Ellis-van Creveld (EVC) syndrome is an autosomal recessive chondrodysplasia. The affected individuals bear a series of skeleton defects, congenital heart septum anomalies, midfacial defects, and dental defects. Previous studies using Evc or Evc2 mutant mice have characterized the pathological mechanism leading to various types of congenital defects. Some patients with EVC have supernumerary tooth; however, it is not known yet if there are supernumerary tooth formed in Evc or Evc2 mutant mice, and if yes, what is the pathological mechanism associated. In the present study, we used Evc2 mutant mice and analyze the pattern of molars in Evc2 mutant mice at various stages. Our studies demonstrate that Evc2 loss of function within the dental mesenchymal cells leads to abnormal molar patterning, and that the most anterior molar in the Evc2 mutant mandible represents a supernumerary tooth. Finally, we provide evidence supporting the idea that both compromised Hedgehog signaling and elevated WNT signaling due to Evc2 loss of function contributes to the supernumerary tooth formation.

Ellis-van Creveld syndrome (EVC; MIM ID #225500) is a rare congenital disease with an occurrence of 1 in 60,000. It is characterized by remarkable skeletal dysplasia, such as short limbs, ribs and polydactyly, and orofacial anomalies. With two of three patients first noted as being offspring of consanguineous marriage, this autosomal recessive disease results from mutations in one of two causative genes: EVC or EVC2/LIMBIN. The recent identification and manipulation of genetic homologs in animals has deepened our understanding beyond human case studies and provided critical insight into disease pathogenesis. This review highlights the utility of animal-based studies of EVC by summarizing: (1) molecular biology of EVC and EVC2/LIMBIN, (2) human disease signs, (3) dysplastic limb development, (4) craniofacial anomalies, (5) tooth anomalies, (6) tracheal cartilage abnormalities, and (7) EVC-like disorders in non-human species.

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplastic disorder. Affected patients present a wide spectrum of symptoms including short stature, postaxial polydactyly, and dental abnormalities. We previously disrupted Evc2, one of the causative genes for EvC syndrome, in mice using a neural crest-specific, Cre-mediated approach (i.e., P0-Cre, referred to as Evc2 P0 mutants). Despite the fact that P0-Cre predominantly targets the mid-facial region, we reported that many mid-facial defects identified in Evc2 global mutants are not present in Evc2 P0 mutants at postnatal day 8 (P8). In the current study, we used multiple Cre lines (P0-Cre and Wnt1-Cre, respectively), to specifically delete Evc2 in neural crest-derived tissues and compared the resulting mid-facial defects at multiple time points (P8 and P28, respectively). While both Cre lines indistinguishably targeted the mid-facial region, they differentially targeted the anterior portion of the skull base. By comprehensively analyzing the shapes of conditional mutant skulls, we detected differentially affected mid-facial defects in Evc2 P0 mutants and Evc2 Wnt1 mutants. Micro-CT analysis of the skull base further revealed that the Evc2 mutation leads to a differentially affected skull base, caused by premature closure of the intersphenoid synchondrosis (presphenoidal synchondrosis), which limited the elongation of the anterior skull base during the postnatal development of the skull. Given the importance of the skull base in mid-facial bone development, our results suggest that loss of function of Evc2 within the skull base secondarily leads to many aspects of the mid-facial defects developed by the EvC syndrome.

EvC syndrome is a type of autosomal-recessive chondrodysplasia. Previous case studies in patients suggest abnormal craniofacial development, in addition to dwarfism and tooth abnormalities. To investigate how craniofacial development is affected in EvC patients, surface models were generated from micro-CT scans of control mice, Evc2 global mutant mice and Evc2 neural crest-specific mutant mice. The anatomic landmarks were placed on the surface model to assess the morphological abnormalities in the Evc2 mutants. Through analyzing the linear and angular measurements between landmarks, we identified a smaller overall skull, shorter nasal bone, shorter frontal bone, and shorter cranial base in the Evc2 global mutants. By comparing neural crest-specific Evc2 mutants with control mice, we demonstrated that the abnormalities within the mid-facial regions are not accounted for by the Evc2 mutation within these regions. Additionally, we also identified disproportionate length to width ratios in the Evc2 mutants at all levels from anterior to posterior of the skull. Overall, this study demonstrates a more comprehensive analysis on the craniofacial morphological abnormalities in EvC syndrome and provides the developmental insight to appreciate the impact of Evc2 mutation within the neural crest cells on multiple aspects of skull deformities. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:46-55, 2018. © 2017 Wiley Periodicals, Inc.

Ellis-van Creveld (EvC) syndrome is an autosomal-recessive skeletal dysplasia, characterized by short stature and postaxial polydactyly. A series of dental abnormalities, including hypomorphic enamel formation, has been reported in patients with EvC. Despite previous studies that attempted to uncover the mechanism leading to abnormal tooth development, little is known regarding how hypomorphic enamel is formed in patients with EvC. In the current study, using Evc2/ Limbin mutant mice we recently generated, we analyzed enamel formation in the mouse incisor. Consistent with symptoms in human patients, we observed that Evc2 mutant mice had smaller incisors with enamel hypoplasia. Histologic observations coupled with ameloblast marker analyses suggested that Evc2 mutant preameloblasts were capable of differentiating to secretory ameloblasts; this process, however, was apparently delayed, due to delayed odontoblast differentiation, mediated by a limited number of dental mesenchymal stem cells in Evc2 mutant mice. This concept was further supported by the observation that dental mesenchymal-specific deletion of Evc2 phenocopied the tooth abnormalities in Evc2 mutants. Overall, our findings suggest that mutations in Evc2 affect dental mesenchymal stem cell homeostasis, which further leads to hypomorphic enamel formation.

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. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc.