ALO is an immune cell hub. (A) UMAP plot of ALO scRNAseq data highlighting blood and immune cell clusters. (B) Dot plot showing the relative expression of genes used to identify and characterize blood and immune cell clusters. (C–Q) Confocal + DIC (C, D, G–I, L, M, N, and Q) or confocal only (E, F, J, K, O, and P) micrographs of ALOs from adult immune cell transgenic reporter zebrafish. Images include ALO overviews (C, H, and M), side views of the dermal cortex and medulla (D, I, and N), and higher magnification images of individual cells (E, F, J, K, O, and P). Images show Tg(mpeg1:EGFP)^gl22-positive macrophages (C–G), Tg(cd79b:EGFP)^fcc89-positive B cells (H–L), and Tg(lck:EGFP)^cz2-positive T cells (M–Q). (G, L, and Q) Single-plane confocal + DIC images of Tg(mpeg1:EGFP)^gl22-positive macrophages (G), Tg(cd79b:EGFP)^fcc89-positive B cells (L), and Tg(lck:EGFP)^cz2 T cells (Q) migrating on FRCs in adult ALO medullae (see Videos 6, 7, 8, and 9). (R–U) Confocal micrographs of a 5-wk-old Tg(cd79b:EGFP)^fcc89, Tg(lck:mcherry)^nz107 double transgenic zebrafish with T cells in magenta and B cells in green, showing large numbers of cells migrating between the thymus (white arrow) and the ALO (yellow arrow). Images show an overview of the head (R) and higher magnification images of T cells (S) connecting the thymus and ALO and B cells (T) starting above the thymus and moving down to the ALO, and an overview of the entire fish (U). (V) CellChat plot of likely chemokine signaling in the ALO based on the expression of chemokine ligands and receptors in different ALO scRNAseq clusters. Basal epithelial and FRCs both appear to be hubs for chemokine signaling to immune cells. n = 5 per transgene combination. Scale bars = 50 µm (C, H, and M), 20 µm (D, I, and N), 5 µm (E, F, J, K, O, and P), 10 µm (G, L, and Q), 500 µm (R, S, and T), and 1 mm (U).