基于单细胞转录组测序技术对鄂尔多斯细毛羊次级毛囊形态发生诱导期的研究

doi:10.16431/j.cnki.1671-7236.2022.11.020

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (11): 4296-4306.doi: 10.16431/j.cnki.1671-7236.2022.11.020

基于单细胞转录组测序技术对鄂尔多斯细毛羊次级毛囊形态发生诱导期的研究

赫雪^1,2, 谷英³, 斯登丹巴³, 德德玛³, 窦骁宁², 吴怡², 雷志惠², 潘和平¹, 岳耀敬^1,2

1. 西北民族大学生命科学与工程学院, 兰州 730030;
2. 中国农业科学院兰州畜牧与兽药研究所, 兰州 730050;
3. 鄂尔多斯市农牧业科学研究院, 鄂尔多斯 017100

收稿日期:2022-03-25 出版日期:2022-11-05 发布日期:2022-11-04
通讯作者: 岳耀敬 E-mail:yueyao-jing@caas.cn
作者简介:赫雪,E-mail:15293100105@163.com。
基金资助:
国家重点研发计划"绵羊高效育种与繁殖关键技术研发"（2021YFD1300901）；中国农业科学院科技创新工程重大科研任务"优质高效肉羊新品种培育与产业化"（CAAS-ZDRW202106）；国家绒毛用羊产业技术体系育种技术与方法岗位（CARS-39-02）；内蒙古自治区科技计划项目"鄂尔多斯细毛羊细型新品种培育"（2021GG0320）

Induction of Secondary Hair Follicle Morphogenesis in Ordos Fine Wool Sheep Based on Single-cell Transcriptome Sequencing Technology

HE Xue^1,2, GU Ying³, SIDENG Danba³, DE Dema³, DOU Xiaoning², WU Yi², LEI Zhihui², PAN Heping¹, YUE Yaojing^1,2

1. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China;
2. Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China;
3. Erdos Institute of Animal Husbandry Science, Erdos 017100, China

Received:2022-03-25 Online:2022-11-05 Published:2022-11-04

摘要/Abstract

摘要： 【目的】试验旨在解析鄂尔多斯细毛羊胚胎期次级毛囊诱导期形态发生过程中主要细胞类型的分子特征和分化过程。【方法】对采集的3只鄂尔多斯细毛羊（胎龄87 d）体侧部（肩胛骨后缘处）的皮肤样本进行HE染色，鉴定毛囊的发育时期；部分皮肤样本经过混样后进行单细胞转录组测序（scRNA-Seq），应用t-分布随机近邻嵌入（tSNE）分析细胞簇，分别使用胶原Ⅰ型α1链（Col1a1）和角蛋白15（Krt15）鉴定真皮谱系细胞和表皮谱系细胞，并使用皮肤组织不同细胞的标记基因进行细胞类型分析；对测序数据进行拟时序分析，探究其分化过程中差异基因的表达；通过GO功能富集分析进一步验证基因的功能。【结果】 HE染色结果发现，鄂尔多斯细毛羊在胎龄87 d处于次级毛囊诱导期。通过scRNA-Seq在胎龄87 d的细毛羊体侧部皮肤细胞样品中获得10 603个细胞和18 704个基因的scRNA-Seq数据可用于下游分析。tSNE分析发现，皮肤组织中共有15个细胞簇；Col1a1和Krt15标记基因鉴定表明，真皮细胞和表皮细胞具有高度异质性。拟时序分析构建毛囊形态发生过程中真皮/表皮细胞谱系细胞的分化轨迹和基因动态表达图谱表明，在真皮谱系细胞由成纤维细胞（Fb）向成熟真皮聚凝物（DC）的分化过程中，多个不同阶段的标记基因FST重组蛋白（Fst）、抑制素亚基βα（Inhba）和转录阻遏物GATA结合1（Trps1）均在拟时序轨道上特异性表达，并富集了Wnt、Noggin和骨形态发生蛋白（BMP）等与毛囊形态发生相关的信号通路；在表皮谱系细胞分化过程中，基质和毛囊间表皮（IFE）的标记基因角蛋白10（Krt10）、同源基因C13（HOXC13）和音猬因子信号（SHH）在表皮谱系细胞拟时序轨道的2个分支上均特异性表达，且富集在细胞增殖和细胞黏附等相关通路。【结论】在细毛羊次级毛囊诱导期，真皮谱系细胞由成纤维细胞分化至真皮聚凝物，表皮谱系细胞处于基质和毛囊间表皮细胞增殖分化阶段，结果可加深人们对细毛羊次级毛囊形态发生过程的了解，为鄂尔多斯细毛羊育种研究提供有力的理论和技术支持。

