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Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis

ABSTRACT: The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes, which constitute the most abundant cell type, perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterial-rich environment. It is unknown if these tasks are equally performed by all enterocytes or whether they are spatially zonated along the villus axis. Here, we performed whole-transcriptome measurements of laser-capture-microdissected villus segments to extract a large panel of landmark genes, expressed in a zonated manner. We used these genes to localize single sequenced enterocytes along the villus axis, thus reconstructing a global spatial expression map. We found that most enterocyte genes were zonated. Enterocytes at villi bottoms expressed an anti-bacterial Reg gene program in a microbiome-dependent manner, potentially reducing the crypt pathogen exposure. Translation, splicing and respiration genes steadily decreased in expression towards the villi tops, whereas distinct mid-top villus zones sub-specialized in the absorption of carbohydrates, peptides and fat. Enterocytes at the villi tips exhibited a unique gene-expression signature consisting of Klf4, Egfr, Neat1, Malat1, cell adhesion and purine metabolism genes. Our study exposes broad spatial heterogeneity of enterocytes, which could be important for achieving their diverse tasks.

ORGANISM(S): Mus musculus

PROVIDER: GSE109413 | GEO | 2018/02/07

REPOSITORIES: GEO

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Project description:Gene expression along the crypt-villus (C-V) axis was analyzed using cryostat sectioning to isolate fractions representing the crypts (bottom) and villus tops (top). These fractions were used for analyzing gene expression in iron replete Wistar rats (++), iron deficient Wistar rats (low iron), and in iron deficient Wistar rats fed iron for 3 and 6 days (iron-fed). Differences were observed between the crypts and villus tops in the expression of genes associated with Wnt and BNP signaling, cell proliferation and apoptosis, lipid and iron transport and metabolism. Gene expression in villus crypts and tops was also compared between Wistar and Belgrade rats (bb) and Belgrade rats fed iron (iron-fed) particularly as related to iron absorption and metabolism to define the affects of the mutation in DMT1 in the Belgrade rat on the expression of genes related to iron absorption and metabolism and the response to iron feeding. Keywords: iron stress response A colony of Wistar strain rats (++) and Belgrade (bb) rats on a Wistar background maintained in our animal quarters was used for this study. Twelve one-month old ++ rats were separated into 3 groups of four rats each. Group 1 rats (samples 1-8) were fed a normal diet and served as control animals. Group 2 (samples 9-16) and Group 3 rats (samples 17-22) were fed a low iron diet and bled 2-3 ml every three days for 4 weeks and served as iron deficient rats (low-iron). Group 3 rats, of which one rat died during anesthesia while undergoing phlebotomy, were fed iron supplementation in drinking water (50 ÂµM Ferric ammonium citrate) for the three days before sacrifice (iron-fed). Rats were fasted overnight, killed by injection of pentobarbital sodium and a 1-cm duodenal segment 1 cm distal from the pylorus was cryostat sectioned at the right angle to the crypt-villus axis and the sections representing the top one-third of villus (top) and the bottom one-third of the villus were pooled for RNA isolation. For the study of Belgrade rats, eleven bb rats were divided into 3 groups with the 4 rats in Group 1 (samples 23-30) maintained on a normal diet, the 4 rats in Group 2 (samples 31-38) fed iron for 3 days prior to sacrifice, and the 3 rats in Group 3 (samples 39-44) fed iron for 6 days prior to sacrifice. All procedures for the bb rats were the same as described above for the ++ rats. An additional ++ rat (samples 45-46) was similarly analyzed in a separate experiment.

Project description:Genes encoding transcription factors function as hubs in gene regulatory networks because they encode DNA-binding proteins, which bind to promoters that carry their binding sites. In the present work we have studied gene regulatory networks defined by genes with transcripts belonging to different mRNA abundance classes in the small intestinal epithelial cell. The focus is the rewiring that occurs in transcription factor hubs in these networks during the differentiation of the small intestinal epithelial cell while it migrates along the crypt-villus axis and during its development from a fetal endodermal cell to a mature adult villus epithelial cell. We have generated transcriptome data for mouse small intestinal villus, crypt and fetal intestinal epithelial cells. In addition we have generated metabolome data from crypt and villus cells. Our results show that the intestinal crypt transcription factor hubs that are rewired during differentiation are involved in the cell cycle process (E2F, NF-Y) and stem cell maintenance (c-Myc). In contrast the villi are dominated by a HNF-4 villus hub, which is rewired during differentiation by the addition of network genes with relevance for lipoprotein synthesis and lipid absorption. Moreover, we have identified a villus NF-kB hub, which was revealed by comparison of the villus and endoderm transcriptomes. The rewiring of the NF-kB villus hub during intestinal development reflects transcriptional activity established by host and microflora interactions. To aid in the mining of our results we have developed a web portal (http://gastro.imbg.ku.dk/mousecv/) allowing easy linkage between the transcriptomic data, biological processes and functions. Experiment Overall Design: Four different sample categories were analyzed. Experiment Overall Design: 1) Small intestinal crypts isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 2) Small intestinal villi isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 3) Embryonic day 12 mesenchyme. These samples are in quadruplicate. each sample is derived from a pool of mesenchymes (10-40) Experiment Overall Design: 4) Embryonic day 12 endoderm. These samples are in quadruplicate. each sample is derived from a pool of endoderms (10-40)

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Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis

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