EVC2: A novel modulator in tooth development

描述(申请人提供)：Ellis-van Creveld(EVC)综合征是一种伴有侏儒症的常染色体隐性遗传性软骨发育不良。EVC患者的嘴唇短，由系带与牙槽脊结合，也被描述为伴有浅唇沟的肾增生，这是该综合征所特有的。他们也有几个主要的牙齿异常，包括新生儿牙齿、部分缺牙、钉形牙齿、恒牙延迟萌出和早期龋齿。由于这些牙齿表现不是简单地由发育迟缓引起的，对这种特殊综合征的研究将为理解牙齿发育提供新的见解。现在很清楚，EVC综合征是由EVC或EVC2基因突变引起的，这两个基因都以头对头的形式位于人类染色体4p上。在日本褐牛中发现的引起牛软骨发育不良侏儒症(BCD)的基因Limbin后来被鉴定为EVC2的牛同源基因，表明EVC2蛋白功能的重要性。为了研究EVC患者侏儒症/牙齿发育的病理生理机制，通过在EVC患者和BCD牛中发现突变，在外显子12引入提前终止密码子，产生了Evc2突变小鼠。由于在EVC综合征中发现的大多数突变都是无义突变，它们会导致多肽提前终止，从而导致功能丧失。这些纯合子突变小鼠表现出与EVC患者相似的严重侏儒症和牙齿异常，表明可能是这种疾病的动物模型。在我们的初步鉴定中，我们在突变小鼠中发现了几种主要的牙齿表型。所有牙齿在釉质形成过程中普遍发育不良。虽然切牙能够持续生长，但长度较短，上切牙在成人期萌出较少(有时不萌出)，方向异常。胚胎18.5天，成釉细胞分化明显受阻，细胞极化消失，未见明显的微管，釉原分泌减少，基质金属蛋白酶-2表达改变。体外研究表明，野生型(WT)Evc2定位于成釉细胞系LS8的高尔基体，而突变型Evc2定位于细胞质。此外，来自Evc2突变小鼠的小鼠胚胎成纤维细胞表现出较差的细胞迁移，并且与WT小鼠相比，这些细胞中JNK的磷酸化水平显著降低。这些初步数据使我们假设，在牙齿发育过程中，EVC2对于成釉细胞微管的形成至关重要，成釉细胞微管控制着细胞外基质(ECM)/基质金属蛋白酶蛋白的运输和细胞的迁移。为了检验这一假说，提出了以下具体目标：研究Evc2基因突变小鼠牙齿发育的特点。AIM2.目的探讨人EVC2基因突变对成釉细胞功能的影响。Aim3.通过EVC2突变挽救成釉细胞表型。本研究为进一步研究EVC2在成釉细胞中的病理生理功能和牙齿发育的分子机制提供了新的思路。 与公共卫生相关：埃利斯-范-克里夫尔德综合征(EVC)患者表现为口腔/牙齿异常，包括缺牙/小牙。为了探讨EVC2基因突变导致EVC患者牙齿异常的机制，我们建立了EVC2突变小鼠。我们的研究目的是确定这些小鼠何时以及如何发育出异常牙齿，研究Evc2突变对牙齿细胞运动的影响，并挽救Evc2突变的影响。

DESCRIPTION (provided by applicant): 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.