A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness

Abstract

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. 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.

Keywords: Intestinal epithelium; Musashi1; RNA binding protein; Stem cells.

Figure 1. Increased MSI1 expression in v-Msi1 intestine

Anti-MSI1 antibodies were used to analyze the expression pattern of MSI1 along the epithelial axis of the small intestine (A, B) and colon (C, D) of v-Msi1 (B, D) and WT (A, C) animals. Pictures show the merging of nuclear (blue) and MSI1 (red) staining. Bar=15μm. (E) Analysis of MSI1 protein levels by WB in v-Msi1 and WT animals. Actin was used as the loading control. The Ctrl lane (positive control) corresponds to lysate from Cos7 cells transfected with a Msi1-expressing vector.

Figure 2. Morphological and proliferative properties of the v-Msi1 intestinal mucosa

A, B) Morphological analysis of the distal small intestine from WT (A) and v-Msi1 (B) mice. C) Quantification of the number of cells per crypt axis, as indicated by the black dotted double arrows in A and B. Approximately 40 axes were scored under the microscope from at least four mice per genotype; histograms represent the mean ± SD. **: P<0.01, in comparison with WT, by Student’s t-test. (D–F) Ki67 immunolabeling of proliferating cells in intestinal sections from WT (D) or v-Msi1 mice (E, F). Images show merged Ki67 immunolabeling (red) and nuclear staining (blue). G) Quantification of the percentage of Ki67 positive cells per crypt. Approximately 40 crypts were scored under the microscope from at least four mice per genotype; histograms represent the mean ± SD. **: P<0.01, in comparison with WT, by Student’s t-test. Bar=15μm. Black double arrows in A and B define the length of the vertical crypt-villus axis. Dotted double black arrows in A and B show the size of the crypts. White bars in D and E define the limit of the proliferative Ki67-positive cells and dotted double white arrows show the size of the proliferating zone. White arrows in F indicate some Ki67-positive cells in villi.

Figure 3. Pathways and functions affected by Msi1 altered expression

A) Enriched KEGG pathways for genes up-regulated in v-Msi1 samples. Only pathways presenting adjusted P-value < 0.05 were selected. B) Increased expression of genes implicated in cell cycle, DNA replication and repair in v-Msi1 compared with WT intestine. Gene pathway data was obtained from DAVID (http://david.abcc.ncifcrf.gov/). Cytoscape (http://www.cytoscape.org/) was used for pathway visualization.

Figure 4. CCND1 and CDK6 are direct targets of MSI1

A) UCSC genome browser plots showing the position (x-axis) and count (y-axis) of iCLIP reads overlapping the 3′ UTR of CCND1 (top) and CDK6 (bottom). Highlighted are the sequences of two regions in each UTR exhibiting particularly high density of iCLIP reads and concordance between replicates. Each region shows the presence of several UAG and GUAG oligomers, the known core of the Msi1 binding site. B) CDK6 and CCND1 immunolabeling in intestinal sections from WT or v-Msi1 mice as indicated. Images show merged specific labeling (red) and nuclear staining (blue). Bar=15μm; insets in CDK6 panels=7μm. C) RIP-PCR performed with anti-MSI1 and control antibodies in 293T cells showing that CCND1 and CDK6 mRNAs are highly associated with MSI1 protein. D) RT-qPCR showing the impact of MSI1 knockdown on CCND1 and CDK6 mRNA levels. E) Representative western blot showing the impact of MSI1 knockdown on CCND1 and CDK6 protein levels. F) MSI1 knockdown affects CCND1 and CDK6 mRNA decay.

Figure 5. Stem cell marker expression is affected in v-Msi1 mice intestine

Stem cell gene markers defined by Muñoz et al. [10] show altered expression in v-Msi1 mice according to RNA-seq analysis.

Figure 6. SOX4 is a direct target of Msi1

A) UCSC genome browser plot showing the position (x-axis) and count (y-axis) of iCLIP reads overlapping the 3′ UTR of SOX4 mRNA. Highlighted are the sequences of two regions exhibiting particularly high density of iCLIP reads and concordance between replicates. Each region shows the presence of UAG and GUAG oligomers, the known core of the Msi1 binding site. B) SOX4 immunolabeling in intestinal sections from WT or v-Msi1 mice as indicated. Images show merged specific labeling (red) and nuclear staining (blue). Bar=15μm; insets=7μm. C) RIP-qPCR performed with anti-MSI1 and control antibodies in 293T cells shows that SOX4 mRNAs are highly associated with MSI1 protein. D) RT-qPCR showing the impact of MSI1 knockdown on SOX4 mRNA levels. E) Representative western blot showing the impact of MSI1 knockdown on SOX4 protein levels. F) MSI1 knockdown affects SOX4 mRNA decay.

Figure 7. Increased growth potentialities of v-Msi1 cultured crypts

A) Crypts were prepared from WT or v-Msi1 intestine as indicated and maintained in culture for several days, allowing complex organoid development and structuration. Pictures in (A) have been taken under inverted microscope at the indicated days after the start of the culture, and are representative of two independent experiments, each conducted on six replicates. Bar=50μm. B) The number of simple structure (spheres) or organoids of increasing complexity (1 or 2 buds, more than 3 buds) were scored under the inverted microscope during the first four days of culture. Multilayered histograms in the upper panels represent the mean ± SD, n=6, of each counted structure in the cultured crypts of indicated genotype. Histograms in the lower panels show the direct comparison of the number of spheres and that of complex organoids depending on the genotype. *: P<0.05, **: P<0.01, in comparison with WT, by Student’s t-test. C) RT-qPCR analysis of indicated stem cell markers and MSI1 targets in organoids of different genotype. Values represent fold change ± SD, n=4, after normalization to WT organoids. *: P<0.05, **: P<0.01 and ***: P<0.001, in comparison with WT, by Student’s t-test.