EVC2: A novel modulator in tooth development

描述（由申请人提供）：Ellis-van Creveld (EVC) 综合征是一种常染色体隐性遗传性软骨发育不良伴侏儒症。 EVC患者的唇部由系带至牙槽嵴相连，也被描述为系带增生伴浅唇沟，这是该综合征所特有的。他们还有几种主要的牙齿异常，包括新生牙、部分无牙、钉状牙、恒牙萌出延迟和早期龋齿。由于这些牙齿表现不仅仅是由发育迟缓引起的，因此对这种特殊综合征的研究将为了解牙齿发育提供新的见解。现在已经明确，EVC 综合征是由 EVC 或 EVC2 基因突变引起的，这两个基因均位于人类染色体 4p 上，呈头对头配置。在日本褐牛中发现的牛软骨发育不良性侏儒症 (bcd) 致病基因 LIMBIN 后来被鉴定为 EVC2 的牛直系同源基因，表明了 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，通过在外显子 12 中引入提前终止密码子来生成 Evc2 突变小鼠，模仿 EVC 患者和 bcd 牛中发现的突变。由于 EVC 综合征中发现的大多数突变都是无义突变，因此它们会导致肽过早终止，从而导致“功能丧失”。这些纯合突变小鼠表现出与 EVC 患者类似的严重侏儒症和牙齿异常，表明该疾病的潜在动物模型。在我们最初的表征过程中，我们在突变小鼠中发现了几种主要的牙齿表型。所有牙齿的牙釉质形成普遍发育不全。虽然门牙能够持续生长，但长度较短，成年期上门牙萌出不良（有时不萌出），方向异常。在胚胎第 18.5 天，成釉细胞的分化明显受到抑制，缺乏细胞极化，没有明显的微管、牙釉蛋白分泌不良和基质金属蛋白酶 (MMP)-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“错误”定位于细胞质。此外，与WT小鼠相比，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性，并且这些细胞中的JNK磷酸化水平显着降低。这些初步数据使我们推测 EVC2 对于成釉细胞微管的形成至关重要，微管控制牙齿发育过程中的细胞外基质 (ECM)/MMP 蛋白运输和细胞迁移。为了检验这一假设，提出以下具体目标；目标1。表征 Evc2 突变小鼠的牙齿发育。目标2。研究人类 EVC2 突变对成釉细胞功能的影响。目标3。通过 EVC2 突变拯救成釉细胞表型。这项研究获得的数据将为EVC2在成釉细胞功能中的病理生理功能和牙齿发育的分子机制提供新的见解。 公众健康相关性：患有 Ellis-van Creveld (EVC) 综合征的人表现出口腔/牙齿异常，包括牙齿缺失/小。为了研究EVC2基因突变引起的EVC患者牙齿异常的机制，我们培育了Evc2突变小鼠。我们研究的目的是确定这些小鼠何时以及如何产生异常牙齿，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.