Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts

Wnt/b-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (PorcnDel/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of PorcnDel organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable, and that stromal production of Wnts can fully support normal murine intestinal homeostasis. Microarray was performed on samples enriched for stromal or epithelial cells from small intestine from Porcn(Del)/Villin-Cre and Porcn(WT)/Villin-Cre male C57Bl/6 mice.Wnt/β-连环蛋白信号通路（Wnt/β-catenin signaling）通过调控肠隐窝内的细胞增殖维持肠道稳态。潘氏细胞（Paneth cells）与其他肠道上皮细胞、肠道基质细胞均可表达多种Wnt蛋白。体外实验中，当通过外源性补充R-脊椎蛋白1（R-Spondin 1，RSPO1）增强Wnt信号通路活性时，潘氏细胞分泌的Wnt蛋白可支持肠道干细胞的存活与增殖。然而在体内环境中，干细胞微环境中Wnt蛋白的具体来源仍未明确。通过基因敲除Porcn——一种定位于内质网的O-酰基转移酶（endoplasmic reticulum resident O-acyltransferase），其对所有脊椎动物Wnt蛋白的分泌与活性均不可或缺——，研究人员证实了肠道上皮来源的Wnt蛋白在体外培养体系中的作用。令人意外的是，缺失上皮Wnt活性的PorcnDel/Villin-Cre小鼠，其肠道增殖与分化过程均维持正常，且在辐射损伤后可顺利完成组织修复，这表明上皮来源的Wnt蛋白并非上述生理过程所必需。这与基质细胞在肠隐窝微环境中发挥关键作用的认知一致：可内源性表达Wnt蛋白与R-脊椎蛋白3（Rspo3）的肠道基质细胞，无需额外补充RSPO1即可在体外支持PorcnDel类器官的生长。反之，通过药物增强PORCN抑制效果（同时作用于基质细胞与上皮细胞），可减少Lgr5标记的肠道干细胞（Lgr5 intestinal stem cells）数量、抑制辐射损伤后的肠道修复，且在最高给药剂量下可完全阻断肠道细胞增殖。综上，本研究证实上皮来源的Wnt蛋白并非必需，而基质细胞产生的Wnt蛋白足以维持正常小鼠的肠道稳态。本研究对来自Porcn(Del)/Villin-Cre与Porcn(WT)/Villin-Cre雄性C57Bl/6小鼠小肠的基质细胞富集样本与上皮细胞富集样本进行了基因芯片（microarray）检测。