Figure 3. Paneth cells produce Wnt3 and other essential niche signals for Lgr5 stem cells.
a: Heatmap of two independent microarray expression experiments (1/2 and 3/4) performed with dye-swap (1 vs 2 and 3 vs 4) from sorted Paneth cells vs Lgr5 stem cells. b: Wnt3 is expressed by Paneth cells at crypt bottoms as analyzed by in situ hybridization, c-h: Localized Wnt production regulates crypt-villus morphogenesis in culture. c: Freshly isolated crypts from Axin2-lacZ mouse were cultured in standard EGF/Noggin/Rspondin1 medium (ENR) medium for 4 days. LacZ response is only seen near the bottoms of the two crypts. d: Intestinal adenoma samples from Apcmin mice were cultured in ENR medium in the absence of R-spondin for 7 days. e: Axin2-LacZ crypts grown in ENR medium. f: As in e) with the addition of porcupine inhibitor IWP1 at 1 μM. g: Crypts from Axin2-LacZ mouse cultured in ENR medium plus Wnt3a h: same as g) with the addition of IWP1. Insets in e-h depict Axin2-LacZ expression (blue). e, g, h: 6 days culture. f: 3 days culture after which the organoids disintegrate. See also Suppl Fig 2. i: Plating efficiency of Lgr5 stem cell-Paneth doublets, Lgr5 stem cell doublets, single Paneth cells and single Lgr5 stem cells with (grey) or without (black) Wnt-3A at 100 ng ml-1. Assays were read out as budding organoids at 14 days after sorting. The values are depicted as mean±standard error of the mean (SEM) from three independent experiments. N.D.; not detected. See also Suppl Fig 3 and online methods for detail of doublet isolation and culture.