Six1 regulates mouse lower incisor development by coordinating Dlx1/2/5 [scRNA-seq]

Tooth development is a complex process orchestrated by intricate gene regulatory networks, involving both odontogenic epithelium and ectomesenchyme. Six1, a pivotal transcription factor (TF), is involved in the development of the lower incisor. However, its precise role during incisor development and the molecular mechanisms underpinning its regulatory functions remain poorly understood. This study employs Six1 deletion mouse models to elucidate the critical regulatory role of Six1 in governing dental mesenchyme development. By performing single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive transcriptome atlas of tooth germ development from the bud to bell stage. Our analyses suggest that the dental follicle (DF) and the dental papilla (DP) are differentiated from dental ectomesenchyme (DEM), and identify the key transcription factors (TFs) underlying these distinct states. Notably, we show that Dlx1, Dlx2, and Dlx5 (Dlx1/2/5) may function as the key TFs that promote the formation of DP. We further show that the deletion of Six1 perturbs dental mesenchyme development by impeding the transitions from DEM to DP states. Importantly, SIX1 directly binds to the promoters of Dlx1/2/5 to promote their co-expression, which subsequently leads to widespread epigenetic and transcriptional remodeling. In summary, our findings unveil Six1's indispensable role in incisor development, offering key insights into transcription factor-driven regulatory networks that govern dental mesenchyme cell fate transitions during tooth development. Single-cell transcriptomes were obtained from the digestion of the incisor germs at E14.5, E15.5, and E16.5.牙齿发育是受精密基因调控网络统筹的复杂过程，涉及牙源性上皮与外胚间充质。Six1作为关键转录因子（transcription factor, TF），参与下切牙的发育。然而，其在切牙发育过程中的精准作用，以及支撑其调控功能的分子机制，目前仍不甚明晰。本研究利用Six1敲除小鼠模型，阐明了Six1在调控牙间充质发育中的关键作用。通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），我们构建了从蕾状期到钟状期的牙胚发育全转录组图谱。分析结果显示，牙囊（dental follicle, DF）与牙乳头（dental papilla, DP）均源自牙源性外胚间充质（dental ectomesenchyme, DEM），并鉴定出维持这些不同细胞状态的关键转录因子。值得注意的是，我们发现Dlx1、Dlx2与Dlx5（Dlx1/2/5）可作为促进牙乳头形成的关键转录因子。进一步研究表明，Six1敲除会通过阻断牙源性外胚间充质向牙乳头细胞状态的转化，扰乱牙间充质发育。尤为重要的是，SIX1可直接结合Dlx1/2/5的启动子区域以促进其共表达，进而引发广泛的表观遗传与转录组重塑。综上，本研究揭示了Six1在切牙发育中不可或缺的作用，为解析调控牙齿发育过程中牙间充质细胞命运转化的转录因子驱动型调控网络提供了关键见解。 本研究的单细胞转录组数据来源于对E14.5、E15.5及E16.5时期的切牙胚进行消化后获取的样本。