Project description:Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer’s patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that M cells are rare but M-like cells positive for lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM16-2-4 are induced by oral administration of cholera toxin in the duodenal villous epithelium. Here, we determined the gene expression of PP M cells, villous M-like cells, and intestinal epithelial cells (IECs) isolated by a novel approach using FACS. Specific expression of glycoprotein 2 (GP2) and MARCKS-like protein (MLP) by PP M cells and not villous M-like cells was confirmed by additional mRNA and protein analyses. Comprehensive gene profiling also suggested that villous M-like cells share traits of both PP M cells and IECs, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology. Keywords: Cell type comparison
Project description:M cells in the follicle-associated epithelium (FAE) of Peyer’s patches (PPs) serve as a main portal for external antigens. Limitation in the number of M cells has hampered identification of their specific molecules until recently. Efforts by us as well as others has gradually unraveled the molecular mechanisms for the development and differentiation of M cells. However, molecular mechanisms of antigen transcytosis and M cell differentiation were not fully elucidated. Recent studies reported that small non-coding RNAs including microRNA (miRNA) regulate gene expression that controls various biological processes such as cellular differentiation and functions. In fact, intestinal epithelial miRNAs play a critical role in goblet cell differentiation and maturation. However, the expression and function of miRNAs in FAE including M cell that function as the sentinels in the mucosal immune system are largely unknown. To address this notion, we performed microarray analysis to characterize expression profiles of miRNA in intestinal villous epithelium (VE) and FAE of PP. We also generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC), in which intestinal phenotypes including M-cell differentiation and epithelial morphology were examined. The microarray analysis identified that 72 miRNAs were up-regulated whereas 11 miRNAs were down-regulated, in FAE compared to VE. DicerΔIEC mice displayed a prominent decrease in mature M cells, suggesting an essential role of miRNAs in maturation of the cell type. Furthermore, electron micrographs clearly showed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake in M cells was impaired in DicerΔIEC mice compared to control floxed Dicer mice. These results raised the possibility that miRNAs play a significant role in the regulation of M cells maturation, and consequently secure mucosal immune homeostasis. Follicle associated epithelium or villous epithelium was isolated from Peyer's patch(PP). PP was soaked Hank's balanced salt solution (HBSS;GIBCO) containing 30mM EDTA. After incubation at 4℃ for 20 min, FAE or VE was isolated by manipulation with fine needle under stereomicroscopic monitoring. The isolated epithelial cell sheets were kept in ice-cold HBSS until RNA extraction(n=2).
Project description:Separate populations of M cells have been detected in the follicle-associated epithelium of Peyerâs patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that M cells are rare but M-like cells positive for lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM16-2-4 are induced by oral administration of cholera toxin in the duodenal villous epithelium. Here, we determined the gene expression of PP M cells, villous M-like cells, and intestinal epithelial cells (IECs) isolated by a novel approach using FACS. Specific expression of glycoprotein 2 (GP2) and MARCKS-like protein (MLP) by PP M cells and not villous M-like cells was confirmed by additional mRNA and protein analyses. Comprehensive gene profiling also suggested that villous M-like cells share traits of both PP M cells and IECs, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology. Experiment Overall Design: Epithelial cells in duodenal Peyer's patches or villi of BALB/c mice treated with or without cholera toxin were dissociated and stained with NKM16-2-4 monoclonal antibody (mAb)-FITC, lectin UEA-1-PE, and anti-CD45 mAb-APC-Cy7. Naive PP M cells (NKM16-2-4+, UEA-1+, and CD45- cells), CT-induced villous M-like cells (NKM16-2-4+, UEA-1+, and CD45- cells), and naive intestinal epithelial cells (NKM16-2-4-, UEA-1-, and CD45- cells) were isolated by FACS. Affymetrix GeneChip Mouse Genome 430 2.0 Array was used in triplicate for each cell fraction.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Translational research is commonly performed in the C57B6/J mouse strain, chosen for its genetic homogeneity and phenotypic uniformity. Here, we evaluate the suitability of the white-footed deer mouse (Peromyscus leucopus) as a model organism for aging research, offering a comparative analysis against C57B6/J and diversity outbred (DO) Mus musculus strains. Our study includes comparisons of body composition, skeletal muscle function, and cardiovascular parameters, shedding light on potential applications and limitations of P. leucopus in aging studies. Notably, P. leucopus exhibits distinct body composition characteristics, emphasizing reduced muscle force exertion and a unique metabolism, particularly in fat mass. Cardiovascular assessments showed changes in arterial stiffness, challenging conventional assumptions and highlighting the need for a nuanced interpretation of aging-related phenotypes. Our study also highlights inherent challenges associated with maintaining and phenotyping P. leucopus cohorts. Behavioral considerations, including anxiety-induced responses during handling and phenotyping assessment, pose obstacles in acquiring meaningful data. Moreover, the unique anatomy of P. leucopus necessitates careful adaptation of protocols designed for Mus musculus. While showcasing potential benefits, further extensive analyses across broader age ranges and larger cohorts are necessary to establish the reliability of P. leucopus as a robust and translatable model for aging studies.
Project description:BackgroundCopy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously.ResultsWe found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR).ConclusionThe analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.
Project description:BackgroundLong terminal repeat (LTR) retrotransposons make up a large fraction of the typical mammalian genome. They comprise about 8% of the human genome and approximately 10% of the mouse genome. On account of their abundance, LTR retrotransposons are believed to hold major significance for genome structure and function. Recent advances in genome sequencing of a variety of model organisms has provided an unprecedented opportunity to evaluate better the diversity of LTR retrotransposons resident in eukaryotic genomes.ResultsUsing a new data-mining program, LTR_STRUC, in conjunction with conventional techniques, we have mined the GenBank mouse (Mus musculus) database and the more complete Ensembl mouse dataset for LTR retrotransposons. We report here that the M. musculus genome contains at least 21 separate families of LTR retrotransposons; 13 of these families are described here for the first time.ConclusionsAll families of mouse LTR retrotransposons are members of the gypsy-like superfamily of retroviral-like elements. Several different families of unrelated non-autonomous elements were identified, suggesting that the evolution of non-autonomy may be a common event. High sequence similarity between several LTR retrotransposons identified in this study and those found in distantly-related species suggests that horizontal transfer has been a significant factor in the evolution of mouse LTR retrotransposons.