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

doi:10.7518/hxkq.2024.2023374

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (4): 435-443.doi: 10.7518/hxkq.2024.2023374

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

徐珏¹(), 刘双¹, 符宏高², 邵美瑛¹, 陈美玲², 黄镇²^,³()

^1.四川大学华西公共卫生学院/华西第四医院，成都 610041
^2.福建师范大学生命科学院，福州 350007
^3.福建中医药大学科学创新与转化中心，福州 350108

收稿日期:2023-11-08 修回日期:2024-04-28 出版日期:2024-08-01 发布日期:2024-07-17
通讯作者: 黄镇 E-mail:xujue1212@qq.com;zhuang@fjtcm.edu.cn
作者简介:徐珏，副主任医师，博士，E-mail：xujue1212@qq.com
基金资助:
国家自然科学基金面上项目(82170918);四川省自然科学基金(2024NSFSC0549)

Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice

Xu Jue¹(), Liu Shuang¹, Fu Honggao², Shao Meiying¹, Chen Meiling², Huang Zhen²^,³()

^1.West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China
^2.College of Life Sciences, Fujian Normal University, Fuzhou 350007, China
^3.Innovation and Transformation Center, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China

Received:2023-11-08 Revised:2024-04-28 Online:2024-08-01 Published:2024-07-17
Contact: Huang Zhen E-mail:xujue1212@qq.com;zhuang@fjtcm.edu.cn
Supported by:
National Nature Science Foundation of China(82170918);Natural Science Foundation of Sichuan Province(2024NSFSC0549)

摘要/Abstract

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;R26R^mTmG及Pax2-Cre;R26R^mTmG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;R26R^Ai9和Wnt1-Cre;R26R^Ai9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;R26R^mTmG和Pax2-Cre;R26R^mTmG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。

关键词: 颅颌面部神经嵴细胞, 第一鳃弓, Wnt1-Cre, Pax2-Cre, 单细胞测序

Abstract:

Objective This 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. Methods The embryos of Wnt1-Cre;R26R^mTmG and Pax2-Cre;R26R^mTmG at 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;R26R^Ai9 and Wnt1-Cre;R26R^Ai9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26R^mTmG and Pax2-cre;R26R^mTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes. Results Wnt1-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. Conclusion Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Key words: cranial neural crest cells, first branchial arch, Wnt1-Cre, Pax2-Cre, single-cell RNA sequencing

中图分类号:

Q257

徐珏, 刘双, 符宏高, 邵美瑛, 陈美玲, 黄镇. 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[J]. 华西口腔医学杂志, 2024, 42(4): 435-443.

Xu Jue, Liu Shuang, Fu Honggao, Shao Meiying, Chen Meiling, Huang Zhen. Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice[J]. West China Journal of Stomatology, 2024, 42(4): 435-443.

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基因型	正向引物（5’-3’）	反向引物（3’-5’）	产物大小/bp
Pax2-Cre	TGCCAGGATCAGGGTTAAAG	GCTTGCATGATCTCCGGTAT	400
Wnt1-Cre	CTCATTGTCTGTGGCCCTGACC	ACGCCTGGCGATCCCTGAA	450
R26R^mTmG	CTCTGCTGCCTCCTGGCTTCT	R1：CGAGGCGGATCACAAGCAATA	Mut：250
		R2：TCAATGGGCGGGGGTCGTT	Wt：330
R26R^Ai9（wild type）	AAGGGAGCTGCAGTGGAGTA	CCGAAAATCTGTGGGAAGTC	Wt：297
R26R^Ai9（mutant）	GGCATTAAAGCAGCGTATC	CTGTTCCTGTACGGCATGG	Mut：196

基因	正向引物（5’-3’）	反向引物（3’-5’）
Tbx2	CCGATGACTGCCGCTATAAGT	CCATCCACTGTTCCCCTGT
Prrx1	GAGCGTGTCTTTGAGCGGA	CATGTGGCAGAATAAGTAGCCAT
Prrx2	CGTGGCACCAAACGAAAGAAG	GTAGTGTGTGCGCTCAAATACA
Msx1	TGCTGCTATGACTTCTTTGCC	GCTTCCTGTGATCGGCCAT
Msx2	CAAGAAGCCGCCCAAGGAAT	TGCTCCGTCTTCGGAATTTTC
Pitx1	GCCTTCAAGGGAGGCATGAG	GATTCGCTGGCGGAGTTCTC
Gja1	AATTTACTGGGTGGCATCCTAGA	GGGAAAGCATCATCGTAACAGAT
Gnas	CAGAGCCTCCATTGGGGTC	GCTTCTCGCTCAACTGGGG
Igf2	GTCCTACTCAAGTCCGGCTAC	CATATTCAGCCAACAGCCCAT
Cited1	AACCTTGGAGTGAAGGATCGC	GTAGGAGAGCCTATTGGAGATGT
Dlk1	AGTGCGAAACCTGGGTGTC	GCCTCCTTGTTGAAAGTGGTCA
Cxcl12	TGCATCAGTGACGGTAAACCA	TTCTTCAGCCGTGCAACAATC
Apoe	CTGACAGGATGCCTAGCCG	CGCAGGTAATCCCAGAAGC
Nr2f2	TCAACTGCCACTCGTACCTG	CCATGATGTTGTTAGGCTGCAT
Rap2c	ATGAGGGAATACAAGGTAGTGGT	ACTTCGATCTCTTTGCGGTAGA
Tpm1	CAGAAGGCAAATGTGCCGAG	TCCAGCATCTGGTGCATACTA
Cnn2	AGAACCGGCTCCTGTCCAA	GTGTGCATAGGATAACCCCATC

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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

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