Figure 2.

CD45⁻CD90⁺CD31⁺ LECs in the small intestine produce R-Spo3. (a) After IECs were harvested from the small intestine of naïve B6D2F1 mice by incubating with EDTA, CD45⁺ hematopoietic cells and CD45⁻ non-IEC non-hematopoietic cells were purified. The relative expressions of R-Spo genes normalized to that of 18S rRNA in each population was measured by quantitative PCR. Data from two independent experiments with similar results were combined and shown as the means ± SE of n-fold difference relative to the expression of R-Spo3 of non-IEC non-hematopoietic cells (n = 12/group). (b) Representative FACS plot of CD31 and CD90 expression on CD45⁻ cells from the small intestine. (c) Flow cytometric analysis of surface markers on CD45⁻CD31⁺CD90⁻ VECs (blue histograms) and CD45⁻CD31⁺CD90⁺ LECs (red histograms) from the small intestines. Shaded histograms represent unstained controls. (d) Expression of CD31 and CD90 was evaluated on gated CD45⁻Lyve-1⁺ cells in the small intestine. (e) Expression levels of R-Spo3 in each population shown in (b). Data from one of two independent experiments with similar results are shown as the means ± SE (n = 4/group). (f,g) Lamina propria (LP) and the serosal layer (SL) of the small intestine were mechanically separated. Representative dot plots (f) and percentage (g) of LECs among CD45⁻ non-IEC non-hematopoietic cells are shown. Data from two independent experiments with similar results were combined and shown as the means ± SE in (f) (n = 6/group). (h) LECs were sorted from the small intestines. Immunofluorescent staining of Lyve-1 (green) and R-Spo3 (red) with nuclear staining with DAPI (blue) was performed. CD45⁻Lyve-1⁻ cells were stained as controls. Scale bar, 10 μm. (i) The concentration of R-Spo3 in the cell lysates from 3 × 10⁵ sorted VECs, and LECs was measured using ELISA. Data from one of three independent experiments with similar results are shown as the means ± SE (n = 4/group). *P < 0.05, **P < 0.01.