Wnt1-Cre和Pax2-Cre标记的小鼠第一鳃弓颅颌面部神经嵴细胞异质性研究
Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice
作者：徐珏, 刘双, 符宏高, 邵美瑛, 陈美玲, 黄镇
Author：Xu Jue, Liu Shuang, Fu Honggao, Shao Meiying, Chen Meiling, Huang Zhen
收稿日期：2023-11-08 年卷（期）页码：2024,42(4):435-435-443
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：颅颌面部神经嵴细胞,第一鳃弓,Wnt1-Cre,Pax2-Cre,单细胞测序,
Key words：cranial neural crest cells,first branchial arch,Wnt1-Cre,Pax2-Cre,single-cell RNA sequencing,
基金项目：国家自然科学基金面上项目(82170918);四川省自然科学基金(2024NSFSC0549)
中文摘要
目的 利用Wnt1-Cre和Pax2-Cre小鼠特异性标记颅颌面神经嵴细胞（CNCs）迁移到第一鳃弓时的分化异质性及机制。 方法 分别收取胚胎期（E）8.0~E9.25 Wnt1-Cre;R26RmTmG及Pax2-Cre;R26RmTmG小鼠胚胎进行整体荧光观察，利用石蜡切片免疫荧光对E15.5的Pax2-Cre;R26RAi9和Wnt1-Cre;R26RAi9小鼠所标记的CNCs在颅面部主要组织器官中的谱系分化情况进行比较分析，最后对E10.5的Wnt1-Cre;R26RmTmG和Pax2-Cre;R26RmTmG小鼠的第一鳃弓组织中CNCs进行单细胞测序分析，并对差异基因进行荧光定量聚合酶链反应（q-PCR）验证。 结果 Pax2-Cre和Wnt1-Cre小鼠特异性标记的CNCs均在E8.0自神经板开始迁移，但Pax2-Cre小鼠仅标记迁移到第一鳃弓的CNCs，而Wnt1-Cre同时标记了迁移到第一和第二鳃弓的CNCs；在分化谱系示踪方面，二者皆标记了CNCs分化形成的颅颌面部组织器官的间充质，但Wnt1-Cre在上腭和舌中标记CNCs更多；在第一鳃弓间充质中，Pax2-Cre所标记的CNCs特异性表达基因主要参与了成骨，而Wnt1-Cre所标记的CNCs特异性表达基因主要参与了肢体发育、细胞迁移和成骨，q-PCR结果也证实了两者高表达差异基因参与了以上功能。 结论 本研究结果提示Pax2-Cre小鼠可特异性用于第一鳃弓CNCs及其衍生组织成骨方面的研究。
英文摘要
ObjectiveThis study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice.MethodsThe embryos of Wnt1-Cre;R26RmTmGand Pax2-Cre;R26RmTmGat embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26RAi9and Wnt1-Cre;R26RAi9mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26RmTmGand Pax2-cre;R26RmTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes.ResultsWnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre-marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis.ConclusionPax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.
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