(A) Both slc2a15b and gjb8 are predicted to be expressed in pre-migratory xanthoblast cells. (B–G) Quantitative single-molecule in situ hybridization (hybridization chain reaction [HCR]) confirms expression of these genes to aox5 expressing xanthoblasts. (B) Schematic of 24 hr post fertilization (hpf) embryos labeling region of embryos shown in subsequent panels. (C) Quantification of RNA expression (corrected total cell fluorescence [CTCF]) in neural crest cells (NCCs) from sections shows novel marker genes slc2a15b and gjb8 are strongly co-expressed with aox5. Sections were taken every 15 µM and analyses were limited to sections over the yolk extension. Each dot represents a cell (slc2a15b = 3 embryos, 9 slides, 49 cells; gjb8 = 3 embryos, 9 slides, 58 cells). (D–G) Representative whole mount (D, F) and sections (E, G) show expression of slc2a15b (D, E) and gjb8 (F, G) in 24 hpf embryos. DAPI (blue, E, G) labels nuclei. Arrows point to NCCs co-expressing xanthoblast marker genes, while arrowheads point to NCCs that lack expression of these same genes. Note that a number of aox5/slc2a15b/gjb8 NCCs are pre-migratory in the dorsal neural tube (NT). (H) Both fgf13a and cxcr4b are predicted to be expressed in Rohon–Beard neurons (RBs). (I–P) HCR confirms expression of these genes to RBs. (I) Schematic labeling region of trunk shown in subsequent panels. (J) Percent of HNK-1-positive RBs also expressing cxcr4b and fgf13a indicates that these genes are co-expressed in RB cells. The number of HNK-1, cxcr4b, and fgf13a-positive cells in the dorsal NT was counted. Note almost all fgf13a/cxcr4b expressing cells co-localize to HNK-1-positive RB cells. Sample size reported above bars (#cells/#embryos). (K, L) Representative confocal images of whole-mount in situ hybridization for elavl3, fgf13a, and cxcr4b show expression of fgf13a and cxcr4b as two rows of cells in the dorsal NT (arrows). Elavl3 marks the NT, and DAPI (gray) labels nuclei. Note ventral NT expression of cxcr4b (arrowheads in L’). (M, N) Dual immunolabeling for HNK-1 and in situ hybridization for fgf13a (M) or cxcr4b (N) shows gene expression overlaps with HNK-1 labeling in RBs in the dorsal NT (arrows). Arrowheads mark ventral expression of cxcr4b in panel (N). (O, P) While fgf13a and cxcr4b expression was never observed in NCCs, a subset of putative RB cells express the tg(sox10:tagRFP) transgene (gray in panels O and P). Shown are whole-mount 3D projections (O, P) and single slice through z-stack (O’, P’) showing fgf13a/cxcr4b/elavl3-positive cells co-labeled with the sox10 transgene. Note that sox10-positive cells are positioned topologically similar to sox10-negative RB cells. Scale bars for all panels are 20 µM.
