Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Msx1-/- tooth mesenchyme Transcriptomes

Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1-/- mice exhibit developmental arrest of all tooth germs at the bud stage, however, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1-/- and Bmp4ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1-/- and Bmp4ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of Dkk in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice. Furthermore, whereas inhibition of Dkk alone was insufficient to rescue tooth morphogenesis in Msx1-/-mice, pharmacological inhibition of Dkk in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1-/- mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway drives tooth organogenesis by activating Wnt signaling via regulation of the secreted Wnt antagonists. E14 mouse embryos tooth germs were micro-dissceted by LCM, mandibular molar and maxillary molar were seperated, 3 pairs of control and mutant samples were pooled for the RNA extractionMSX1基因突变可导致人类与小鼠出现包括牙齿发育不全在内的颅面部发育缺陷。既往研究显示，Msx1可在发育中的牙齿间充质中激活Bmp4的表达，以驱动早期牙齿器官发生。Msx1敲除（Msx1-/-）小鼠的全部牙胚均停滞于牙蕾期发育，然而，神经嵴特异性灭活Bmp4的小鼠（Bmp4ncko/ncko，该模型在发育中的牙齿间充质中缺失Bmp4表达）仅表现为下颌磨牙发育停滞。我们近期的研究证实，敲除Osr2——该基因编码一种在发育中的牙蕾间充质中呈舌侧-颊侧梯度表达的锌指转录因子——可挽救Msx1-/-与Bmp4ncko/ncko小鼠的磨牙牙形态发生。 本研究通过对突变体与野生型胚胎的发育牙齿间充质进行RNA测序（RNA-seq）分析，发现Msx1与Osr2对牙蕾间充质内多种分泌型Wnt拮抗剂的表达具有相反调控作用。值得注意的是，Dkk2与Sfrp2均呈现Osr2依赖性的牙蕾间充质舌侧优先表达，且在Msx1-/-与Bmp4ncko/ncko突变体胚胎中，这两个基因的表达均被上调并扩散至整个牙蕾间充质。 我们证实，通过氯化锂（LiCl）处理或宫内抑制Dkk来药理学激活经典Wnt信号通路，足以挽救Bmp4ncko/ncko小鼠的下颌磨牙牙形态发生。此外，仅抑制Dkk无法挽救Msx1-/-小鼠的牙形态发生，但将Dkk药理学抑制与Sfrp2、Sfrp3的基因灭活相结合，则可挽救Msx1-/-小鼠的上颌磨牙形态发生。 综上，上述数据揭示了一种全新的机制：Bmp4-Msx1通路通过调控分泌型Wnt拮抗剂的表达激活Wnt信号通路，进而驱动牙齿器官发生。本研究取材于E14小鼠胚胎的牙胚，通过激光捕获显微切割（LCM）进行显微解离，分离下颌磨牙与上颌磨牙，将3对对照与突变体样本混合后开展RNA提取。