A, Comparative syntenic analysis of human, mouse, medaka, fugu, and zebrafish orthologous loci, depicting the conserved presence of two genes, fgf13a and zic3, in the nearest vicinity of three CNEs (red, green, and purple vertical lines). Genes are color‐coded. The arrow direction depicts the direction of gene transcription. The vertical line depicts the position of the CNE. The horizontal blue line depicts the scale. B, Human and zebrafish alignment of CNE12030 and CNE12032, highlighting the highly similar sequences between them and graphical representation of the transcription factor binding motif that was identified by MEME. Each of the motifs was further screened against the TRANSFAC library to mark binding sites for multiple developmentally important transcription factors. C, Schematic representation of the reporter gene cassette that was used for the in vivo characterization of CNEs. Brown vertical boxes represent different lengths of noncoding DNA that were selected for the transgenic assays. D, Embryos injected with Tol2–c‐fos–EGFP backbone without a CNE (left side embryos, negative) and with a CNE12032‐containing reporter construct (right side embryos, positive) at 48 hpf in the transient transgenic assay. Both pictures are lateral views with the dorsal side toward the right. Scale bar = 500 μm. Chr, chromosome; CNE, conserved noncoding element; EGFP, enhanced green fluorescent protein; MEME, multiple EM for motif elicitation

Sites of GFP signals that were recorded in zebrafish embryos that were transiently transfected with a full‐length construct. The reporter genes that were induced by individual CNEs (indicated by the name) are depicted in schematic representations of day 2 (48 hpf) or day 3 (72 hpf) embryos. Categories of a cell type that was positive for a given element are color‐coded, with a GFP‐expressing cell. The bar graphs (y‐axis) show the percentage of GFP‐expressing embryos that presented expression in each tissue category for a given element. The percentage of GFP‐expressing embryos per CNE is indicated beneath each schematic (EE = %). The bar graphs use the same color code as the schematics for each cell type. B, The injected fish were raised to adulthood and crossed with wild‐type fish to screen the germ‐line transmission of enhancers. Each GFP‐positive fish was further studied in situ to compliment the GFP signals and anti‐GFP to confirm GFP expression. CNE, conserved noncoding element; EE, GFP‐expressing embryos; GFP, green fluorescent protein; hpf, hours postfertilization

Green fluorescent protein expression in stable F1 line at 48 hpf. A, Green fluorescent protein expression in live embryos is indicated by arrowheads in the left and right habenula. B, The same GFP expression from the same embryo in a closer view. C, Green fluorescent protein expression from the lateral side. D and E, Schematics that show GFP expression in the habenula at 48 hpf in the dorsal and lateral views. F, RNA whole‐mount in situ hybridization using a probe against GFP, confirming its expression in the habenula at 30 hpf. G, Whole‐mount anti‐GFP immunohistochemistry, confirming GFP expression in the habenula at 48 hpf. H‐K, RNA whole‐mount in situ hybridization against GFP, showing GFP expression in the habenula at 30 hpf (H, I) and 48 hpf (J, K). L and M, RNA whole‐mount in situ hybridization using pou4f1 probe labeling in the left and right habenula at 48 hpf. N and O, Whole‐mount in situ hybridization of zic3 expression at 48 hpf. The photomicrographs in (H‐O) were taken using a Nomarski contrast microscope with a high‐sensitivity monochromatic camera (Zeiss AxioCam MRm). Scale bars = 50 μm. e, eye; GFP, green fluorescent protein; Lhb, left habenula; Rhb, right habenula

Robust expression pattern driven by CNE12032. Live fluorescent images of CNE12032 stable transgenic zebrafish embryo on day 2. Green‐fluorescent protein‐expressing regions in live embryos are shown, indicated by arrowheads. A, Confocal Z‐stack images of the dorsal view. The white arrowhead indicates GFP‐expressing cells in the olfactory pit. The yellow arrows show the optic tectum. B, Merged channel, highlighting colocalized DiI with EGFP in olfactory sensory neurons. B′, DiI fluorescence in olfactory neurons in the same embryo in a closer view. C, Light‐sheet Z‐stack images in the lateral view, anterior to the left. The white arrow indicates GFP signals in the eye. D, Light‐sheet Z‐stack images in the dorsal view, anterior to the left. The white arrowheads indicate GFP signals in the spinal cord. The blue arrowheads indicate GFP signals in the pectoral fin. Scale bars = 20 μm in (C) and 50 μm in (D). CNE, conserved noncoding element; e, eye; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; l, lens; op, olfactory pit; ot, optic tectum; pf, pectoral fin; sc, spinal cord