Fig. 4: Site-specific chicken enteroids demonstrate in vivo multicellular composition and architecture.

a–l Confocal images of embryonic chicken enteroids at 2 days of culture grown from the duodenum, jejunum and caeca. Stained for a, e, i Lysozyme C (green, Paneth cells), b, f, j Muc5AC (green, goblet cells), c, g, k Sox9 (green, proliferating cells), and d, h, l Chromogranin A (green, enteroendocrine cells). All counterstained with DAPI (blue). b, f, j stained to detect F-actin-expressing brush border (red). Scale bar: 20 µm. Images are representative of data from at least 3 independent cultures, each containing 2–3 embryos. m Enteroids were cultured for 2 days and the number of villus-crypt like structures or buds, i.e. 0, 1, 2, 3 or more per enteroid, were counted. After 2 days of culture there were statistically significant differences in numbers of buds between the different regions of the small intestine. The caecal enteroids more often lack buds (0) or only have 1 bud compared to duodenal and jejunal enteroids, whereas jejunal enteroids have more buds (3+) in comparison to duodenal and caecal enteroids. n The length of the buds resembled the in vivo architecture with jejunal and duodenal buds measuring significantly longer than caecal buds. Box and whisker plot data derived from 3 independent experiments each containing 2–3 embryos, at least 260 enteroids and 150 buds measured per location for m and n, respectively. Statistics performed using a Kruskal–Wallis test; m 0 budding, **p 0.001, H = 14.06, df = 2; 1 budding, **p 0.001, H = 13.42, df = 2; and more than 3 budding, **p 0.001, H = 13.62, df = 2, enteroids between the duodenal, jejunal and caecal cultures. n ***p 0.0001, H = 110.08, df = 2, with post hoc Mann–Whitney U-tests (two-sided); duodenum–jejunum, ***p 0.0001, W = 23230, 95% CI for difference (−6.820, −3.266); duodenum–caecum, ***p 0.0001, W = 29697.5, 95% CI for difference (13.914, 9.780); jejunum–caecum, ***p 0.0001, W = 46327.5, 95% CI for difference (7.249, 10.707).