In order to define the miRNA transcriptome of the small intestinal epithelium, small RNAs from purified jejunal epithelia of adult wild-type mice were isolated and quantified using ultra-high throughput sequencing. The resulting sequence reads were aligned to known miRNA precursor genes, obtained from miRBase 17
, in order to assess the abundance of each mature miRNA. Next, we verified that these sequence reads represented miRNAs and not degraded mRNAs by aligning them to the RefSeq database. As shown in , less than 20% of reads in the miRNA size range aligned to mRNAs, while more than 90% matched to precursor miRNAs, indicating that our small RNA preparation was highly enriched for true miRNAs. In total, we generated 15.3 million trimmed reads in the range of 19–25 nts that aligned to precursor miRNAs. Using these reads, we found evidence for expression of 606 of the 1,094 (55.4%) known or predicted mature miRNAs that represent 573 distinct families and cover 399 of 574 (69.5%) pre-miRNAs. (, Supplemental Table 1
). Included in the 573 miRNA families expressed in the small intestinal epithelium are those with known functions in intestinal disease, such as miR-194, and the miR-200 and let-7 families () 11, 14, 18
The miRNA transcriptome of murine small intestinal epithelium
The extraordinary dynamic range (about six orders of magnitude) of the technology used allowed us to detect and quantify miRNAs present in a few copies per million as well as those that contribute up to ~30 percent of the total miRNA pool, i.e. mmu-miR-192. Because of technical limitations of prior efforts, many of the miRNAs identified here had been missed in previous studies 16
. As the intestinal epithelium is made up of multiple cell types of varying abundance, without detailed in situ hybridization analysis for all 606 intestinal miRNAs we cannot determine if they are expressed uniformly in all intestinal epithelial cells, or whether they might be differentially expressed in rarer cell-types such as enteroendocrine cells or stem cells. Nevertheless, our complete miRNA profile of the small intestinal epithelium dramatically extends prior knowledge in this field, and represents a rich resource for future investigations.
Next we searched for evidence of novel miRNAs by analyzing clusters of ten or more reads aligned to the genome that did not overlap with known small RNA genes, e.g, tRNAs, SNORDS, etc. However, none of these clusters appeared to represent novel miRNAs. This suggests that miRBase is approaching coverage of all miRNAs. We did, however, find support for 94 mature miRNAs (based on a minimum of two trimmed reads in our collection) that have been predicted by their precursor miRNAs, but are not presently included in mirBase. In addition, when we analyzed the 187 pre-miRNAs with at least 100 aligned trimmed reads, 113 (60%) showed evidence of miRNA editing, predominantly at the 3′ end (data not shown).
Next, we wanted to determine if and to what degree miRNAs contribute to the differentiation and viability of the intestinal epithelium. We derived mice lacking functional miRNAs in the intestinal epithelium by crossing Dicer1loxP/loxP
conditional mutant mice to Villin-Cre transgenic mice 5, 19
. qPCR analysis confirmed the effective deletion of Dicer1
in the small intestinal epithelium at both three and ten weeks of age (). In addition, we confirmed the ablation of Dicer1
at the functional level by determining the abundance of two intestinal miRNAs, mmu-miR-21 and mmu-let-7b (). Both were dramatically but not completely reduced, reflecting residual Dicer1
and miRNA expression in cell populations where the Villin-Cre transgene is silent, such as mesenchymal or immune cells. This notion is supported by the fact that expression of mmu-let-7b, which is thought to be present in all cell types, is reduced by a smaller percentage than mmu-miR-21, which is enriched in epithelial cells, in our mutant mice 20
. Collectively, these results indicate that Dicer1
is functionally ablated in the intestinal epithelium of Dicer1loxP/loxP
; Villin-Cre mice.
Mice with conditional ablation of Dicer1 in the intestinal epithelium display impaired growth, fat absorption, and water retention
; Villin-Cre mutants appeared normal at birth and were born in the expected Mendelian ratio (data not shown). Some severely affected mutants, approximately 10% of the total, did not survive to weaning and displayed severely stunted growth (data not shown). Mutant mice that survived fed normally but were significantly smaller than their littermate controls beginning at p10 (). This growth impairment continued through weaning (p21), when the food source is switched from the mother’s milk to starch-based chow. After approximately two weeks on normal chow, surviving mutants began to catch up in weight with their control littermates, becoming indistinguishable in size by seven weeks of age (). In conjunction with impaired growth, pre-weaned pups had noticeably pale and loose stool. Oil-red-O staining on fecal smears from pre-weaned (p19) mutants showed the presence of large fat droplets, which were absent from control stool (). Once weaned and subsisting on normal mouse chow, which has only 13.5% kcal from fat as compared to the 80% kcal from fat in mouse milk 21
, mutant mice no longer produced fatty stool (data not shown). However, when we placed Dicer1loxP/loxP
; Villin-Cre mice on a “Western Diet” of 45% kcal from fat, mutants again had markedly increased levels of fat in their stool as compared to controls, even after only two days on the diet (). Thus, Dicer1loxP/loxP
; Villin-Cre mutants have a severely impaired ability to process dietary triglycerides. In addition, adult mutants have ~20% higher percent mass of water in their stool as compared to controls, indicative of decreased water absorption in the colon ().
