Koyama Eiki, Wu Changshan, Shimo Tsuyoshi, Pacifici Maurizio
Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Dev Dyn. 2003 Jan;226(1):149-54. doi: 10.1002/dvdy.10217.
Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4-5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.
目前，小鼠牙胚发育主要通过三种方法进行研究：在野生型和突变小鼠品系中研究、将牙胚移植到异位部位后进行研究以及在器官培养中进行研究。体内研究方法最为贴近生理状态，但无法对牙胚进行进一步的实验操作。器官培养虽然易于操作，但无法维持牙胚的完全发育，适合进行短期分析。因此，我们试图建立一种新方法，将实验可及性与持续发育相结合。我们将含有早期芽阶段牙胚的胚胎第12天小鼠胚胎第一鳃弓片段植入4-5日龄鸡胚翼芽的侧方间充质中进行体内培养。将鸡蛋重新孵化，并随着时间的推移通过组织化学和原位杂交对植入组织进行检查。牙胚经历了看似正常的生长、分化和形态发生。它们分别在大约3天、6天和10天达到帽状期、钟状期和冠期，惊人地模拟了自然的时间模式。为了研究调节牙胚发育的机制，我们首先植入牙胚片段，用针对关键信号分子音猬因子（Shh）的中和抗体对其进行显微注射，并随着时间的推移对其进行检查。牙胚发育明显延迟，表现为形态发生不良以及缺乏显示出如细胞形状拉长、核重新定位和成釉蛋白基因表达等特征性性状的成熟成釉细胞和成牙本质细胞。进一步孵育后，这些表型变化开始逆转。数据表明，肢体芽代表了一种用于发育牙胚生长和分析的有效、实验可及且经济的系统。本文结合本研究组和其他研究组先前提出的该信号通路的作用，讨论了Shh中和抗体处理的抑制作用。
