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Single cell RNA sequencing reveals critical functions in tooth development

June 26th, 2023
Single cell RNA sequencing reveals critical functions in tooth development
Single cell RNA sequencing analysis of maxillary processes from wild-type and Cd271 knockout mice. We identified 21 clusters of cells, and the single cell RNA sequencing data were characterized. A: Schematic representation of the experimental workflow. The Cd271 knockout and wild-type maxillary processes were collected from mouse embryos at 16.5 d. After genotyping, cells were suspended as single cells, and the cDNA library was constructed and sequenced; B: Cells extracted from the samples were subjected to Uniform Manifold Approximation and Projection hierarchical clustering and color-coded by cluster grouping, predominant cell type, and sample origin; C: Heatmap showing expression levels of differentially expressed genes in each cluster; D: Dot plot showing prominent marker genes for each cell type (mesenchymal cells, epithelial, muscle cell, macrophage, glial, T cell, endothelial, and perivascular); E: Pie chart showing the number of each cell type; F: Histogram showing the number and proportion of cells from wild-type and Cd271 knockout mice. UMAP: Uniform Manifold Approximation and Projection; KO: Knockout; WT: Wild-type. Credit: World Journal of Stem Cells (2023). DOI: 10.4252/wjsc.v15.i6.589

Accumulating evidence suggests that the maxillary process, to which cranial crest cells migrate, is essential to tooth development. Emerging studies indicate that Cd271 plays an essential role in odontogenesis. However, the underlying mechanisms have yet to be elucidated.

A team of researchers from Wenzhou Medical University in China used p75NTR knockout (Cd271-/-) mice (from American Jackson laboratory) to collect the maxillofacial process tissue of p75NTR knockout mice, and the wild-type maxillofacial process of the same pregnant mouse wild was used as control. After single cell suspension, the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system. Finally, the sequencing data in Fastq format were obtained.

The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data. The gene expression matrix is read by R software, and Seurat is used to control and standardize the data, reduce the dimension and cluster. The researchers sought marker genes for subgroup annotation by consulting literature and database; explore the effect of p75NTR knockout on mesenchymal stem cells (MSCs) gene expression and cell proportion by cell subgrouping, differential gene analysis, enrichment analysis and protein-protein interaction network analysis; understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis. Last, they verified the findings single cell sequencing in vitro.

The researchers identified 21 cell clusters, and they re-clustered these into three subclusters. Importantly, they revealed the cell–cell communication networks between clusters. The team clarified that Cd271 was significantly associated with the regulation of mineralization. The research is published in the World Journal of Stem Cells.

This study provides comprehensive mechanistic insights into the maxillary- process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

More information:
Yan-Yan Zhang et al, Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development, World Journal of Stem Cells (2023). DOI: 10.4252/wjsc.v15.i6.589

Provided by World Journal of Stem Cells

Citation: Single cell RNA sequencing reveals critical functions in tooth development (2023, June 26) retrieved 30 November 2024 from https://sciencex.com/wire-news/449252034/single-cell-rna-sequencing-reveals-critical-functions-in-tooth-d.html
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