Background & Aims: The stem/progenitor cells of the developing intestine arebiologically distinct from their adult counterparts. Here we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor, Delta-Like Protein 1 (DLK1).Methods: mRNAseq analyses of intestinal mesenchymal cells (IMC) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel co-culture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR and immunocytochemistry. The intestinal phenotype of littermate Dlk1 +/+ and Dlk1 -/- mice was compared using immunohistochemistry.Results: Using transcriptomic analyses we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate, DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth and shifted to the intestinal epithelium in adulthood. In co-culture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knock-down in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1 -/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1 +/+ embryos.Conclusions: Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.

中文翻译：

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基质 DLK1 在发育过程中促进肠上皮增殖并抑制分化


背景和目的：发育中的肠道干/祖细胞在生物学上不同于成年的干细胞/祖细胞。在这里，我们检查调节胚胎干/祖细胞群的微环境线索，重点关注 Notch 通路因子 Delta 样蛋白 1 (DLK1) 的作用。方法：对从胚胎第 14.5 天收集的肠间充质细胞 (IMC) 进行 mRNAseq 分析（使用 E14.5）或成人 IMC 以及与 E14.5 肠上皮类器官的新型共培养系统。添加重组 DLK1 (rDLK) 或 Dlk1 siRNA (siDlk1) 后，使用成像、重铺效率测定、qPCR 和免疫细胞化学比较上皮特征。使用免疫组织化学比较同窝 Dlk1 +/+ 和 Dlk1 -/- 小鼠的肠道表型。结果：通过转录组分析，我们鉴定了源自胚胎间充质的形态发生素，它们可能调节发育中的上皮细胞，重点关注 Notch 家族候选者 DLK1。免疫组织化学表明，DLK1仅在新生绒毛顶部E14.5的肠间质中表达，出生后下降，并在成年后转移到肠上皮。在共培养实验中，向成体 IMC 中添加 rDLK1 会抑制类器官分化，而胚胎 IMC 中的 Dlk1 敲除则会增加上皮向分泌谱系细胞的分化。与 Dlk1 +/+ 胚胎相比，Dlk1 -/- 小鼠绒毛基部 Ki67+ 细胞受到限制，分泌谱系细胞增多。结论：间充质来源的 DLK1 在促进上皮干/前体扩张和阻止分化为上皮干细胞中发挥重要作用。发育中的肠道中的分泌谱系。