Intestinal epithelial cells (IECs) absorb nutrients, respond to microbes, provide barrier function and help coordinate immune responses. We profiled 53,193 individual epithelial cells from mouse small intestine and organoids, and characterized novel subtypes and their gene signatures. We showed unexpected diversity of hormone-secreting enteroendocrine cells and constructed their novel taxonomy. We distinguished between two tuft cell subtypes, one of which expresses the epithelial cytokine TSLP and CD45 (Ptprc), the pan-immune marker not previously associated with non-hematopoietic cells. We also characterized how cell-intrinsic states and cell proportions respond to bacterial and helminth infections. Salmonella infection caused an increase in Paneth cells and enterocytes abundance, and broad activation of an antimicrobial program. In contrast, Heligmosomoides polygyrus caused an expansion of goblet and tuft cell populations. Our survey highlights new markers and programs, associates sensory molecules to cell types, and uncovers principles of gut homeostasis and response to pathogens.

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV crosslinking-immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression.The distinct isoforms expressed from metazoan genes through alternative splicing can be important in development, differentiation and disease 1 . For example, the pyruvate kinase gene produces two distinct tissue-specific spliced isoforms that differ in their enzymatic activity, allosteric regulation and ability to support tumor growth 2 . Conservative estimates predict 2-12 mRNA isoforms for most mammalian genes ( Supplementary Fig. 1), though some genes, including neurexins, may express more than 1,000 isoforms each 3 .© 2010 Nature America, Inc. All rights reserved.Correspondence should be addressed to E.M.A. (airoldi@fas.harvard.edu) or C.B.B. (cburge@mit.edu). AUTHOR CONTRIBUTIONS Y.K., development of MISO model and software, analyses involving MISO, writing of main text and methods; E.T.W., hnRNP H CLIP-seq experiments and associated computational analyses, CUGBP1 knockdown RNA-seq experiments and associated computational analyses; E.M.A., development of model and statistical analysis, writing of methods; C.B.B., development of MISO model, contributions to computational analyses, writing of main text. Recently, high-throughput sequencing of short cDNA fragments, RNA-seq, has emerged as a powerful approach to characterizing the transcriptome. RNA-seq data have recently been used to show that the vast majority of human genes are alternatively spliced and that most alternative exons show tissue-specific regulation 4 . To date, RNA-seq analysis methods have focused mostly on estimation of gene expression levels and discovery of novel exons and genes 4-6 , assembly and annotation of mRNA transcripts 5,7 , and estimation of the expression levels of alternative exons 4 . Two recent methods, Cufflinks and Scripture, can produce de novo annotations of transcripts in metazoan genomes using RNA-seq data alone [8][9][10] . COMPETING FINANCIAL INTERESTSAccurate quantification of alternative-exon abundance and detection of differentially regulated exons and isoforms remain challenging. Paired...

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