The Dicer1-deficient intestine differs from that of controls not only functionally but also morphologically. In the small intestine, the villi appeared normal; however, the crypt zone was markedly expanded and the lamina propria appeared more cellular (). In the colon, the regular crypt structure was disorganized in Dicer1-deficient mice, and a more densely packed lamina propria was present between crypts (). There was also a four-fold decrease in the number of mucus filled goblet cells in the colon, which was verified by Alcian blue staining (). Thus, epithelial miRNAs are necessary to maintain normal intestinal architecture and goblet cell number. There was also a drastic increase in the number of apoptotic cells as shown by TUNEL staining in the lower crypt zone of both the small and large intestine of Dicer1 mutants as compared to controls ().
Intestine-specific Dicer1 mutants display an expanded crypt zone in the small intestine, an increase in apoptotic cells and a reduction in goblet cell number in the colon
Next, we aimed to link the intestinal phenotype of Dicer1
-mutants to dysregulation of specific classes of mRNAs. While miRNAs affect protein translation, in many cases they also regulate mRNA abundance 1, 2
. We employed oligonucleotide microarray analysis to identify differentially expressed protein-coding genes. We identified 3,156 differentially expressed genes in the small intestinal epithelium of Dicer1 mutants (see Experimental Procedures for details, and Supplemental Fig. 2
). Next, we identified enriched KEGG Pathways using Gene Set Enrichment Analysis (GSEA), and grouped the genes into categories (, Supplemental Fig. 2
. The most significantly changed pathways in each category are listed in Supplemental Fig 3
. Surprisingly, differentially expressed genes in immune pathways were the largest category of genes affected in Dicer1
Increased inflammation in the intestine of Dicer1 mutants
Based on our mRNA expression profiling data, we decided to further investigate immune pathways in Dicer1
mutants. An important component of intestinal defense against luminal pathogens are neutrophils in the lamina propria. There was an increase in the number of neutrophils in the lamina propria in both the small and large intestine, with a more dramatic phenotype in the large intestine (). Low magnification images demonstrate a dramatic increase in lymphoid nodules in the large intestine of mutants as compared to controls (). In the large intestine of Dicer1
mutant mice there was an increase in neutrophil number in the lamina propria at the base of the crypts as compared to controls, in addition to a small number seen infiltrating the colonic epithelium (). Increased inflammation has been linked previously to loss of goblet cells 25, Makkink MK, 2002 #50
and is a likely explanation for the four-fold decrease in goblet cells in Dicer1
mutants described above.
To investigate potential causes for the redistribution and increase in immune cells in Dicer1
-deficient mice, we next analyzed epithelial maintenance and tight junctions in the small intestine of the mutants, as these act as part of the physical barrier that prevents luminal pathogens from entering the blood stream. Claudin-7, a component of tight junctions, was localized to the basolateral membrane of epithelial cells in the control large intestine, as has been described previously () 26
. Claudin-7 was mislocalized in the large intestine of Dicer1loxP/loxP
; Villin-Cre mutants, with all cell surfaces staining positive for the protein (). An important aspect of the intestinal barrier function is proper epithelial cell arrangement and organization. In the control large intestine, the epithelial nuclei are uniformly aligned parallel to the apical surface (; insert). This was not the case, however, in the mutant colon, where the epithelial layer was disorganized and the nuclei distributed on different planes throughout the epithelium (; insert). In addition to Claudin-7, we investigated the localization and expression of Claudin-4, a functional component of tight junctions in the small intestine. In the control small intestine Claudin-4 is expressed in puncta, a majority of which line the apical membrane, with a few distributed on the intra-cellular and basolateral membranes (). In contrast, in the small intestine of mutants there was a decrease in Claudin-4 positive tight junctions ().
Dicer1 mutants exhibit a disorganized epithelium and decreased tight junctions leading to increased intestinal permeability
In order to determine the consequence of the observed epithelial disorganization in the Dicer1
mutants, we measured the intestinal paracellular permeability. Lactulose and mannitol are non-digestable carbohydrates that cross the epithelium via paracellular routes and are thus useful tools to measure intestinal barrier function 27, Travis S, 1992 #48
. We fed radioactively labeled mannitol and lactulose to three month old mice by gastric gavage and determined their rate of transfer to circulation by periodic sampling of peripheral blood. As shown in , the rate of epithelial crossing of both lactulose and mannitol was dramatically increased in Dicer1
-deficient mice, clearly demonstrating decreased intestinal epithelial barrier function in these mice.