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. 2009 Dec 21;206(13):3089–3100. doi: 10.1084/jem.20091586

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© 2009 Chorro et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — LCs expand in the epidermis by proliferating during the first week of life to establish an LC network. (A, left) Kinetic of the number of CX₃CR1⁺ cells per square millimeter of epidermis of CX₃CR1-GFP Rag^−/− γc^−/− mice at various ages (E14, E18, P0, P1, and P2). CX₃CR1⁺ cells appear slowly in the epidermis between E18 and P2. Cells were enumerated as indicated in Materials and methods. Each dot represents an individual mouse. (right) Kinetic of the number of langerin-GFP⁺ cells per square millimeter of the epidermis of langerin-GFP CCR2^+/− or langerin-GFP CCR2^−/− mice at various ages (P2, P4, P5, and P7). The epidermis was studied by intravital microscopy in live animals, as indicated in Materials and methods. LCs rapidly expand between P2 and P7. Each dot represents an individual mouse. Representative micrographs of maximal intensity projections of Z-stacks obtained at various time points are displayed below the graph. (B, top) Micrographs of epidermal sheets of CX₃CR1-GFP Rag^−/− γc^−/− mice at various ages (E18, P0, and P2). CX₃CR1⁺ cells are labeled in green and Ki67 is labeled in red. Images are optical slices from Z-stacks (optical zoom factor 2). (bottom) The graph shows the percentage of CX₃CR1⁺ cells expressing the nuclear proliferation marker Ki67 at various ages. For each mouse, a range of 100–500 cells was analyzed. Each dot represents an individual mouse. (C) Same as in B. Langerin-GFP CCR2^+/− or langerin-GFP CCR2^−/− mice at various ages (P2, P4, and P7) were analyzed. Horizontal lines represent means. (D) LC mitosis within the epidermis. Micrographs of langerin-GFP⁺ cells (green) expressing Ki67 (red) from epidermal sheets of P4 langerin-GFP mice. Pictures are optical slices from Z-stacks (optical zoom factor 2). Results are representative of six mice. For more information, see Fig. S3 and Video 1. (E) Cell-cycle analysis by PI staining of sorted P4 and P7 LCs by flow cytometry. Dot plots show the gate used for GFP-expressing LC sorting. Percentages of GFP⁺ cells among all events are indicated. Histograms show the cell-cycle analysis. Percentages of gated singlet PI⁺ events in the S/G2/M phase are indicated. Means of three and two independent experiments ± SD at P4 and P7, respectively, are indicated. (F) Estimate of the duration of the langerin⁺ cell-cycle during the first week of life. Using a nonlinear regression model (sigmoidal curve) based on data from A (R-squared value of the trend line = 0.98) and cell-cycle data from E, we formulated a polynomial regression model to extrapolate the percentage of cells proliferating at each day after birth (0–7 d), and we calculated the total number of cells per square millimeter over time that would be obtained considering one (red line), two (yellow line), or three (green line) divisions per day per dividing cell. Experimental data are represented by blue diamonds. The graph shows that experimental data fit with a model of in situ proliferation of LCs in which the length of the cell cycle is between 12 and 24 h. SSC, side scatter.