Ellis-van Creveld (EVC) syndrome is an autosomal recessive chondrodysplasia with dwarfism. The EVC patients have a short lip bound by frenum to alveolar ridge, which is also described as hyperplastic frena with a shallow labial sulcus, unique to this syndrome. They also have several major dental anomalies including neonatal teeth, partial anodontia, peg-shaped teeth, delayed eruption of permanent teeth and early involvement with caries. Since these dental manifestations are not simply caused by developmental delay, an investigation in this particular syndrome will provide a new insight into the understanding of tooth development. It is now clear that EVC syndrome is caused by mutations in either EVC or EVC2 gene, both of which are located on human chromosome 4p in a head-to-head configuration. A causative gene, LIMBIN for bovine chondrodysplastic dwarfism (bcd) found in Japanese brown cattle was later identified as the bovine ortholog of EVC2, indicating the significance of EVC2 protein function. In order to investigate the pathophysiological mechanism of dwarfism/tooth development seen in EVC patients, Evc2 mutant mice were generated by introducing a premature stop codon in exon12, mimicking mutations found in EVC patients and bcd cattle. As the majority of mutations identified in EVC syndrome are non-sense mutations, they result in premature termination of the peptide causing 'loss of function'. These homozygous mutant mice showed severe dwarfism with dental anomalies similar to EVC patients, indicating a potential animal model of this disease. During our initial characterization, we found several major dental phenotypes in the mutant mice. All teeth were generally hypoplastic in enamel formation. Although incisors were able to grow continuously, they were short in length and upper incisors were poorly erupted (sometimes not erupted) with abnormal direction at the adult stage. At embryonic day 18.5, differentiation of ameloblasts was clearly arrested lacking cell polarization with no apparent microtubules, poor amelogenin secretion and altered matrix metalloproteinase (MMP)-2 expression. The in vitro studies demonstrated that wild type (WT) Evc2 is localized at the Golgi in an ameloblastic cell line, LS8, whereas mutant Evc2 is """"""""mis""""""""localized at the cytoplasm. Moreover, mouse embryonic fibroblasts derived from the Evc2 mutant mouse exhibited poor cell migration and the level of JNK phosphorylation in these cells was markedly reduced when compared to those from WT mouse. These preliminary data led us to hypothesize that EVC2 is critical for the formation of microtubules of ameloblasts that control extracellular matrix (ECM)/MMP protein trafficking and cell migration during tooth development. In order to test this hypothesis, the following specific aims are proposed;
Aim1. To characterize the tooth development in Evc2 mutant mice.
Aim2. To investigate the effects of human EVC2 mutations on ameloblast cell function.
Aim3. To rescue the ameloblast phenotypes by EVC2 mutation. The data obtained from this study will provide new insights into the pathophysiological function of EVC2 in ameloblast cell function and the molecular mechanism for tooth development.

Public Health Relevance

People suffering from Ellis-van Creveld (EVC) syndrome show oral/dental abnormalities including missing/small teeth. In order to investigate the mechanism of dental abnormalities in EVC patients caused by a mutation in EVC2 gene, we have generated Evc2 mutant mice. The goal of our study is to determine when and how these mice develop abnormal teeth, to investigate the effect of Evc2 mutation on the cell movement in teeth, and to rescue the effect of Evc2 mutation.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
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Special Emphasis Panel (ZRG1-MOSS-K (09))
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Scholnick, Steven
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Boston University
Schools of Dentistry
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Kwon, Edwin K; Louie, Ke'ale; Kulkarni, Anshul et al. (2018) The Role of Ellis-Van Creveld 2(EVC2) in Mice During Cranial Bone Development. Anat Rec (Hoboken) 301:46-55
Kulkarni, Anshul K; Louie, Ke'ale W; Yatabe, Marilia et al. (2018) A Ciliary Protein EVC2/LIMBIN Plays a Critical Role in the Skull Base for Mid-Facial Development. Front Physiol 9:1484
Zhang, H; Takeda, H; Tsuji, T et al. (2017) Loss of Function of Evc2 in Dental Mesenchyme Leads to Hypomorphic Enamel. J Dent Res 96:421-429
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Badri, Mohammed K; Zhang, Honghao; Ohyama, Yoshio et al. (2016) Ellis Van Creveld2 is Required for Postnatal Craniofacial Bone Development. Anat Rec (Hoboken) 299:1110-20
Zhang, Honghao; Takeda, Haruko; Tsuji, Takehito et al. (2015) Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation. Genesis 53:612-626
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