hrh1 expression in Turku wt fishes and the mutation site of hrh1^−/− fish. a In situ hybridization of hrh1 in Turku wt zebrafish at 3 dpf (lateral and dorsal view), 5 dpf (lateral and dorsal view). Scale bars: 50 µm. b qRT-PCR results of hrh1 mRNA expression at 5 hpf, 10 hpf, 24 hpf, 3 dpf, 6 dpf, and 12 dpf. N = 6. Mean ± SEM. c The mutation site of hrh1^−/− zebrafish and genomic DNA sequencing results. d Predicated 3D modeling of hrh1^+/+ and hrh1^−/− protein by using SWISS-model online. e Brightfield image of larval zebrafish at 6 dpf. Scale bar: 200 µm.

Histaminergic, dopaminergic, and hcrt systems in hrh1^−/− zebrafish. a Representative pictures (ventral view) for immunohistochemistry of histamine and Th1 in hrh1^+/+ and hrh1^−/− 6 dpf zebrafish brains. Anterior to the left. Scale bars: 100 µm. b Histaminergic neuron numbers. N = 6. c Number of Th1-expressing neurons in group 10. N = 6. d–f qPCR results of th1, th2, chata in hrh1^+/+ and hrh1^−/− in 6 dpf whole zebrafish. N (hrh1^+/+) = 5, N (hrh1^−/−) = 6. g–h Representative pictures (ventral view) for in situ hybridization of hcrt in hrh1^+/+ and hrh1^−/− 6 dpf zebrafish brain and cell numbers. Anterior to the top. Scale bar 50 µm. N = 6. i qPCR results of hcrt in hrh1^+/+ and hrh1^−/− in 6 dpf whole zebrafish. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All histograms are mean ± SEM, unpaired t-test, two-tailed. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001

Neurogenesis markers in hrh1^−/− zebrafish. a Representative pictures for in situ hybridization of nes in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf in both dorsal and lateral view. Scale bar: 100 µm. Anterior to the left. N (hrh1^+/+) = 5, N (hrh1^−/−) = 6. c Representative pictures (dorsal view) for in situ hybridization of notch1a in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N (hrh1^+/+) = 8, N (hrh1^−/−) = 8. b, d, e qPCR results of nes, notch1a, sox2 in whole larval zebrafish at 6 dpf. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All bar graphs show mean ± SEM, unpaired t-test, two-tailed. **0.001 < P ≤ 0.01

Proliferation and differentiation markers in hrh1^−/− zebrafish. a Representative pictures (ventral view) for immunohistochemistry of HuC/D in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N = 6. c Representative pictures (dorsal view) for immunohistochemistry of PCNA in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N = 6. i Representative pictures (ventral view) for in situ hybridization of pax2a and pax6a in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. b, d–h qPCR results of elavl3, pcna, olig2, slc1a3a, gfap, and isl1 in whole larval zebrafish at 6 dpf. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All histograms are exhibit as mean ± SEM, unpaired t test, two tailed. **0.001 < P ≤ 0.01

Larval behavior of hrh1^+/+ and hrh1^−/− fish. a Total distances moved of hrh1^+/+ and hrh1^−/− larvae at 6 dpf in an open field for 15 min. b The frequency of zebrafish entering the center zone of an open field. c The duration of zebrafish staying in the center zone of open field. d Dark flash responses of hrh1^+/+ and hrh1^−/− zebrafish. Each dark period lasted 2 min. Distance moved during every 30-s period is indicated on the Y axis. e Distance moved/s of hrh1^+/+ and hrh1^−/− in the first dark response period. f The index of the first dark response (distances moved in the dark (30 s) minus distances moved in the light (30 s)). a–fN (hrh1^+/+) = 17, N (hrh1^−/−) = 27. g Sleep-like behavior from 5 to 6 dpf of hrh1^+/+ and hrh1^−/− zebrafish. Distance covered during every 30-min period is indicated on the Y axis. N (hrh1^+/+) = 16, N (hrh1^−/−) = 14. h–i Mean values for maximum velocity during 10 acoustic/vibrational stimuli with a 20-s interstimulus interval (ISI) (h) and 30 acoustic/vibrational stimuli with 1 s ISI. N (hrh1^+/+) = 14, N (hrh1^−/−) = 15. Mean ± SEM, two‐way ANOVA

Adult behavior of hrh1^+/+ and hrh1^−/− zebrafish. a Total distances moved in an open-field tank in 15 min in total by adult hrh1^+/+ and hrh1^−/− fish. b Duration of hrh1^+/+ and hrh1^−/− fish in the border zone. N = 10. c Total distances moved in the novel tank in 6 min in total. N (hrh1^+/+) = 13, N (hrh1^−/−) = 10. d Representative tracking map of novel tank for hrh1^+/+ and hrh1^−/− adult zebrafish. e Statistics of cumulative durations in the bottom zone for hrh1^+/+ and hrh1^−/− adult zebrafish. f–g Statistics of the distances between subjects and cumulative duration in proximity for hrh1^+/+ and hrh1^−/− adult zebrafish. N = 20. Mean ± SEM, unpaired t test, two tailed. *0.01 < P ≤ 0.05; no stars mean no significant differences

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.

Unillustrated author statements