The licensing state defines distinct proliferative zones in intestinal crypts. (A) Representative image of an extracted intestinal crypt isolated after a 1-h EdU pulse in vivo (green) and stained with Hoechst (blue) and antibodies against Mcm2 (red) and pH H3 (white). Bars: (top) 10 µm; (bottom) 1 µm. Costaining shows distinct cell-cycle phases (bottom), licensed cells committed in G₁ with Mcm2⁺ and EdU⁻; early Mcm2⁺ and EdU⁺ to late Mcm2⁻ and EdU⁺ in the S phase, and mitotic cells, pH H3⁺. Negative cells represent deeply quiescent (G₀), terminally differentiated cells or cells in G₁ that have not made a proliferative-fate decision, remaining unlicensed. The crypt base is to the left of the displayed image. (B) Nuclear volume was estimated in cells at the distinct cell-cycle phases identified previously: negative (G_0/G₁/G₂, n = 115); G₁ licensed Mcm⁺ and EdU⁻ (n = 38); S phase Mcm⁺ and EdU⁺ (n = 24); and late S/G₂ Mcm⁻ and EdU⁺ (n = 26). (Top) Representative examples of each cell-cycle phase and the associated 3D rendered nuclei. Bars, 10 µm. The means ± SEM are displayed. There was a significant difference in the size of licensed G₁, S, and late S/G₂ nuclei (t test, P < 0.0001). (C) Representative images of intestinal crypts isolated after a 1-h EdU pulse (green) in vivo. Displayed are 3D projections of extracted and unextracted crypts stained with Hoechst (blue) and an antibody against Mcm2 (red). The crypt base is to the left of the displayed image. (D) Comparison between cells expressing Mcm2 protein and DNA-bound Mcm2 along the crypt–villus axis between unextracted (n = 101 crypts) and extracted (n = 109 crypts; taken from 3 mice) cells. Data are displayed as the mean percentage of cells per set distance bin. (E) All cells were divided into four distinct groups based on Mcm2 and EdU intensities. These groups represent distinctive cell-cycle phases as defined by their total (unextracted, n = 101 crypts) or licensed (extracted, n = 109 crypts) Mcm2 content: extracted, (1) Unlicensed Mcm2⁻ and EdU⁻, (2) G₁ licensed Mcm2⁺ and EdU⁻, (3) early/mid S phase Mcm2⁺ and EdU⁺, and (4) late S/G₂ Mcm2⁻ and EdU⁺. The data are represented as the population means of the total cells per distance bin. Means ± SEM; ***, P < 0.001; **, P < 0.01. (F) The distance of the most distal Mcm2⁺ and EdU⁺ cells to the crypt base was compared in extracted and unextracted crypts. Data were scored manually for 10 representative crypts per condition. Licensed Mcm2⁺ cells were significantly closer to the crypt base than were EdU⁺ cells (t test, P = 0.0015. Cells expressing Mcm2 protein extended significantly above the last EdU⁺ cell (t test, P < 0.0003). (G) Representative images of crypts isolated 17 h after administration of EdU (green). 3D projections of extracted and unextracted crypts stained with Hoechst (blue) and an antibody against Mcm2 (red) are shown. (H) Cells were divided into four distinct groups as in E (n = 51 crypts).