The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. In order to undertake a detailed study of Musashi1’s function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1’s association with gut stem cell signature, cell cycle, DNA replication and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6 and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle and DNA synthesis and repair.
The intestinal epithelium self-renews rapidly and continuously throughout life, due to the presence of crypt stem cells. Two pools of these cells have been identified in the small intestine, which differ in position ("14" or the bottom of the crypts), expression of specific markers (Bmi1/ mTert or Lgr5/Ascl2), and cell cycle characteristics. Interestingly, the RNA-binding protein Musashi1 is expressed in both populations and therefore a potential marker for both stem cell types. In order to locate, isolate, and study Musashi1-expressing cells within the intestinal epithelium, we generated transgenic mice expressing GFP fluorescent protein under the control of a 7-kb Msi1 promoter. The expression pattern of GFP in the intestinal crypts of both small and large intestines completely overlapped that of Musashi1, validating our model. By using fluorescenceactivated cell sorting, cellular, and molecular analyses, we showed that GFP-positive Msi1-expressing cells are divided into two major pools corresponding to the Lgr5-and mTert-expressing stem cells. Interestingly, monitoring the cell cycle activity of the two sorted populations reveals that they are both actively cycling, although differences in cell cycle length were confirmed. Altogether, our new reporter mouse model based upon Musashi1 expression is a useful tool to isolate and study stem cells of the intestinal epithelium. Moreover, these mice uniquely enable the concomitant study of two pools of intestinal stem cells within the same animal model.
The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.