Volume 146, Issue 3, Pages 754-764.e3 (March 2014) Distinct Cellular Origins for Serotonin-Expressing and Enterochromaffin-like Cells in the Gastric Corpus  Hui Joyce Li, Brian Johnston, Daniel Aiello, Daniel R. Caffrey, Maryann Giel–Moloney, Guido Rindi, Andrew B. Leiter  Gastroenterology  Volume 146, Issue 3, Pages 754-764.e3 (March 2014) DOI: 10.1053/j.gastro.2013.11.048 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Contribution of NeuroD-expressing cells to endocrine cells of the stomach. (A) Expression of tdTom in ChgA+ cells in the antrum and corpus of NeuroD-cre;ROSAtdTom mice. Absence of tdTom (green arrows) in many corpus ChgA+ cells. (B) Lineage tracing of NeuroD+ cells in gastric endocrine cell subtypes in NeuroD-cre;ROSAtdTom or NeuroD-Cre;ROSA26R-LacZ mice. Yellow arrows indicate endocrine cells coexpressing tdTom or LacZ, and green arrows indicate endocrine cells that do not coexpress tdTom. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Expression of mast cell–associated genes in serotonin cells of the corpus. (A) Tph1-CFP BAC transgene. (B) Expression of CFP in the stomach of Tph1-CFP mice stained with CFP and either serotonin (5HT) or ChgA. Arrows indicate double-positive cells. (C) FACS enrichment of endocrine cells from the corpus of Tph1-CFP and Neurog3cre;ROSAEYFP mice. (D) RT-PCR analysis of FACS-enriched cells from the corpus of Tph1-CFP compared with Neurog3Cre;ROSAEYFP or Hdc-CFP mice. (E) Coexpression of Mcpt1 (arrowhead) and c-Kit (arrow) in mucosal serotonin cells. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Gastric corpus serotonin cells are bone marrow–derived mast cells. (A) Corpus from wild-type (WT) and c-KitWsh/Wsh mutant mice immunostained for 5HT or HDC (arrowhead). (B) Presence of endocrine cell subtypes in c-KitWsh/Wsh mutant mice. Results are shown as a percentage of the total number of epithelial cells ± SD for at least 3 mice. (C and D) Corpus from irradiated mice that underwent transplant with EGFP donor bone marrow. (C) Separate and merged images showing coexpression of EGFP in serotonin cells. (D) Absence of EGFP expression in HDC, ghrelin, or somatostatin cells. Green arrows denote EGFP+ cells, whereas red arrows point to cells expressing hormones. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Enrichment and characterization of HDC+ ECL cells. (A) Stomach of Hdc-CFP mouse showing CFP expression in HDC+ and ChgA+ cells, denoted by arrow. Red arrowheads point to ChgA+CFP- cells. (B) FACS analysis of CFP+ cells from the corpus of HDC-CFP mice with the indicated cell surface markers. (C) RT-PCR analysis of NeuroD gene expression in FACS-enriched HDC-CFP cells. The NeuroD+ STC1 enteroendocrine cell line was used as a positive control. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 RNA-seq analysis of enriched ECL cells. (A) Smear plot showing distribution of up-regulated and down-regulated genes between Hdc-CFP+ and CFP− cells. (B) Heat map representation of genes differentially expressed between CFP+ and CFP− (NEG) cells. Scaled expression values of all 1449 genes are shown for each group. (C) Variance normalized read counts for selected hormone and receptor genes in HDC+ and CFP– cells. (D) Transcription factors highly enriched in HDC+ cells. (E) Limited overlap of transcription factor expression in intestinal endocrine cells and HDC+ cells in the stomach. Only 9 ECL-enriched TF genes were up-regulated in intestinal enteroendocrine cells. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 1 Contribution of Neurog3-expressing cells to gastric serotonin, ghrelin, and somatostatin cells in Neurog3Cre;ROSAtdTom mice. (Top row) Coexpression of tdTom with 5HT (B) in the antrum but (A) not in the corpus. (Bottom row) tdTom coexpression in the majority of (C) ChgA+ cells and (D) ghrelin cells in the corpus. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 2 Correlation between RNA-seq variance normalized read counts and relative gene expression measured by quantitative PCR. Expression measured by quantitative PCR for 6 genes with normalized read counts between 1000 and 60,000. Logarithmic scale. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 3 Coexpression of somatostatin (Sst) and ghrelin in some HDC+ cells from HDC-CFP mice. Green arrows, CFP+ cells; yellow arrows, double-positive cells; red arrows, ghrelin+ or Sst+ cells; red arrowhead, CFP− cell with strong ghrelin staining. Gastroenterology 2014 146, 754-764.e3DOI: (10.1053/j.gastro.2013.11.048) Copyright © 2014 AGA Institute Terms and Conditions