Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb

Abstract

The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate beta-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(-/-) mice. Consistent with the ligand role of R-spondins in the Wnt/beta-catenin signaling pathway, expression of Axin2 and Sp8, targets for beta-catenin signaling, within AER was greatly reduced in Rspo2(-/-) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(-/-) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/beta-catenin signaling.

A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis

“…Animals-Mice carrying the Rspo2 null (Rspo2 Ϫ/Ϫ ) allele were described previously (20). A second mutant allele of the Rspo2 (Rspo2 ⌬ZN ) gene in which the LacZ and neo gene cassettes were removed by Flp-dependent recombination was generated.…”

“…WNT reporter (TopGAL) mice (21) were obtained from The Jackson Laboratory (Bar Harbor, ME). The Rspo2 null and Rspo2 ⌬ZN alleles and TopGAL transgene were genotyped by polymerase chain reaction (PCR) as described (20) and according to protocols available from The Jackson Laboratory, respectively. Mice were housed in a pathogen-free air barrier facility, and animal handling and procedures were approved by the Maine Medical Center Institutional Animal Care and Use Committee.…”

“…Whole Mount in Situ Hybridization and ␤-Galactosidase Staining-Whole mount in situ hybridization was performed as described (20). To visualize expression of the LacZ gene encoding ␤-galactosidase (␤-GAL), freshly collected embryos were fixed with 0.2% glutaraldehyde for 15 min at room temperature and stained with X-Gal substrate (Invitrogen) overnight at 37°C.…”

Secreted and Transmembrane Wnt Inhibitors and Activators

“…Footless is a mouse mutant carrying a hypomorphic allele of Rspo2 (Rspo2 Tg/Tg ) (Bell et al 2008). Rspo2 knockout mice were generated (Nam et al 2007a;Aoki et al 2008;Bell et al 2008;Yamada et al 2009;Jin et al 2011). All these animals immediately die after birth and display hindlimb defects, severe laryngeal and tracheal malformations, lung hypoplasia, and branching defects.…”

“…In particular, abnormal limb development is manifested through a delayed maturation of the AER. Expression of Axin2 and the TopGAL transgene is significantly reduced in the AER of Rspo2 mutant mice, indicating that Rspo2 activity in the AER is mediated by Wnt/ b-catenin signaling (Nam et al 2007a;Bell et al 2008). Furthermore, double-knockout mutants of Rspo2 and LRP6 (Rspo2 Tg/Tg /LRP6 2/2 ) showed much more severe limb defects than the single-knockouts, indicating that both genes functionally interact to regulate limb development (Bell et al 2008).…”