Colitis is associated with the loss and recovery of nonhematopoietic CD90 ⁺ CD24 ⁺ myenteric cells enriched in neurons. (A) Water content in fecal pellets of 3×DSS- or water-treated WT mice (control, n = 15; DSS, n = 20). (B) Occult blood in fecal pellets of 3×DSS- or water-treated WT mice (control, n = 15; DSS, n = 20). (C) Colon length in 3×DSS- or water-treated WT mice (control, n = 6–10; DSS, n = 5–8). (D) FC gating strategy to identify CD45⁺ immune cells (including MMs) and CD45^– nonhematopoietic stromal cells (including myenteric neurons) in colonic muscularis. (E) Intracellular expression of pan-neuronal marker PGP9.5 by four CD45^– nonhematopoietic subsets identified based on their co-expression of CD90 and CD24, shown by FC. (F) Relative expression by qPCR of pan-neuronal genes Elavl3 (HuC) and Elavl4 (HuD) by FACS-purified CD45^– subsets defined in E, normalized to Actb (n = 3 per group, n—independent cell sorting). (G) Transgenic mice used in H and I. (H) Confocal images of colonic myenteric plexus isolated from control R26-STOP^fl/WTtdTomato (Cre^–, top) and Actl6b^CreR26-STOP^fl/WTtdTomato (Cre⁺, bottom) mice at steady state, stained with Hu (neuronal somas), tdTomato (Baf53b(Actl6b):tdTomato⁺ neurons), and βIII-tubulin (nerve fibers) antibodies. Scale bars, 100 μm. (I) FC plots showing gating strategy and percentage of Actl6b:tdTomato⁺ neurons among total CD45^–CD90⁺CD24⁺ cells (left, Cre^– and Cre⁺) or percentage of CD90⁺CD24⁺ cells among total CD45^–Actl6b:tdTomato⁺ neurons (right Cre⁺) in colonic muscularis isolated from control R26-STOP^fl/WTtdTomato (Cre^–) and Actl6b^CreR26-STOP^fl/WTtdTomato (Cre⁺) mice at steady state. All graphs show the mean ± SEM, n—mouse unless stated differently. Data are representative of at least two independent experiments with similar results. Statistical analyses: unpaired Student’s t test (A, C, and F), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.