Presence and role of Enteroendocrine Cells Residing at the Intestinal Crypt Base

肠内分泌细胞占胃肠道上皮细胞的约1%，是体内最大的产酶细胞群。 肠内分泌细胞与其他非内分泌细胞谱系共享共同谱系，并且起源于肠隐窝中的原始肠干细胞。 有两种互补的干细胞群，自我更新干细胞群和静止干细胞群。 自我更新干细胞高度表达Lgr 5，并位于隐窝基底的潘氏细胞之间，在那里Wnt信号传导是活跃的，而静止干细胞表达Bmi 1，并位于位置4。 因此，谱系特异性前体细胞被认为可以立即与自我更新的Lgr 5+细胞分化。 内分泌前体细胞分化为成熟的分泌细胞，这些细胞通过表达特定的肽激素被分为至少15种不同的终末分化谱系（或亚群）。 虽然调节内分泌前体细胞分化的分子机制尚未完全表征，但关键转录因子（Pax 4、Pax 6、BETA 2/NeuorD、Pdx 1、Gfi、Nkx 2.2和Sox 9）与肠内分泌细胞分化有关。 肠内分泌细胞的空间定向与其分化过程有关。先前的报告使用BrdU掺入，形态学和免疫组织化学方法表征的内分泌细胞分化表明，大多数的肠内分泌细胞完成的分化过程中的隐窝和迁移向上沿着绒毛成熟的分泌细胞。然而，一小群肠内分泌细胞向下迁移到隐窝底部。 肠内分泌细胞由亚群组成，其分化由调节激素的特异性共表达及其在隐窝-绒毛轴内的位置的特异性转录因子决定。 由Wnt、Hedgehog、Notch、BMP和EphB/ephrin介导的分化信号传导途径在空间上沿着小肠上皮和间充质中的隐窝-绒毛轴受到限制。在隐窝中的位置+4以下（自我更新的Lgr 5+干细胞与潘氏细胞一起驻留的位置），Wnt信号传导特别活跃。 目前，还不清楚是什么决定了肠内分泌细胞的一个子集是否留在Wnt信号丰富的区域，而不是迁移到绒毛，以及为什么。 肠内分泌细胞的这一子集，居住在隐窝基地已成为我们特别感兴趣的，特别是因为我们能够可视化GFP+肠内分泌细胞在隐窝基地的基因工程动物，表达GFP的胆囊收缩素（CCK）基因启动子的控制下。 此外，最近开发的技术，使培养的类器官使我们能够跟踪迁移模式的GFP表达的内分泌细胞沿着隐窝绒毛轴。 通过利用GFP表达，我们分别在新鲜分离的初级隐窝和培养的类器官系统中检测CCK-GFP表达细胞的分化标记物表达和迁移模式，以使用CCK-GFP转基因动物表征隐窝基部的亚群。 我们发现，在隐窝底部的内分泌细胞的子集居住在这个Wnt信号活性区后，其向下迁移的位置+4区域，并表达干细胞和有丝分裂后成熟的内分泌细胞标志物，这个子集至少是表型不同的肠内分泌细胞的其他子集向上迁移到绒毛。

The enteroendocrine cells, which comprise approximately 1% of epithelial cells in the gastrointestinal tract, represent the largest population of hormone-producing cells in the body. The enteroendocrine cells share a common lineage with other non-endocrine cell lineages and originate from primitive intestinal stem cells in the intestinal crypts. There are two complementary populations of stem cells, a self-renewing stem cell population and a quiescent stem cell population. The self-renewing stem cells highly express Lgr5 and reside between Paneth cells at the crypt base where Wnt signaling is active whereas quiescent stem cells express Bmi1 and reside at the position 4. Thus the lineage-specific precursor cells are thought to differentiate immediately from the self-renewing Lgr5+ cells. Endocrine precursor cells differentiate toward mature hormone-producing endocrine cells that are classified into at least 15 different terminally differentiated lineages (or subsets) by their expression of specific peptide hormones. Although the molecular mechanisms that regulate the differentiation from the endocrine precursor cells have not been fully characterized, key transcription factors have been implicated in enteroendocrine cell differentiation (Pax4, Pax6, BETA2/NeuorD, Pdx1, Gfi, Nkx2.2 and Sox9). There is a relationship between spatial orientation of the enteroendocrine cells and their differentiation process. Previous reports on characterization of entroendocrine cell differentiation using BrdU incorporation, morphological and immunohistochemical methods demonstrated that the majority of enteroendocrine cells complete the differentiation process within the crypt and migrate upward along the villus as mature hormone-producing cells. However, a small population of enteroendocrine cells migrates downward to the bottom of the crypt. Enteroendocrine cells are comprised of subsets, whose differentiation is determined by specific transcription factors that regulate both the specific co-expression of hormones and their location within the crypt-villus axis. The differentiation signaling pathways mediated by Wnt, Hedgehog, Notch, BMP and EphB/ephrin are restricted spatially along the crypt-villus axis in the epithelium and the mesenchyme of the intestine. Below position +4 (where the self-renewing Lgr5+ stem cells reside with Paneth cells) in the crypt, Wnt-signaling is especially active . Presently, it is unclear what determines whether a subset of enteroendocrine cells remains in the Wnt signaling rich area instead of migrating up to the villi and why. This subset of enteroendocrine cells that resides at the crypt base has become our particular interest especially since we are able to visualize GFP+ enteroendocrine cells at the crypt base in the genetically engineered animals that express GFP under the control of the cholecystokinin (CCK) gene promoter. In addition, the technique recently developed to make cultured organoids enables us to trace migration patterns of GFP-expressing endocrine cells along the crypt-villus axis. By taking advantage of GFP expression, we examined CCK-GFP-expressing cells for expression of differentiation markers and migration patterns in freshly isolated primary crypts and cultured organoid system, respectively, to characterize the subset at the crypt base using the CCK-GFP transgenic animals. We found that the subset of endocrine cells at the crypt base resides at this Wnt signaling-active zone after its downward migration from around position+4 region and expresses both stem and post-mitotic mature endocrine cell markers and that this subset is at least phenotypically different from other subsets of enteroendocrine cells which migrate upward to the villi.