Molecular Patterning of Mammalian Dentition

描述（申请人提供）：长期以来，牙齿发育一直是研究人类牙齿发育异常分子机制调控器官发生和致病机制的有力模型系统。牙发育的一个关键步骤是神经嵴源性牙间质的成牙电位激活，它负责驱动牙芽期的牙齿形态发生，并且当与非牙上皮细胞重组时足以诱导牙齿器官发生。在缺乏Msx1转录因子的小鼠中，不能激活间充质成牙潜能与牙芽发育停滞有关。MSX1的突变也与人类牙齿发育有关。先前的研究表明Bmp4是Msx1下游重要的牙源性信号。Msx1-/-牙间质中Bmp4 mRNA表达下调。然而，我们发现神经嵴特异性失活Bmp4基因的小鼠仅表现出下颌磨牙芽发育停滞，而上颌磨牙和门牙发育为矿化牙。我们对微解剖的牙芽间质进行了RNAseq分析，发现下颌磨牙间质比上颌磨牙间质表达更高水平的分泌Wnt拮抗剂。我们发现？-catenin是早期牙齿间质中典型Wnt信号的必要换能器，它导致了牙芽发育停滞，这表明下颌磨牙间质中较高水平的Wnt拮抗剂导致了Bmp4突变小鼠上颌和下颌磨牙缺陷的显著差异。此外，我们最近发现Osr2转录因子失活导致小鼠从口腔舌上皮到臼齿细菌形成多余的牙齿。Osr2以梯度模式表达，并沿发育中的牙间质颊舌轴抑制间质成牙电位域。Msx1-/-突变体小鼠的牙齿发育在萌芽阶段就停止了，而Msx1-/- osr2 -/-双突变体小鼠的第一磨牙形态发生几乎正常，但没有产生多余的牙齿发育。此外，我们发现Msx1和Osr2对发育中的牙齿间质分泌的Wnt拮抗剂的表达具有拮抗作用。综上所述，这些数据提示了涉及Bmp、Msx1、Osr2和Wnt信号通路调控间充质成牙潜能的新机制。本项目的主要目的是利用多方面的实验方法，包括体内药物救援、基于纳克尺度rnaseq的基因表达谱、外植体器官培养和遗传功能分析，识别、表征和整合结合这些因素和途径调节早期牙形成的分子机制。该项目的数据将大大提高目前对牙齿发育和多种人类出生缺陷的分子机制的理解，并将促进新的治疗策略的发展。

DESCRIPTION (provided by applicant): Tooth development has long been used as a powerful model system for studying the molecular mechanisms regulating organogenesis and the pathogenic mechanisms of tooth developmental anomalies in humans. A critical step in tooth development is the activation of odontogenic potential in the neural crest-derived tooth mesenchyme, which is responsible for driving tooth morphogenesis from the tooth bud stage and is sufficient to induce tooth organogenesis when recombined with non-dental epithelium. Failure to activate mesenchymal odontogenic potential is associated with tooth bud developmental arrest, as seen in mice lacking the Msx1 transcription factor. Mutations in MSX1 are also associated with tooth agenesis in humans. Previous studies suggested that Bmp4 is an important odontogenic signal downstream of Msx1. Bmp4 mRNA expression was downregulated in Msx1-/- tooth mesenchyme. However, we found that mice with neural crest specific inactivation of the Bmp4 gene exhibit developmental arrest of only the mandibular molar buds but their maxillary molars and incisors developed to mineralized teeth. We carried out RNAseq analyses of microdissected tooth bud mesenchyme and found that the mandibular molar mesenchyme expresses much higher levels of secreted Wnt antagonists than does the maxillary molar mesenchyme. We found that tissue- specific inactivation of ?-catenin, the obligatory transducer of canonical Wnt signaling, in the early tooth mesenchyme caused tooth bud developmental arrest, suggesting that the higher levels of Wnt antagonists in mandibular molar tooth mesenchyme contribute to the dramatic differences in maxillary and mandibular molar tooth defects in the Bmp4 mutant mice. Moreover, we discovered recently that inactivation of the Osr2 transcription factor caused supernumerary tooth formation from oral epithelium lingual to the molar tooth germs in mice. Osr2 is expressed in a gradient pattern and suppressed the domain of mesenchymal odontogenic potential along the buccolingual axis of the developing tooth mesenchyme. Whereas Msx1-/- mutant mice had tooth development arrested at the bud stage, Msx1-/-Osr2-/- double mutants exhibited nearly normal first molar morphogenesis but did not initiate supernumerary tooth development. Moreover, we found that Msx1 and Osr2 regulate antagonistically the expression of secreted Wnt antagonists in the developing tooth mesenchyme. Together, these data suggest novel mechanisms involving Bmp, Msx1, Osr2, and Wnt signaling pathways in the regulation of mesenchymal odontogenic potential. The major aims of this project are to identify, characterize, and integrate the molecula mechanisms combining these factors and pathways regulating early odontogenesis, using multifaceted experimental approaches, including in vivo pharmacological rescue, nanogram scale RNAseq-based gene expression profiling, and combined explant organ culture and genetic functional assays. Data from this project will significantly improve the current understanding of the molecular mechanisms of tooth development and multiple human birth defects and will facilitate development of new therapeutic strategies.