Hasebe Takashi, Fujimoto Kenta, Kajita Mitsuko, Fu Liezhen, Shi Yun-Bo, Ishizuya-Oka Atsuko
Department of Biology, Nippon Medical School, Musashino, Tokyo, Japan.
Department of Molecular Biology, Institute for Advanced Medical Sciences, Nippon Medical School, Kawasaki, Kanagawa, Japan.
Stem Cells. 2017 Apr;35(4):1028-1039. doi: 10.1002/stem.2544. Epub 2016 Nov 25.
In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028-1039.
在非洲爪蟾变态发育过程中的肠道中，幼虫上皮细胞通过凋亡被清除，同时成年上皮干细胞（AE细胞）出现。它们增殖并分化形成成年上皮（Ep）。甲状腺激素（TH）通过调节包括Notch受体在内的各种基因的表达来触发这种重塑，这一点已得到充分证实。为了研究Notch信号通路的作用，我们通过实时逆转录-聚合酶链反应以及原位杂交或免疫组织化学分析了其组成成分（包括配体（DLL和Jag）、受体（Notch）和靶标（Hairy））在变态发育肠道中的表达。我们发现它们在自然变态和TH诱导的变态过程中均以组织特异性方式上调。特别地，Hairy1在AE细胞中特异性表达。此外，用γ-分泌酶抑制剂（GSI）处理蝌蚪可阻止TH对Hairy1和Hairy2b的上调，GSI可抑制Notch信号通路。更重要的是，GSI处理可抑制TH诱导的成年肠道干细胞标志物LGR5的上调。我们的结果表明，Notch信号通路在肠道重塑过程中通过调节Hairy基因的表达在干细胞发育中发挥作用。此外，我们通过器官培养实验表明，蝌蚪肠道长时间暴露于TH加GSI会导致分泌细胞增生和吸收细胞减少。因此，我们的研究结果为Notch信号通路在肠道细胞命运决定中的进化保守作用提供了证据，但更重要的是首次揭示了Notch信号通路在脊椎动物发育过程中成年肠道干细胞形成中的重要作用。《干细胞》2017年；35卷：1028 - 1039页
