Fig 2: Representative photomicrographs of protein localization of CHGA, GSTA3 and PRSS2 in endometrial tissue from RB and non-RB cows on Day 15 of estrous cycle. Protein localization of (a, b, c and d) CHGA, (e, f, g and h) GSTA3 and (i, j, k and l) PRSS2 in endometrial tissue from RB (a, b, e, f, i and j) and non-RB (c, d, g, h, k and l) cows was detected by immunohistochemistry. Seven-micrometer sections of bovine endometrial tissues of ipsilateral uterine horns on Day 15 of estrous cycle were immunostained with anti-human CHGA, anti-human GSTA3 and anti-bovine PRSS2 polyclonal antibodies. Positive staining of CHGA and PRSS2 were found in the uterine luminal epithelium, uterine stroma and glandular epithelium of both RB and non-RB cows. GSTA3 was detected in the uterine luminal, uterine stroma and glandular epithelium in RB cows, whereas positive staining was not observed in non-RB cows. No signal was detected in the negative control sections using normal rabbit IgG (inserted panels). LE, luminal epithelium; US, uterine stroma; GE, glandular epithelium. Scale bars = 50 µm

Fig 3: Loss of epithelial Sprouty2 results in expansion of colonic tuft and goblet cell numbers.a Deletion of Sprouty2 in Spry2IEKO mice was confirmed by qPCR and western blot of colonic homogenates. b Tuft (Dclk1, Trpm5, and Il25), c enteroendocrine (Chga), d goblet (Muc2 and Tff3), e stem (Lgr5 and Lrig1), and f absorptive enterocyte (Car2 and Aqp8) markers were measured in Spry2IEKO colonic homogenates by qPCR. g Tuft (Dclk1+; in red), h enteroendocrine (ChgA+; in red), and i goblet (Muc2+; in red) cells were quantified by immunofluorescence staining of distal colonic sections. Green = E-cadherin. Scale bar = 25 µm. j Proliferation (Ki67+), k apoptosis (TUNEL+), and l crypt length were quantified in distal colons. Data are presented as mean ± SEM. Analyzed by two-sided t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Exact p values where appropriate are as follows b Dclk1 p = 0.0065; Trpm5 p = 0.0203; Il25 p = 0.0046; d Muc2 p = 0.0124; Tff3 p = 0.0099; g p = 0.0002. Number of mice analyzed, from five independent litters, in each panel as follows: a 16 per group; b Dclk1 19 FF, 14 KO; Trpm5 16 FF, 12 KO; Il25 15 FF, 13 KO; c 14 FF, 9 KO; d Muc2 17 FF, 13 KO; Tff3 17 FF, 13 KO; e, f 14 FF, 9 KO; g 10 FF, 8 KO; h 14 FF, 8 KO; i 7 FF, 7 KO; and j–l 7 FF, 6 KO.

Fig 4: Markers to identify the secretory cell lineage.Identification of secretory cells in porcine small intestine and colon are shown. Immunostaining for enteroendocrine cells, the hormone secreting cells important to gut homeostasis, using antibodies against CgA, SST, GLP-1 and GAST, demonstrated staining within the cytoplasm of positive cells of the small intestine. A similar pattern of staining was identified in the colon for CgA, SST and GLP-1. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.

Fig 5: Morphology and features of human iPS cell-derived intestinal organoids by suspension culture (A) Day 10 of differentiation induction. Spheroids of the differentiation cells cultured for 3 days on EZSPHERE plates (B, C) Day 34 of differentiation induction. Spheroid of (A) transferred to ultralow attachment plates and cultured in Advanced DMEM/F12 containing 3% Matrigel suspension (B) without or (C) with small-molecule compounds at the end of differentiation (day 34) (A–C) Scale bar = 500 µm (D) Relative mRNA expression of the HIOs cultured without [HIO (A) group] and with [HIO (B) group] small-molecule compounds. SI: total RNA from human adult small intestine samples (five donors), Caudal-type homeobox 2 (CDX2), villin 1 (VIL1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), olfactomedin 4 (OLFM4), mucin 2 (MUC2), chromogranin A (CHGA), vimentin (VIM), and actin alpha 2, smooth muscle (ACTA2) are markers of the hindgut, absorptive epithelial cells, intestinal stem cells, goblet cells, enteroendocrine cells, mesenchymal cells, and smooth muscle cells, respectively. The mRNA expression levels of SI were defined as 1. Data were shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01 [HIO (A) vs. HIO (B)] (E) Fluorescence immunostaining of HIOs. Ki67 and E-cadherin (E-cad) are markers of cell proliferation and epithelial cells, respectively. Villin, OLFM4, Ki67, CDX2, E-cad, and CHGA were highly expressed in the luminal-like structures, and vimentin (Vime) and a-smooth muscle actin (a-SMA) were expressed outside them. Scale bar = 100 µm.