Data for the fluorescence-time conversion dataset were collected in intervals of 45 min between recorded z stacks of individual organoids in 3 independent experiments. Ct values were calculated using the second derivative method. By continuing you agree to the, https://doi.org/10.1016/j.cell.2018.12.029, Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping, View Large To corroborate the validity of our findings, we chose 9 candidate genes (. Numbers indicate unique transcripts + 0.1. Single CCK-eGFP-positive cells isolated from the duodenum of transgenic mice were sorted individually into 96-well plates and subjected to QPCR analysis for Cck, Sct, Ghrl, and Gip gene expression without preamplification. (E) Relative frequency of individual mature EE cell types in crypt and villus. As expected based on the previously well-characterized coexpression of GLP-1 with PYY, PYY-containing cells were ablated to an almost similar extent. 2008 Jun;377(4-6):523-7. doi: 10.1007/s00210-007-0200-8. Three fatty acids had a distinct specific structure containing one double bond, whereas the other two had two double bonds, nearby an oxo residue. (G) Expression of CHGA in all EE populations of the high sensitivity EE single cell dataset (min. Epub 2007 Oct 31. A total number of 6906 cells were sorted and processed following the SORT-Seq method (. Ab, Antibody. Humanized Renilla green fluorescent protein (Ghrelin-hrGFP) (line hrGFP10) mice were generated as previously described (23). April 21, The results of the present study, as well as, for example, the studies by Roth et al. In this review, we will present an overview of the latest advances for roles of EECs in maintaining intestinal homeostasis. 5 and see Fig. For identification of antibodies see Supplemental Table 2. Vertical panel II, Immunohistochemical localization of hormones (red) using primary antibodies against secretin (horizontal lane A), ghrelin (lane B), neurotensin (lane C), somatostatin (lane D), gastric inhibitory polypeptide (GIP) (lane E), GLP-1 (lane F), and peptide YY (PYY) (lane G). and VENI fellowship ( NWO-ZonMW, 016.166.140 to K.K.). (I) Projection of calculated differentiation time on tSNE map. Enteroendocrine cells switch hormone expression along the crypt-to-villus BMP signalling gradient. About 15 different types of enteroendocrine cell types have been identified, and a similar, large number of peptide hormones have been assigned to each distinct cell type. Epub 2010 Oct 16. Because differential gene expression analysis among mature populations alone (. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Ab, Antibody. (left) cluster identity (right) cell origin. USA.gov. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Statistically significant differences between wild-type and knockout clones were determined using an unpaired two-tailed Student’s t test. Panel C, Colocalization of CCK (red, rabbit Ab) and secretin (green, goat Ab) in human duodenum. Distribution of eGFP-positive enteroendocrine cells in the GI tract of CCK-eGFP transgenic mice. (B) Structure of Neurog3Chrono reporter on DNA level and protein products. Enteroendocrine cells. J.B. provided reagents and conceptional input, K.K. Basic helix-loop-helix transcription factors and enteroendocrine cell differentiation. (C) Venn diagram of population-specific genes (p < 0.01, log2FC > 1.5). Tissue sections were permeabilized with PBST [PBS-Ca-Mg (pH 7.4) + 0.1% triton] three times for 2 min. generated the. O’Morain C, Bishop AE, McGregor GP, et al. 2B). Table S1. Ct values less than 40 were counted as positive, and non-RT samples were included to validate the specificity of the assay. For detailed information on DNA library preparation, sequencing, mapping to the mouse reference genome and quantification of transcript abundance please refer to (, Bulk sequencing libraries were analyzed using the DESeq2 package (, For single cell analysis, cell clusters and expression comparisons between clusters were generated using the RaceID2 algorithm (. Gophna U, Sommerfeld K, Gophna S, et al. Secretin- and GIP-producing cells display an approximately 50–60% colocalization with CCK-eGFP. We did not detect any costaining of CCK-eGFP with antibodies directed against somatostatin, which is in agreement with the QPCR data. 4,000 unique transcripts/cell) (top-left), time projection on tSNE (top-right), expression levels of. Gray cells stem from whole SI preparations. Fluorescence raw data was imported into R and normalized per cell after background subtraction (based on the background signal of each individual frame).

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