Blue-light-stimulated bPAC⁺ larvae show strengthened stress reactions.

(a) Schematic showing the expected amplification of cAMP-dependent hypothalamic signalling and Ca²⁺-dependent hormone release in pituitary corticotrophs in presence of bPAC. bPAC⁺ larvae are expected to show an amplified level of pituitary corticotroph cell activity and subsequent cortisol release in response to blue light, as compared with bPAC^- larvae. Bottom, dorsal view of bPAC expression in pituitary corticotrophs as detected by fused tdTomato fluorescence. Scale bar, 500 µm. Modified from ref. 28. (b) The Ca²⁺-sensitive photoprotein GFP-Aequorin is expected to produce higher Ca²⁺-dependent photo-emission in corticotroph cells as a result of a blue-light-dependent bPAC activation. Top, Co-expression (right) of fluorescent tdTomato (left) and GFP (center). Scale bar, 50 µm. Bottom left, Representative post-blue-light Ca²⁺-dependent photo-emission time curves of bPAC⁺/GFP-Aequorin⁺ and bPAC^-/GFP-Aequorin⁺ larvae pre-incubated in the Aequorin substrate coelenterazine, whose oxidation results in photon emission. Bottom right, After a brief exposure to blue light (blue square), bPAC⁺/GFP-Aequorin⁺ larvae on average showed a twofold increase in photo-emission, that is, area under the photons s^-1-time curve, as compared with bPAC^-/GFP-Aequorin⁺. This was not the case with a yellow-light exposure (yellow square), reflecting a blue-light-dependent Ca²⁺ increase as a blue-light-specific response (***P<0.001 after one-sample t-tests against a fold change of ′1′ (dashed line), P value after a two-tailed t-test). (c) Top, groups and protocol for testing responses of briefly dark-adapted bPAC⁺ and bPAC^- larvae to a squared pulse of blue light. Bottom, individual motion (mean±s.e.m., in mm per 5 s) before, during and after a squared pulse of blue light of either low (left) or high (right) power. (d) Blue-light-mediated motion (top) and cortisol (bottom) change in bPAC⁺ and bPAC^- larvae as a function of light power. *P<0.05, **P<0.01, ***P<0.001 after one-sample t-tests against a fold change of ′1′ (red lines), P values indicate results of Bonferroni′s tests after a two-way ANOVA. (b,d) Sample size in parentheses. (e) Correlation between motion and cortisol fold change from c and d, respectively.

The interrenal gland in zebrafish larvae.

a-c, The interrenal gland consists of steroidogenic cells expressing star (a) and the ACTH-binding mc2r (b), as shown by fluorescence in situ hybridization, and catecholaminergic chromaffin cells expressing TH (c), as shown by immunohistochemistry. d-f, The star-expressing steroidogenic cells coexpress mc2r (d), and are intermingled with the TH-expressing chromaffin cells (e), which do not co-express mc2r (f). Scale bar: 50 µm.