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

肠内分泌细胞约占胃肠道上皮细胞的1%，是体内最大的激素分泌细胞群。肠内分泌细胞与其他非内分泌细胞谱系有共同的谱系，起源于肠隐窝中的原始肠道干细胞。有两个互补的干细胞群体，一个是自我更新的干细胞群体，另一个是静止的干细胞群体。自我更新干细胞高表达Lgr5，位于Wnt信号活跃的隐窝基底部的Paneth细胞之间，而静止干细胞表达Bmi1，位于第4位。因此，谱系特异性前体细胞被认为是立即从自我更新的Lgr5+细胞分化出来的。内分泌前体细胞分化为成熟的产生激素的内分泌细胞，根据其表达特定的多肽激素，内分泌细胞被分为至少15个不同的终末分化谱系(或亚群)。尽管调控内分泌前体细胞分化的分子机制尚未完全确定，但关键的转录因子(Pax4、Pax6、Beta2/NeuorD、Pdx1、GFI、Nkx2.2和Sox9)与肠内分泌细胞的分化有关。 肠内分泌细胞的空间定位与其分化过程有关。以往用BrdU掺入、形态和免疫组织化学方法鉴定内分泌细胞分化的报道表明，大多数肠内分泌细胞在隐窝内完成分化过程，并作为成熟的激素产生细胞沿绒毛向上迁移。然而，一小部分肠内分泌细胞向下迁移到隐窝的底部。肠内分泌细胞由亚群组成，其分化由特定的转录因子决定，这些转录因子调节激素的特定共表达及其在隐窝绒毛轴中的位置。由Wnt、Hedgehog、Notch、BMP和EphB/Ephin介导的分化信号通路在肠上皮和间质中沿隐窝绒毛轴空间受限。在隐窝中的+4位置(自我更新的Lgr5+干细胞与Paneth细胞共同居住的位置)以下，Wnt信号尤其活跃。目前，还不清楚是什么决定了肠内分泌细胞亚群是否保留在Wnt信号丰富区，而不是迁移到绒毛，以及为什么。 这种位于隐窝底部的肠内分泌细胞亚群已经成为我们特别感兴趣的细胞，特别是因为我们能够在CCK基因启动子的控制下，在表达GFP的基因工程动物的隐窝底部观察到GFP+的肠内分泌细胞。此外，最近开发的制作培养类器官的技术使我们能够追踪表达GFP的内分泌细胞沿隐窝-绒毛轴的迁移模式。 通过利用GFP的表达，我们分别在新鲜分离的原代隐窝和培养的器官系统中检测了CCK-GFP表达细胞的分化标志物和迁移模式，以利用CCK-GFP转基因动物来表征隐窝底部的亚群。我们发现，隐窝底部的内分泌细胞亚群从+4区附近向下迁移后，位于Wnt信号活动区，表达干细胞和有丝分裂后成熟内分泌细胞标志物，这一亚群至少在表型上不同于向上迁移到绒毛的其他肠道内分泌细胞亚群。

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.