Expansion of Intestinal Epithelial Stem Cells during Murine Development

Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

小鼠小肠隐窝的发育始于出生后第一周。隐窝形成后不久，其总数通过一种名为隐窝分裂（crypt fission）的分叉过程持续增加，该过程被认为由肠道干细胞（intestinal stem cells, ISCs）的发育性增殖扩增所驱动。近期研究证据表明，成年肠道内存在异质性的肠道干细胞群体。活跃增殖的肠道干细胞可由Lgr5、Ascl2及Olfm4标记；而缓慢增殖或静息态的肠道干细胞则以Bmi1与mTert作为标志物。本研究旨在将上述标志物的表达，与发育过程中肠道干细胞扩增的间接检测手段（包括隐窝分裂量化与侧群细胞（side population, SP）分选）进行关联分析。在出生后第14天至第21天期间，隐窝分裂比例与侧群细胞占比均出现显著变化，这与肠道干细胞扩增的特征相符。针对各类肠道干细胞标志物mRNA的实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, RT-PCR）检测结果显示，各标志物的表达模式存在显著差异：mTert的表达最早出现峰值，即在出生后第一周隐窝初始形成阶段；而Lgr5与Bmi1的表达峰值则出现在出生后第14天；Olfm4与Ascl2的表达模式则相对多变。为评估此阶段表达Lgr5的细胞数量与分布情况，研究人员对Lgr5-EGFP转基因小鼠的肠道组织切片进行了定量分析。出生时及出生后第一周，Lgr5-EGFP的表达水平较低。隐窝形成后，每个隐窝中Lgr5-EGFP阳性细胞的总数与占比在整个发育阶段直至成年期均保持稳定。上述结果在野生型小鼠的Lgr5原位杂交（in situ hybridization）实验中得到了验证。综上，通过侧群细胞分选与mRNA表达检测的结果证实，异质性肠道干细胞群体在不同发育时间点均存在扩增现象。