Fig. 3: Multicellular composition of chicken enteroids.

Confocal images of a–d embryonic jejunum, e–h 6 week old chicken jejunum, and i–l, o embryonic chicken enteroids at 2 days of culture. All cells are counterstained with DAPI (blue). The cells are 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). i–l Chicken enteroids stained to detect F-actin-expressing brush border (red). m Transmission electron microscopy of chicken enteroids (4 h in culture) demonstrates an enterocyte (closed arrow) and Paneth cell (open arrow). n TEM of chicken enteroids (7 days in culture) demonstrates a goblet cell. o Confocal image of chicken enteroid stained for villin (green, epithelial microvilli). p TEM of chicken enteroid 7 days in culture, enterocyte basal lamina (closed arrow) and microvilli (open arrow). Scale bar: a–l, o 20 µm. m, n, p 2 µm. Images a–p are representative of data from at least 3 independent cultures each containing 2–3 embryos. q Expression of intestinal epithelial cell lineage-specific genes in freshly isolated villi (0 h) and enteroids at 3 and 7 days of culture compared by RNA sequencing analysis. Heat maps show the relative expression levels (log2 counts per million reads) of a range of mammalian epithelial cell lineage-related genes. TA: transit amplifying cells, ECepr: early enterocyte precursor cells, EClpr: late enterocyte precursor cells. RNA sequencing data is representative of 3 independent experiments, each comprising of 2 technical replicates, each containing 3 embryos.