Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a
Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a
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Wnt信号通路通过Wnt5a调控鸡胚胎干细胞向精原干细胞的分化
DOI：
10.1002/jcb.26329
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发表时间：
2018-02-01
影响因子：
4
通讯作者：
Li, Bichun
中科院分区：
文献类型：
--
作者：
He, Nana;Wang, Yilin;Li, Bichun
本研究探讨了鸡胚胎干细胞（ESCs）向精原干细胞（SSCs）分化的信号转导机制。基于先前的RNA测序结果鉴定了Wnt信号通路，并且被证明是参与ESC向SSC分化的关键信号通路。在维甲酸（RA）体外诱导实验中，我们发现Wnt 5a-shRNA抑制了Wnt信号通路的表达，导致SSC中相应标志基因C-kit、Cvh、integrin 6和integrin 1的表达降低，而RA处理显著促进了其表达。免疫荧光检测显示，RA治疗组和Wnt 5a过表达组C-kit、Cvh、整合素6和整合素1阳性细胞百分比均显著高于Wnt 5a信号转导干扰组。荧光激活细胞分选分析（FACS）结果也显示，转染后第4天和第12天，生殖样细胞的比例分别降低了14.3%（从18.3%降至4.0%）和15.4%（从18.6%降至3.2%）。在体内实验中，shRNA-Wnt 5a在受精鸡胚中稳定表达，并显著降低生殖细胞形成11.3%（从21.7%降低到10.4%）和3.7%（从10.1%降低到6.4%）。定量PCR（qRT-PCR）和western blot检测结果显示，Wnt 5a信号转导干扰后，某些特定的生殖细胞标志基因整合素6和整合素1的表达明显受到抑制。综上所述，我们的研究表明Wnt信号通路可能通过Wnt 5a正向调节鸡胚胎干细胞向精原干细胞的分化。
In this study, we investigated the mechanism of signaling pathway-mediated differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) in chicken. The Wnt signaling pathway was identified based on previous RNA Sequencing results and was proven a crucial signaling pathway that participates in the differentiation of ESCs into SSCs. In retinoic acid (RA) induction experiments in vitro, we found that Wnt signaling expression was inhibited by Wnt5a-shRNA, resulting in decreased expression of corresponding marker genes in SSCs, C-kit, Cvh, integrin 6 and integrin 1, but it was significantly promoted by RA treatment. Immunofluorescence assay showed that percentage of C-kit, Cvh, and integrin 6 and integrin 1-positive cells in RA treatment group and Wnt5a overexpression group was significantly higher than that in Wnt5a signaling interference group. Results of fluorescence-activated cell sorting analysis (FACS) also showed that proportion of germ-like cells was reduced by 14.3% (from 18.3% to 4.0%) at day 4 and 15.4% (from 18.6% to 3.2%) at day 12 after transfection, respectively. In experiments in vivo, shRNA-Wnt5a was stably expressed in fertilized chicken embryos and significantly reduced germ cell formation by 11.3% (from 21.7% to 10.4%) and 3.7% (6.4% from 10.1%). Results of quantitative PCR (qRT-PCR) and western blot assays showed that the expression of some specific germ cell marker genes, integrin 6 and integrin 1, was significantly suppressed following Wnt5a signaling interference in vivo. Taken together, our study suggests that Wnt signaling pathway could regulate positively the differentiation of chicken ESCs into SSCs through Wnt5a.