Elucidation of Cellular and Molecular Pathogenesis of Maxillo-Mandibular and Facial Malformation and Disorders : Use of Gene-Modified Mice

我们的长期策略是阐明在颌面区发生的各种畸形的发病机制。为此，本项目旨在检查小鼠胚胎发育过程中表型表达（和改变）和相关遗传网络的时空运作。我们特别关注：(1)腭裂形成，这是人类各种畸形中最常见的，(2)梅克尔软骨和下颌骨的发育和异常，(3)颞下颌关节（TMJ）的早期发育和发育不全，(4)内皮素- 1 （i -）小鼠舌头发育和发育不全的细胞事件。利用Trowell器官培养系统进行了体外培养，以监测腭发生和舌形态发生的细胞事件。培养标本用DMEM/Fl2（1:1）培养基在5% C0_2下37℃的加湿培养箱中保存。2004年，采用RT-PCR和real-time PCR技术，用80个探针检测了次上颚和舌头在体内和体外的基因表达模式。我们还继续分析了特定细胞类型的基因表达，即腭发育过程中内侧边缘上皮细胞中的snail和Rho家族，以及通过激光捕获显微解剖分离的舌原基中肌源性物质基因（pax3, MyoD, myogenin）。采用TGF-β、δEF1、内皮素1纯合子（i -）和Klotho （i -）转基因小鼠进行TMJ老化研究。结合三维重建技术，采用组织学和x射线μCT观察转基因小鼠的表型特征。在资助期间收集的影像数据已通过去年购买的计算机服务器在我们的数据库中进行了积累和制表。我们目前正在把它们展示在网站（http://www.ndu.ac.jp/~pathhome）上。

Our long-term strategy is to elucidate pathogenesis of varieties of malformations taking place in the maxillo-facial region. To this objective, the present project aimed to inspect phenotypic expression (and alterations) and relevant genetic network operating temporo-spatially during mouse embryonic development. We specifically focused attention on: (1) palategenesis and cleft formation, which is most frequently encountered among various malformations in humans, (2) development and anomalies of Meckel's cartilage and mandible, (3) early development and hypoplasia of temporomandibular joint (TMJ), and (4) cellular events involved in tongue development and its hypoplasia in Endothelin1 (-I-) mice. Cultures using the Trowell organ culture system were conducted to monitor in vitro cellular events in palatogenesis and tongue morphogenesis. Specimens in culture were maintained in a humidified incubator at 37℃ under 5% C0_2 using DMEM/Fl2 (1:1) medium. In 2004, gene expression patterns in secondary palate and tongue in vivo and in vitro were surveyed using 80 probes for RT-PCR and real-time PCR. We also continued analysis of gene expression in specific cell types, namely, snail and Rho family in medial edge epithelial cells during palategenesis and myogenic mater genes (Pax 3, MyoD, myogenin) in tongue primordium, which were isolated by laser-capture microdissection. Transgenic mice used were homozygous (-I-) for TGF-β, δEF1 or endothelin1, as well as Klotho (-I-) used for investigation of TMJ aging. Phenotypic features of these transgenic mice were scrutinized by means of histology and x-ray μCT in combination of 3D-reconstruction techniques. Imaging data collected during the funded period have been accumulated and tabulated in our database with the aid of a computer server, which was purchased in the last year. We at present are in the process to display them on the Web-site (http://www.ndu.ac.jp/~pathhome).