthew L. Warman

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Figure 4

Overexpression of zWisp3 in zebrafish inhibits BMP signaling.

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Overexpression of zWisp3 in zebrafish inhibits BMP signaling. (A) Wester...
(A) Western blot of zWisp3 protein extracted from deyolked embryos and larvae injected with 150 pg zwisp3 RNA. Protein (40 μg) was separated by 10% SDS-PAGE under reducing conditions and detected using WISP3-C or anti–β-tubulin antibody. zWisp3 protein was detectable by 10 hpf when overexpressed. (B) Photographs of live 24-hpf embryos depicting the phenotypic classes of dorsalization. Embryos classified as mild correspond to convergence-extension classes C1 and C2, medium to C3 and C4, and severe to C5, as previously described (30). (C) Phenotype frequencies (shown in B) caused by injection of wild-type, C78R, C145Y, and Q338L zwisp3 RNA. Mutants had reduced biologic activity compared with wild-type zWisp3. (D) Photographs of 60% epiboly embryos oriented with their dorsal domains to the right. Arrowheads indicate extent of strong expression. Expression of flh was strongly expanded in wild-type zwisp3–injected embryos and less dramatically so in zwisp3 C78R–injected embryos. Expression of eve1 was strongly reduced in wild-type zwisp3–injected embryos and less dramatically reduced in C78R-injected embryos. (E) Photographs of live 24-hpf embryos depicting phenotypic classes of ventralization caused by injection of zbmp2b RNA as previously reported (30). (F) Phenotype frequencies (shown in E) caused by injection of zbmp2b RNA plus equimolar amounts of control (luciferase), zwisp3 wild-type, or zwisp3 missense mutant RNAs. C145Y and Q338L did not inhibit BMP signaling, while wild-type and C78R RNAs substantially rescued the defects caused by zbmp2b overexpression.