Song Yiqiang, Zhang Zunyi, Yu Xueyan, Yan Minquan, Zhang Xiaoyun, Gu Shuping, Stuart Thomas, Liu Chao, Reiser Jakob, Zhang Yanding, Chen Yiping
Division of Developmental Biology, Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118, USA.
Dev Dyn. 2006 May;235(5):1334-44. doi: 10.1002/dvdy.20706.
RNA interference (RNAi) has recently become a powerful tool to silence gene expression in mammalian cells, but its application in assessing gene function in mammalian developing organs remains highly limited. Here we describe several unique developmental properties of the mouse molar germ. Embryonic molar mesenchyme, but not the incisor mesenchyme, once dissociated into single cell suspension and re-aggregated, retains its odontogenic potential, the capability of a tissue to instruct an adjacent tissue to initiate tooth formation. Dissociated molar mesenchymal cells, even after being plated in cell culture, retain odontogenic competence, the capability of a tissue to respond to odontogenic signals and to support tooth formation. Most interestingly, while dissociated epithelial and mesenchymal cells of molar tooth germ are mixed and re-aggregated, the epithelial cells are able to sort out from the mesenchymal cells and organize into a well-defined dental epithelial structure, leading to the formation of a well-differentiated tooth organ after sub-renal culture. These unique molar developmental properties allow us to develop a strategy using a lentivirus-mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development. We show that knockdown of Msx1 or Dlx2 expression in the dental mesenchyme faithfully recapitulates the tooth phenotype of their targeted mutant mice. Silencing of Barx1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Barx1 in tooth formation. Our studies have established a reliable and rapid assay that would permit large-scale analysis of gene function in mammalian tooth development.
RNA干扰（RNAi）最近已成为在哺乳动物细胞中使基因表达沉默的强大工具，但其在评估哺乳动物发育器官中的基因功能方面的应用仍然非常有限。在此，我们描述了小鼠磨牙胚的几个独特的发育特性。胚胎磨牙间充质，而非切牙间充质，一旦解离成单细胞悬液并重新聚集，仍保留其牙源性潜能，即一种组织指导相邻组织启动牙齿形成的能力。解离的磨牙间充质细胞，即使接种在细胞培养物中，仍保留牙源性能力，即一种组织响应牙源性信号并支持牙齿形成的能力。最有趣的是，当磨牙胚的解离上皮细胞和间充质细胞混合并重新聚集时，上皮细胞能够从间充质细胞中分离出来并组织成明确的牙上皮结构，在肾下培养后导致形成分化良好的牙齿器官。这些独特的磨牙发育特性使我们能够开发一种策略，使用慢病毒介导的RNAi方法使牙间充质细胞中的基因表达沉默，并评估牙齿发育中的基因功能。我们表明，在牙间充质中敲低Msx1或Dlx2的表达忠实地重现了其靶向突变小鼠的牙齿表型。在牙间充质中沉默Barx1的表达会导致牙齿发育在芽期停滞，证明Barx1在牙齿形成中起关键作用。我们的研究建立了一种可靠且快速的检测方法，可用于大规模分析哺乳动物牙齿发育中的基因功能。
