Reg4+ Deep Crypt Secretory cells function as epithelial niche for Lgr5+ stem cells in colon. Mus musculus

Lgr5+ stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF and Notch signals to neighboring Lgr5+ stem cells. While the colon lacks Paneth cells, Deep Crypt Secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report Reg4 as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon mini-gut formation. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. Stem cells are forced to generate DCS cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche. Overall design: To define a global gene expression signature of DCS cells, we performed RNA-sequencing (RNA-seq) of sorted Reg4-dsRed+ and Lgr5-GFP+ cells from colonic epithelium. Sorting and RNA-seq library preparation was performed twice, to obtain a biological replicate.

Lgr5+干细胞（Lgr5+ stem cells）定殖于小肠与大肠的隐窝底部。小肠潘氏细胞（Paneth cells）可为邻近的Lgr5+干细胞提供Wnt3、表皮生长因子（EGF）与Notch信号。尽管结肠缺乏潘氏细胞，但深层隐窝分泌细胞（Deep Crypt Secretory, DCS）会与隐窝底部的Lgr5+干细胞混杂共存。本研究首次发现Reg4可作为DCS细胞的特异性标志物。
为探究其生态位功能，我们借助敲入小鼠Reg4基因座的白喉毒素受体基因，实现了对DCS细胞的特异性清除。清除DCS细胞会导致结肠隐窝干细胞丢失，并破坏肠道稳态与结肠类器官（mini-gut）的形成。与之相符的是，分选获得的Reg4+ DCS细胞可促进单个Lgr5+结肠干细胞的类器官形成。
体外实验中，联合抑制Notch通路并激活Wnt通路，可诱导干细胞定向分化为DCS细胞。综上，Reg4+ DCS细胞可作为结肠隐窝生态位中潘氏细胞的功能等效细胞。
整体实验设计：为明确DCS细胞的全局基因表达特征，我们对结肠上皮中分选得到的Reg4-dsRed+与Lgr5-GFP+细胞开展了RNA测序（RNA-seq）。分选与RNA-seq文库制备流程各重复两次，以获取生物学重复样本。