Schematic representation of the experimental protocol at 5 days post-fertilization (dpf), 21 dpf, and 6-months old adult fish. Experiments done at the three ages are separated and denoted with purple outline. Blue arrows show timeline of procedures while orange dotted lines represent virtual division of testing arenas into relevant areas. For 5 dpf larval fish (a), general locomotion was observed during a baseline period, followed by PTZ-induced hyperlocomotion. Representative examples of behavioural traces are provided to illustrate typical locomotion (wells A1 and B2), immobility (well B1), and hyperlocomotion (well A2). For 21 dpf fish (b), three traces illustrate preference (top), indifference (middle), and aversion (bottom) to social stimuli. The dotted orange lines mark the social and non-social zone used for calculation of social preference index [SPI]. For adult fish, the dotted orange lines indicate the line between border and center during (c) baseline open field testing, five zones during social preference testing (d) with zone 1 being closest to the shoal [social zone] and zone 5 being the furthest away from the shoal during social behavioural testing, and the division of water column into vertical top, middle and bottom areas (e) during novel tank diving test. Representative traces below show bottom-dwelling vs. exploration in higher water column in the open-field, no preference vs. social preference during the social behaviour test, and exploration of bottom only, middle, and top areas of the novel tank. After the behavioural testing, all fish were (f) fin-clipped under anaesthesia for genotyping. Separate group of experimentally naïve adult fish were used to measure whole-body cortisol levels (g) using enzyme immunoassay

Larval locomotion behaviour was similarly affected in mecp2^−/− fish bred from homozygous parents (top panel) and heterozygous parents (bottom panel). a Baseline locomotion prior to PTZ-treatment for 5 dpf larvae from homozygous breeding showed that mecp2^−/− (n = 117) larvae moved significantly shorter distances than mecp2^+/+ (n = 203). b Distances moved by mecp2^+/+ (blue; n = 33–35) and mecp2^−/− (red; n = 19–20) fish from homozygous parents were similar during baseline (20 min before; lighter colours) and showed similar increases during PTZ-treatment (20 min, darker colours). c Baseline locomotion prior to PTZ-treatment for 5 dpf larvae from heterozygous mecp2^+/− crosses showed that mecp2^−/− (n = 68) larvae moved significantly shorter distances than mecp2^+/+ (n = 90). d The distances moved were similar during baseline (20 min before; lighter colours) and the hyperlocomotion during PTZ-treatment (20 min, darker colours) was also similar in 5 dpf mecp2^+/+, mecp2^+/−, and mecp2^+/+ fish derived from heterozygous breeding. Sample sizes were mecp2^+/+ (blue; n = 41–65), mecp2^+/− (purple; n = 86–90) and mecp2^−/− (red; n = 35–37). Significant effects of PTZ-treatment within genotypes are indicated in grey. ****p < 0.0001 and *p < 0.05. Mean values shown as dotted lines and brackets mark SEM (a and c) and bars correspond to mean ± SEM (b and d)

Social preference index, time spent in the social zone, and total distance moved by 21 dpf zebrafish during baseline and social stimulus phases. Social preference (a) was not altered in mecp2 (n = 77) or mecp2^−/− (n = 37) larvae compared to mecp2^+/+ (n = 23) zebrafish. Additionally, sibling zebrafish of all three genotypes spent similar length of time in the social zone (b) and swam similar distances (c) during the baseline and stimulus phases of the experiment. ****p < 0.0001 ***p < 0.001 **p < 0.01 and *p < 0.05. Mean ± SEM

Anxiety-related behaviours and whole-body cortisol levels were altered in adult mecp2^−/− fish while locomotion and social preference were unaffected. Locomotion in the open-field (a) was not affected but adult mecp2^−/− fish spend significantly less time in the (b) centre zone in the open field compared to the mecp2^+/+ group and (c) male mecp2^−/− fish showed somewhat less time in the periphery, compared to female mecp2^−/− fish. During the presentation of social stimuli, time spent in the social zone (d) was not significantly different in adult mecp2^−/− (n = 43) as compared to mecp2^+/+ (n = 22). While mecp2^−/− fish generally trended towards more time closer to social stimuli than mecp2^+/+ fish across the 15 min (e) the SPI was not different between the two groups of fish (f). In a novel tank diving task, the mecp2^−/− (n = 22) zebrafish made less bottom-to-middle transitions (g) and spent less time in high-mobility state (h) and more time in the immobile state (i) as compared to mecp2^+/+ (n = 10) indicating an increase in bottom-dwelling and freezing behaviours, but overall time spent in bottom, middle, and top areas (j) was not different between the two genotypes. Baseline cortisol levels (k) were higher in experimentally naïve mecp2^−/− (n = 6) fish compared to mecp2^+/+ (n = 6) zebrafish. *p < 0.05 and ****p < 0.0001. Mean ± SEM