Abstract：
OBJECTIVE: This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process.
METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.
RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.
CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.
METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.
RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.
CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.
摘要：
目的：本研究旨在探讨牙齿发育过程中牙间充质细胞定型的调控机制，重点研究成牙本质细胞的分化以及表观遗传调控在这一过程中的作用。
方法：我们对胚胎第14.5天（E14.5）小鼠的牙细胞进行了单细胞RNA测序（scRNA-seq），以了解发育中的牙胚细胞的异质性。进行了包括基因调控网络(GRN)评估的计算分析。我们使用免疫组织化学（IHC）和使用DNA甲基转移酶1（DNMT1）抑制剂Gsk-3484862在从E14.5小鼠牙胚分离的原代牙间充质细胞（DMC）中进行的体外功能丧失分析验证了我们的发现。进行Gsk-3484862处理的DMC的大量RNA-seq以鉴定DNMT1的潜在下游靶标。
结果：scRNA-seq分析揭示了牙胚内不同的细胞群，包括上皮,间充质,免疫，和肌肉细胞。使用单细胞调控网络推断和聚类(SCENIC)，我们确定Dnmt1是早期成牙本质细胞发育的关键调节因子。IHC分析显示DNMT1在牙乳头和上皮中普遍存在。培养的DMC的大量RNA-seq显示Gsk-3484862处理上调成牙本质细胞相关基因，而与细胞分裂和细胞周期相关的基因被下调。使用大量RNA-seq数据与scRNA-seqSCENIC谱的综合分析来鉴定潜在的Dnmt1靶基因。
结论：Dnmt1可能对牙齿发育过程中成牙本质细胞的定型和分化产生负面影响。这些发现有助于更好地了解牙齿发育的分子机制以及硬组织再生疗法的未来发展。
方法：我们对胚胎第14.5天（E14.5）小鼠的牙细胞进行了单细胞RNA测序（scRNA-seq），以了解发育中的牙胚细胞的异质性。进行了包括基因调控网络(GRN)评估的计算分析。我们使用免疫组织化学（IHC）和使用DNA甲基转移酶1（DNMT1）抑制剂Gsk-3484862在从E14.5小鼠牙胚分离的原代牙间充质细胞（DMC）中进行的体外功能丧失分析验证了我们的发现。进行Gsk-3484862处理的DMC的大量RNA-seq以鉴定DNMT1的潜在下游靶标。
结果：scRNA-seq分析揭示了牙胚内不同的细胞群，包括上皮,间充质,免疫，和肌肉细胞。使用单细胞调控网络推断和聚类(SCENIC)，我们确定Dnmt1是早期成牙本质细胞发育的关键调节因子。IHC分析显示DNMT1在牙乳头和上皮中普遍存在。培养的DMC的大量RNA-seq显示Gsk-3484862处理上调成牙本质细胞相关基因，而与细胞分裂和细胞周期相关的基因被下调。使用大量RNA-seq数据与scRNA-seqSCENIC谱的综合分析来鉴定潜在的Dnmt1靶基因。
结论：Dnmt1可能对牙齿发育过程中成牙本质细胞的定型和分化产生负面影响。这些发现有助于更好地了解牙齿发育的分子机制以及硬组织再生疗法的未来发展。
