Van der Heyden C, Allizard F, Sire J-Y, Huysseune A
Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium.
Cell Tissue Res. 2005 Sep;321(3):375-89. doi: 10.1007/s00441-004-1036-x. Epub 2005 Jun 21.
A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.
一种使用无血清培养基进行器官型体外培养的技术，已针对其是否适合模拟双斑半丽鱼（Hemichromis bimaculatus）和斑马鱼（Danio rerio）的正常牙发生过程进行了测试。通过光学显微镜观察连续半薄切片，以收集有关牙齿模式的数据，并使用透射电子显微镜比较体外发育的牙胚与体内情况的细胞和细胞外特征。双斑半丽鱼受精后120小时（hPF）至受精后8.5天（dPF）的头部外植体，以及斑马鱼受精后45 hPF至79 hPF的头部外植体和在培养中保存3、4或7天的成鱼，结果显示，外植体中颊部或咽部上皮明显未分化时，牙胚可在体外发育，并且在植入时若存在牙胚，它们会持续发育直至附着阶段。此外，该培养基能使牙胚发生形态发生和细胞分化，类似于体内观察到的情况，并能建立模仿体内的牙齿模式（牙齿出现和附着的位置及顺序）。因此，无血清条件下的器官型培养为我们提供了研究硬骨鱼类牙齿发育过程中上皮-间充质相互作用的方法，以及分析这些多牙列物种下颌或咽部牙齿发育及替换的遗传控制的方法。重要的是，它能使来自胚胎致死（斑马鱼）突变体或早期致死基因敲低实验的头部发育到胚胎正常死亡点之后。因此，这种无血清条件下的器官型培养可能成为发育研究中的有力工具，并为颅面研究开辟新的前景。
