Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.
Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.
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DOI：
10.1371/journal.pone.0111655
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kubo T
中科院分区：
文献类型：
--
作者：
Tsujioka H;Kunieda T;Katou Y;Shirahige K;Kubo T
器官再生能力取决于动物的种类和发育阶段。然而，可变器官再生能力的分子基础仍然未知。以往的研究已经确定了基因优先表达在各种动物的芽基组织，但转录组分析的分离增殖芽基细胞尚未报道。在本研究中，我们使用RNA测序分析的基因表达谱的分离增殖芽细胞再生非洲爪蟾蝌蚪尾巴。我们使用流式细胞仪分离增殖细胞，和非增殖芽细胞，再生蝌蚪尾巴以及增殖尾芽细胞从尾芽胚胎，后两者被用作对照细胞，基于它们的DNA含量。在通过RNA测序分析确定的28个候选基因中，定量逆转录-聚合酶链反应确定了10个基因，其表达在再生蝌蚪尾巴中富集，与非再生蝌蚪尾巴或来自尾芽胚胎的尾巴相比。其中，整片原位杂交结果显示，类染色体分离1和白细胞介素11在再生尾胚基的大范围内表达，而短蛋白聚糖、赖氨酰氧化酶和角蛋白18主要在再生尾的脊索芽中表达。我们进一步结合整体原位杂交和免疫组织化学的掺入5-溴-2-脱氧尿苷，以确认角蛋白18和白细胞介素11在增殖的尾胚基细胞中表达。基于其同源物在其他动物物种中的拟议功能，这些基因可能在脊索芽中细胞外基质形成（短聚糖和赖氨酰氧化酶）、细胞增殖（染色体分离1样和角蛋白18）以及维持分化中发挥作用再生蝌蚪尾巴中增殖芽基细胞（白细胞介素11）的分化能力。
Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was enriched in regenerating tadpole tails compared with non-regenerating tadpole tails or tails from the tail bud embryos. Among them, whole mount in situ hybridization revealed that chromosome segregation 1-like and interleukin 11 were expressed in the broad area of the tail blastema, while brevican, lysyl oxidase, and keratin 18 were mainly expressed in the notochord bud in regenerating tails. We further combined whole mount in situ hybridization with immunohistochemistry for the incorporated 5-bromo-2-deoxyuridine to confirm that keratin 18 and interleukin 11 were expressed in the proliferating tail blastema cells. Based on the proposed functions of their homologs in other animal species, these genes might have roles in the extracellular matrix formation in the notochord bud (brevican and lysyl oxidase), cell proliferation (chromosome segregation 1-like and keratin 18), and in the maintenance of the differentiation ability of proliferating blastema cells (interleukin 11) in regenerating tadpole tails.
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