Expression patterns of exorh, pax6b, bsx and insm1a in the zebrafish pineal gland at 48 hpf. Schematic representations of the pineal complex are also shown in (e) and (j). Dorsal views (a–e) are oriented with anterior to the top, while frontal views (f–j) are with dorsal up. White arrowheads indicate bsx expression in the parapineal organ (c, h). Scale bar, 30 μm; PRN, photoreceptor cells; PN, projection neurons; PP, parapineal organ; U, undifferentiated/progenitor cells

MO-mediated knockdown of bsx. a Schematic illustration of the target site of bsx MO. b RT-PCR of bsx mRNA in 48-hpf embryos injected with control or bsx MO. Full images of the electrophoreses are shown in Supplementary Fig. 8. c Apparent views of 3.5 dpf larvae injected with control or bsx MO. No gross morphological difference was detected. d–k Effects of bsx MO on the pineal gland. All the larvae are viewed dorsally with anterior to the top. Three images in each row (d, f, h) were taken from the same larva at different stages. d Size reduction of the pineal gland in bsx morphants. White dashed lines indicate the position of the pineal gland. f Reduction of pineal EGFP fluorescence in Tg(exorh:egfp) by Bsx depletion. h Induction of EGFP expression in Tg(rho:egfp) by Bsx depletion (indicated by white arrowheads). e, g, i Quantification of pineal size and fluorescence signals in bsx MO- or control MO-injected larvae. Horizontal bars indicate mean values. The difference between bsx MO-injected and control groups was statistically significant for each case [P < 2.2 × 10⁻¹⁶ (3.5, 6.5, and 9.5 dpf, e), P = 2.8 × 10⁻¹² (3.5 dpf, g), P < 2.2 × 10⁻¹⁶ (6.5 and 9.5 dpf, g), P = 2.0 × 10⁻⁸ (3.5 dpf, i), P = 1.3 × 10⁻⁷ (6.5 dpf, i), P = 0.011 (9.5 dpf, i) by Welch’s two-sided t-test]. j Effects of bsx MO on Tg(exorh:egfp);Tg(rho:ntr-mCherry) double reporter line at 5.5 dpf. In control larva, EGFP (green) and NTR-mCherry (magenta) were localized in the pineal gland and the retina, respectively. In bsx morphant, both EGFP (green) and NTR-mCherry (magenta) signals were detected in the pineal gland. k A confocal image of pineal cells in Bsx-depleted Tg(exorh:egfp);Tg(rho:ntr-mCherry), showing mutually exclusive expression of EGFP (green) and NTR-mCherry (magenta). Scale bars, 1 mm (c), 30 μm (d, f), 100 μm (h,j), 10 μm (k)

Effects of depletion of Bsx, Otx5 or Flh on the pineal gene expression at 48 hpf. a–p Each panel represents head images of embryos (n = 12–32), all showing similar results. Embryos are viewed dorsally with anterior to the top. Arrowheads in (c), (m), and (p) indicate bsx expression in the parapineal organ. Scale bar, 30 μm

Bsx transactivates exorh promoter in vitro and in vivo. a Cooperative transactivation of the 147-bp exorh promoter by Bsx and Otx5. Horizontal bars indicate mean values of relative luciferase activities (n = 3). Significant differences were observed between two groups that do not share the same lowercase letter (P < 0.01 by two-sided, Games-Howell test). b–e EGFP induction in the retina of Tg(exorh:egfp) fish by ectopic bsx expression under the control of rho promoter. Lateral (b, d) and ventral (c, e) views of rho:bsx-injected larva (b, c) or a control larva without injection (d, e) at 6.5 dpf. Arrowheads indicate GFP-positive retinal cells induced by rho:bsx injection, and arrows indicate pineal GFP fluorescence signals originally found in Tg(exorh:egfp) fish. Each of panels (b) and (d) is the montage of two photographs taken at different focal planes. Scale bar, 100 μm

Bsx directly binds to exorh promoter via PIRE sites. a A position-specific score matrix of Bsx recognition sequences obtained by SAAB. See Supplementary Fig. 7 for raw sequence data. b EMSA with BSXRE probe in the presence of GST-Bsx protein. BSXRE probe harbors the 18-bp highest matching sequence determined by SAAB (BSXRE; indicated by capital letters), which is flanked by a pair of short arm sequences (indicated by lowercase letters). Mutated sequences in BSXRE-mut probe were highlighted with dots. c A schematic structure of zebrafish exorh promoter. Gray rectangles represent DNA elements matching the consensus sequence of PIRE (TAATC/T), whereas white ones represent other potential sites for Bsx binding. See (d) for the nucleotide sequences. Arrows indicate the direction of each element. Nucleotide positions are given relative to the translation initiation site. d Competitive EMSA against BSXRE probe. Each competitor oligonucleotide has an 18-bp sequence of interest, which is flanked with the arm sequences shown in (b). Matching scores are calculated as the sum of relative appearance frequencies of nucleotides using the score matrix in (a) at the positions 4 through 15. P2 oligonucleotide contains the 12-bp sequence of PIPE, in which the nucleotide at the 5′-most position overlaps the left arm sequence (indicated by a lowercase letter). e EMSA with exorh promoter sequence (wt147bp) or its mutated ones. Multiple shifted bands possibly represent the variety of higher order structures of protein-probe complex. Full images of the electrophoreses (b, d, e) are shown in Supplementary Fig. 8

Models for specification mechanisms of the zebrafish pineal gland. a A model for Bsx function in the pineal development. b A model for transcriptional network regulating pineal photoreceptor-specific gene expression. The specificity is defined by the combinatorial action of Bsx (pineal specificity) and Otx5 (photoreceptor cell specificity) through multiple PIRE sequences, possibly in cooperation with another uncharacterized factor that binds to PIPE²⁸

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.