关键词: 鄂尔多斯细毛羊; 次级毛囊; 单细胞转录组测序(scRNA-Seq); 毛囊发育

Abstract: 【Objective】 The purpose of this experiment was to analyze the molecular characteristics and differentiation process of main cell types in the morphogenesis of the secondary hair follicles in Erdos fine wool sheep during the embryonic stage.【Method】 HE staining was performed on skin samples from the lateral part (posterior edge of scapula) of 87 fetal age of 3 Ordos fine wool sheep to identify the development period of hair follicles.Single-cell transcriptome sequence (scRNA-Seq) was performed on some skin samples after mixing.Cell clusters were analyzed by t-distributed stochastic Neigh or embedding (tSNE), and dermal lineage cells and epidermal lineage cells were identified by Col1a1 and Krt15 genes, respectively.The marker genes of different skin cells were used for cell type analysis.The sequence data were analyzed to explore the expression of differential genes in the process of differentiation.The function of the gene was further verified by GO function enrichment analysis.【Result】 HE staining results showed that Ordos fine wool sheep were in the secondary hair follicle induction stage at 87 days of gestational age. 10 603 cells and 18 704 genes were obtained by scRNA-Seq in skin cell samples from the body side of fine wool sheep at 87 days of gestational age could be used for downstream analysis.Based on tSNE analysis, 15 cell clusters were found in scRNA-Seq data.The identification of Col1a1 and Krt15 marker genes showed that dermal and epidermal cells were highly heterogeneous.The differentiation trajectories and dynamic gene expression profiles of dermal/epidermal cell lineage in the process of hair follicle morphogenesis were constructed by quasi-sequential analysis, during the differentiation of dermal lineate cells from fibroblast (Fb) to mature dermal condensates (DC), several different stage marker genes FST recombinant protein (Fst), inhibin subunit βα(Inhba) and transcriptional repressor GATA binding 1(Trps1) were specifically expressed on the quasisequential track, and Wnt, Noggin, bone morphogenetic protein (BMP) and other signaling pathways related to hair follicle morphogenesis were enriched.During the differentiation of epidermal lineage cells, the marker genes Krt10, HOXC13 and SHH of matrix and interfollicle epidermis (IFE) were specifically expressed in both branches of the quasi-sequential trajectory of epidermal lineage cells, and the pathways related to cell proliferation and cell adhesion were enriched.【Conclusion】 The results showed that dermal lineage cells differentiated from Fb to DC during the induction of the secondary hair follicles in fine wool sheep.Epidermal lineage cells were in the stage of proliferation and differentiation of epidermal cells between matrix and hair follicle.The results of this study could enrich people's understanding of the morphogenesis of the secondary hair follicles in fine wool sheep, and provide strong theoretical and technical support for the breeding of Ordos fine wool sheep.

Key words: Ordos fine wool sheep; secondary hair follicles; single-cell transcriptome sequence(scRNA-Seq); hair follicle development

中图分类号:

S826.9⁺1

赫雪, 谷英, 斯登丹巴, 德德玛, 窦骁宁, 吴怡, 雷志惠, 潘和平, 岳耀敬. 基于单细胞转录组测序技术对鄂尔多斯细毛羊次级毛囊形态发生诱导期的研究[J]. 中国畜牧兽医, 2022, 49(11): 4296-4306.

HE Xue, GU Ying, SIDENG Danba, DE Dema, DOU Xiaoning, WU Yi, LEI Zhihui, PAN Heping, YUE Yaojing. Induction of Secondary Hair Follicle Morphogenesis in Ordos Fine Wool Sheep Based on Single-cell Transcriptome Sequencing Technology[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4296-4306.

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基于单细胞转录组测序技术对鄂尔多斯细毛羊次级毛囊形态发生诱导期的研究

Induction of Secondary Hair Follicle Morphogenesis in Ordos Fine Wool Sheep Based on Single-cell Transcriptome Sequencing Technology

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