Glucocorticoid reactivity in rx3^+/+ and rx3^-/- zebrafish larvae. Whole-body cortisol in rx3^+/+ (left) and rx3^-/- (right) larvae (all data points shown, mean in red, sample size in parentheses) after exposure to a pH drop (pH), hyperosmotic medium (salt) or stress-evoking fast water flows (flows). Baseline levels are those of control (unexposed) larvae (unexp.), which were equally handled, omitting stressor exposure. Letters indicate results of Bonferroni’s tests (p < 0.001) after one-way ANOVAs (left, rx3^+/+: F(3,59)=42.8, p < 0.0001, right, rx3^-/-: F(3,59)=2.7, p = 0.1), followed by post hoc comparisons.

HPI axis elements in rx3^+/+ and rx3^-/- zebrafish larvae. A-F’’, The hypothalamic part of the HPI axis, illustrated by dorsal (A, B, C, D, E, F) and lateral (A’, B’, C’, D’, E’, F’) views of dorsally imaged IHC stainings of three NPO cell types. Cells producing Avp (A-A’), Oxt (C-C’), or Crh (E-E’) form a cluster in the NPO (arrowheads), and many of their fibers innervate the pituitary (arrows) in rx3^+/+ wild-types (wt) via the hypothalamo-hypophyseal tract (red dotted lines). See also dense innervation in dorsal pituitary closeup views of more examples (A’’, C’’, E’’). In rx3^-/- mutant larvae, cells producing Avp (B-B’), Oxt (D-D’), or Crh (F-F’) cluster less densely in the NPO (arrowheads) and their numbers are reduced. Pituitary innervation is present in some of the stainings (e.g., arrows in B, B’, F, F’) but not in others (e.g., D, D’); generally, the projection patterns appear drastically different. See also complete lack of innervation in dorsal pituitary closeup views of more examples (B’’, D’’, F’’). G-H, The pituitary part of the HPI axis, illustrated by dorsal (G, H) and lateral (G’, H’) images of ISH stainings for pomc. Two pituitary clusters are formed by pomc-positive cells in rx3^+/+ wild-types (G-G’). The rostral cluster is absent, and the caudal cluster appears larger in rx3^-/- mutants (H-H’). Another example is shown for each case as well (G’’, H’’). I-J’, The interrenal part of the HPI axis, illustrated by combined staining for star (I’, J’, ISH, steroidogenic part) and TH (I’’, J’’, IHC, chromaffin part). The interrenal gland is intact and both cell types are present in rx3^+/+ wild-types (I-I’’) and rx3^-/- mutants (J-J’’). Abbreviations: Avp, arginine vasopressin; Crh, corticotropic hormone; Oxt, oxytocin; PO, preoptic area; pomc, proopiomelanocortin; Pit, pituitary; PT, posterior tuberculum; Tel, telencephalon; Th, thalamus. Rostral to the left. Scale bars: 100 µm (A-D’, E-F’), 25 µm (A’’-D’’, E’’, F’’, G-H’’, I, J).

Response to rising temperature in rx3^+/+ and rx3^-/- zebrafish larvae under basal and stressed conditions. (A) Representative 1 s swim paths from larvae showing increased speed and turns near the inlet (bottom right arrow) as the temperature inside the swimming chamber increases faster in zone 1. White dots indicate start positions. Scale bar, 2.5 mm. Adapted from 43. (B, C) Differential speed, i.e., the difference between swim velocity (mm per 40 ms) in zones 1 and 2 (in %), across groups of unexposed (control) and pre-exposed (to either ‘pH’, ‘salt’ or ‘flows’) rx3^+/+(B) and rx3^-/-(C) larvae before and after the onset of temperature rise (see also Methods). Sample size in parentheses. (B, C) Letters and asterisks indicate results of Bonferroni’s tests (P<0.001) after two-way repeated measures ANOVAs; (B) time factor: F(1,56)=215.2, P<0.0001, treatment factor: F(3,56)=7.7, P=0.0002, time × treatment factor: F(3,56)=9.2, P<0.000; (C) time factor: F(1,56)=163.0, P<0.0001, treatment factor: F(3,56)=0.2, P=0.91, time × treatment factor: F(3,56)=0.3, P=0.83.

Response to minute water motions in rx3^+/+ and rx3^-/- zebrafish larvae under basal and stressed conditions. (A) Representative average trace of a wildtype larva’s distance to the stimulus source (top) and swimming activity (bottom) after the onset of mWMs. Red arrow heads in A depict the onset of stimulation. (See also Figure 3 in 50.) (B) Representative examples of consecutive x-y coordinates (swim trajectories) measured every 40 msec from rx3^+/+ and rx3^-/- larvae without (top) and with (bottom) mWMs. Top, 50 x-y coordinates per larva without mWMs (source off) measured over the last 2 s before the onset of mWMs. Bottom, 500 x-y coordinates per larva with mWMs (source on) measured over the last 20 s before the offset of mWMs. The time window in bottom (20 s) is enlarged 10 times relative to top (2 s) with the sole purpose of highlighting the mWMs-derived lack of locomotion observed typically at the end of a 2 min stimulation period. (C, D) Motion level, i.e., the area under the swim velocity-time curve over 120 s, across groups of unexposed and pre-exposed (same groups as in Figure 3B, C) rx3^+/+(C) and rx3^-/-(D) larvae before and during mWMs (see also Methods). Sample size in parentheses. (C, D) Letters and asterisks indicate results of Bonferroni’s tests (P<0.001) after two-way repeated measures ANOVAs; (C) time factor: F(1,56)=359.3, P<0.0001, treatment factor: F(3,56)=1.6, P=0.20, time × treatment factor: F(3,56)=5.6, P=0.002; (D) time factor: F(1,56)=317.0, P<0.0001, treatment factor: F(3,56)=0.03, P=0.99, time × treatment factor: F(3,56)=0.14, P=0.94.