Summary Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome.

Dentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD-II) and dentinogenesis imperfecta (DGI) types II and III. We identified two kindreds with DGI-II exhibiting vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged DPP sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their likely effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice support the hypothesis that all DSPP mutations cause pathology through dominant-negative effects. Noting that Dspp is transiently expressed by preameloblasts during formation of the dentino-enamel junction, we hypothesize that DSPP dominant-negative effects potentially cause cellular pathology in preameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.