Molecular Patterning of Mammalian Dentition

描述（由申请人提供）：牙齿发育长期以来一直被用作研究调节器官发生的分子机制和人类牙齿发育异常的致病机制的强大模型系统。牙齿发育的一个关键步骤是激活神经嵴来源的牙齿间充质中的成牙潜能，其负责从牙芽期驱动牙齿形态发生，并且当与非牙上皮重组时足以诱导牙齿器官发生。未能激活间充质成牙潜能与牙芽发育停滞有关，如在缺乏Msx 1转录因子的小鼠中所见。MSX 1的突变也与人类牙齿发育不全有关。以往的研究表明Bmp 4是Msx 1下游的一个重要的牙源性信号。Bmp 4 mRNA在Msx 1-/-牙间充质中表达下调。然而，我们发现，神经嵴特异性失活的Bmp 4基因的小鼠表现出的发展停滞，只有下颌磨牙芽，但他们的上颌磨牙和门牙发展成矿化的牙齿。我们对显微切割的牙芽间充质进行了RNAseq分析，发现下颌磨牙间充质比上颌磨牙间充质表达更高水平的分泌型Wnt拮抗剂。我们发现，在早期牙齿间充质中，经典Wnt信号的特异性转导子β-连环蛋白的组织特异性失活导致牙芽发育停滞，这表明下颌磨牙间充质中更高水平的Wnt拮抗剂导致Bmp 4突变小鼠中上颌和下颌磨牙牙缺损的显著差异.此外，我们最近发现，Osr 2转录因子的失活导致小鼠从口腔上皮舌到磨牙牙胚的多生牙形成。Osr 2以梯度模式表达，并抑制发育中的牙齿间充质沿沿着颊舌轴的间充质牙源性潜力域。而Msx 1-/-突变小鼠的牙齿发育停滞在芽期，Msx 1-/-Osr 2-/-双突变体表现出几乎正常的第一磨牙形态发生，但没有启动多生牙的发展。此外，我们发现，Msx 1和Osr 2调节拮抗分泌的Wnt拮抗剂在发育中的牙齿间充质的表达。总之，这些数据表明，新的机制，涉及BMP，Msx 1，Osr 2，和Wnt信号通路的调节间充质牙的潜力。该项目的主要目的是识别，表征和整合这些因素和途径相结合的分子机制，调节早期牙形成，使用多方面的实验方法，包括在体内的药理学救援，纳克级RNAseq为基础的基因表达谱，并结合外植体器官培养和遗传功能测定。该项目的数据将显著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，并将促进新治疗策略的开发。

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 ß-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.