Extended Data Fig. 4: Villi formation and parameter description of villi fissions.
a, Total number of villi (projected) in the proximal half of the small intestine based on equal density along the length. Samples from three mice were analysed per time point. Each dot represents a mouse; data are mean ± s.e.m. b, The fold change in villi numbers between the indicated time points based on three samples analysed per time point. Each dot represents an independent sample and lines represent the mean ± s.e.m. c, Villus height at the different time points. The demarcated red lines indicate the interval containing villi with sharing stroma. The length was assessed in 25 villi per mouse and in 3 mice per time point. Dots represent independent measurements and lines represent the mean. d, Quantification of the number of villi sharing stroma in three mice per indicated time point (E16.5 n = 233, P0 n = 412, P5 n = 406, P11 n = 412, adult n = 129 villi were counted). Dots represent the percentage of villi sharing stroma in each independent mouse and the line represents the mean. e, Detection of E-cadherin (red) and PDGFRA (yellow) in whole mounts indicating villi with sharing stroma (arrowhead). Samples from three mice were analysed per time point and representative images are shown. Scale bars, 100 μm. f, Detection of E-cadherin (magenta) and GFP (Lgr5-DTR-eGFP) in E16.5 intestinal whole mount. Boxed area (1) indicates a villus undergoing fission, which is shown at higher magnification (middle). Transverse sections (2 and 3) illustrating villi surrounded by LGR5-expressing cells (2) and villus with shared mesenchyme (3). The arrowhead indicates that pockets formed in a fissioning villus are LGR5-negative, the dashed line outlines the epithelium. Samples from three mice were analysed and a representative image is shown. g, Detection of EdU incorporation (green) in the epithelium (E-cadherin, red) following a 1-h chase in E16.5 intestinal whole mounts. Arrowheads indicate proliferative cells at the edge of putative villi undergoing fission. These are detected in 11 out of 16 structures. Representative pictures from three mice analysed are shown. Scale bars, 50 μm. h, Quantification of EdU intensity in the fissioning areas compared to the surrounding villi at the same height quantified as depicted in the cartoon on the basis of thresholded intensity in the demarcated boxes. n = 16 independent villi sharing stroma were quantified. Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend to minimum and maximum values. Dots represent fluorescent ratio of the independent villi sharing stroma. Paired t-test. i, Height of the proliferative fissioning areas compared to the surrounding intervillus regions were quantified as depicted in the cartoon. n = 11 independent villi sharing stroma were quantified. j, Images showing the start and end points from live imaging of villi undergoing fission (Supplementary Video 6). Image from mT/mG;Villin-cre mice, where the epithelium is shown Green (mG) and remaining cells in red (mT). A representative fission event from five analysed mice is shown. Scale bar, 50 μm.
