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

doi:10.7518/hxkq.2024.2023374

Abstract

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

CLC Number:

Q257

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