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Fig. 3 opn4a Expression Depends on Otp Activity in Areas of Coexpression with otpa(A) Analysis of expression domains of opn4a, otpa, and TH (anti-TH) in 3 dpf wild-type larvae. Top row shows z projections of combined channels (see labels) of confocal stacks recorded from lateral (left) and dorsal (right) views of the brain. Anterior is at left; dorsal is at top for lateral view. Bottom row shows z projections of channel combinations of a confocal stack showing dorsal views of the brain. Scale bars represent 50 μm. opn4a is coexpressed with otpa in the anterior preoptic area (aPO) (asterisk) and posterior tuberculum (PT) (arrowhead). The following abbreviations are also used in (A), (B), and (D): Th, thalamus; pPO, posterior preoptic area; vH, ventral hypothalamus; DC2, dopaminergic group 2; DC4, dopaminergic group 4; ac, anterior commissure; oc, optic chiasm; PI, pineal; PT DA, posterior tuberculum dopaminergic neurons.(B) Expression of opn4a in otpa and otpb mutants. Whole-mount in situ hybridization reveals loss of opn4a expression in the aPO (asterisk) and PT (arrowhead) of otpa and otpa, otpb double mutants (3 dpf). otpb mutants alone did not significantly affect opn4a expression, which is in line with the previously reported compensation of otpb knockdown by otpa activity in A11 DA neuron differentiation [20]. Anterior is at left; dorsal is up. Scale bar represents 50 μm.(C) Increase in activity following decrements in light intensity in enucleated Tg(otpb.A:Gal4)zc67; Tg(UAS:GFP-v2a-opn4)y233 larvae. Data show the difference in mean activity between 2 min after light change and 1 min prior to light change (mean displacement at t2 mean displacement at t1). Enucleated opn4-overexpressing larvae show an increased response to decrements in light intensity (repeated-measures two-way ANOVA; F1,100 = 8.84, p < 0.005; nonexpressing control: 28 larvae; GFP positive in otpb domain: n = 42 larvae). p < 0.001.(D) Schematic summarizing our results suggesting that preoptic opn4a-expressing neurons are deep brain photoreceptors driving dark photokinesis. We eliminated all depicted photoreceptive regions except the PO and found that the VMR response remained intact. Because otpa mutants lack aPO but not pPO opn4a expression, and because mutants without eyes do not show a VMR, the behavior must originate in the opn4a-positive cells in the aPO (pink). The illustrated DA domain (red) only comprises the diencephalic groups 2–6.See also Figure S3.