Early recruitment of a CX₃CR1⁺ CD45⁺ CD115⁺ CD11b⁺ myeloid precursor in the epidermis between E14 and E18. (A, top left) H&E staining and three-dimensional reconstruction of skin from E14 CX₃CR1-GFP Rag^−/− γc^−/− mice. CX₃CR1⁺ cells are labeled in green and nuclei are labeled in blue. Confocal Z-stacks were acquired using a Leica SP5 microscope. Images show a 2–4-µm thick virtual section of the tissue. The dashed line indicates the separation between the dermis and epidermis. CX₃CR1⁺ cells can be detected in the dermis but not in the epidermis. Results are representative of 10 skin samples per mouse (n = 4). (top right and bottom) Tiled micrographs of epidermal sheets of CX₃CR1-GFP Rag^−/− γc^−/− E18 fetuses and pups at P0 (the day of birth) and P2. CX₃CR1⁺ cells are labeled in green. Tiles were obtained by assembling a series of maximal intensity projections of Z-stacks. (insets) Enlarged CX₃CR1⁺ cells (green) and nuclei (white). CX₃CR1-expressing cells are first detected in the epidermis at E18, and acquire a dendritic shape between P0 and P2 (n = 3–4 mice for each time point). (B, top) Flow cytometry analysis of epidermal cells from E18 CX₃CR1-GFP Rag^−/− γc^−/− embryos and 10-wk-old langerin-GFP mice. Cells were labeled with antibodies against CSF-1R (CD115), Flt3 (CD135), c-kit (CD117), I-A, CD11c, CD11b, and Lyc6 and analyzed by multicolor flow cytometry. Dot plots show the gate used for analysis of GFP-expressing LC precursors and adult LCs. Overlaid histograms show the expression of surface markers on gated GFP⁺ cells (red line) and control (fluorescence minus one; black line). Results are representative of three to five experiments with similar results. (bottom) Micrographs display maximal intensity projections of Z-stacks from epidermal sheets of 10-wk-old CX₃CR1-GFP Rag^−/− γc^−/− mice. CX₃CR1-expressing cells are labeled in green and langerin is labeled in red. Results are representative of 12 0.03-mm² Z-stacks per mouse (n = 4). Adult LCs do not express CX₃CR1 (GFP). (C) Flow cytometry analysis of bone marrow MDPs and CDPs. Bone marrow cells from adult CX₃CR1-GFP Rag2^−/− γc^−/− mice were labeled with antibodies against lineage markers (CD11b, NK1.1, CD3, Ter119, and CD19), c-kit (CD117), Flt3 (CD135), CSF-1R (CD115), CD11c, and I-A and analyzed by multicolor flow cytometry. MDPs are defined as CX₃CR1^high (GFP) c-kit⁺ lineage⁻, whereas CDPs are defined as lineage⁻ c-kit^low Flt3⁺. Overlaid histograms show expression of surface markers on gated MDPs and CDPs (red line) over control (black line). Results are representative of three to five experiments with similar results. SSC, side scatter.