Successive positions of objects over time referred to the frame of reference used by the tracker software. The coordinate axis is represented by blue lines. The red circle represents the arena of radius 8.5 mm.

Phenotype evolution of double homozygous larvae (mef2ca^b1086/b1086;mef2cb^fh288/fh288) from 3 to 6 dpf of development. The images were obtained at different times of development showing an increase in cardiac oedema, with a consequent general deformation that leads to complete immobilization of the animal. Scale bars: 600 µm.

Craniofacial cartilage phenotypes in WT, mef2ca single-mutant and homozygous double-mutant larvae. The alcian blue staining technique was used to analyse craniofacial cartilaginous phenotype in zebrafish larvae at 5 dpf. (A–C) Ventral view of WT, mef2ca single-mutant or homozygous double-mutant larvae. (D–F) Lateral view of WT, mef2ca single-mutant or homozygous double-mutant larvae. (G) Lateral view of the whole body of homozygous double-mutant larvae. hs—hyosymplectic cartilage; M—Meckel’s cartilage; ch—ceratohyal; pq—palatoquadrate cartilage; cb—ceratobranchials; oa—occipital arch; tc—trabecula communis. Scale bars: 600 µm.

Craniofacial bone phenotypes in WT, mef2ca single mutants and homozygous double-mutant larvae. The alizarin red S live staining technique was used to analyse craniofacial bone phenotype in zebrafish larvae at 6 dpf. (A–C) Ventral view of WT, mef2ca single-mutant and homozygous double-mutant larvae. (D–F) Lateral view of WT, mef2ca single-mutant and homozygous double-mutant larvae. op—operculum; cl—cleithrum; ps—parasphenoid; ot—otoliths; pt—pharyngeal teeth; not—notochord. Scale bars: 600 µm.

Craniofacial phenotype of mef2ca single mutants through the use of the transgenic line Tg(-4725sox10:GFP). Crossing the transgenic line Tg(-4725sox10:GFP) with the zebrafish mef2ca^b1086 mutant line allowed the observation in vivo of the craniofacial phenotype from these mutants. (A–C,G–I) WT larvae at developmental stages of 6, 9 and 10 dpf in ventral and lateral views, respectively. (D–F,J–L) mef2ca^b1086/b1086 homozygous larvae at developmental stages of 6, 9 and 10 dpf in ventral and lateral views, respectively. Scale bars: 600 µm.

Craniofacial phenotype of double homozygous mutants through the use of the transgenic line Tg(-4725sox10:GFP). Crossing the transgenic line Tg(-4725sox10:GFP) with the zebrafish mef2ca^b1086;mef2cb^fh288 mutant line allowed the observation in vivo of the craniofacial phenotype from these mutants. (A–D,I–L) WT larvae at developmental stages ranging from 3 to 6 dpf, in ventral and lateral views, respectively. (E–H,M–P) mef2ca^b1086/b1086;mef2cb^fh288/fh288 double homozygous larvae at developmental stages from 3 to 6 dpf, in ventral and lateral views, respectively. Scale bars: 600 µm.

Expression analysis of brain-development-related genes, mecp2 and cdkl5, in zebrafish mef2ca and mef2cb single mutants and homozygous double mutants. Gene expression was measured by qPCR in samples obtained from pools of 20 larvae at 3 dpf and sharing the same mutation. Values are normalised to the expression levels of the gapdh gene and expressed as mean ± SD. mecp2—methyl cpg binding protein 2; cdkl5—cyclin dependent kinase like 5. Similar superscript letters indicate no statistically significant difference (p > 0.05). Different superscript letters indicate significant difference within column (p < 0.05).

Comparison between wild-type and mef2c mutant embryos’ motor behaviour. (A) Frequency of coiling in wild-type (N = 46), mef2ca^b1086/b1086 (N = 19), mef2ca^b1086/+ (N = 46), mef2cb^fh288/fh288 (N = 53) and mef2cb^fh288/+ (N = 42) 25 hpf embryos, in a period of 2 min. (B) Double contraction percentage. The multiple contraction events were extracted from the video support for the five groups analysed. Results are presented as a box-and-whiskers graph. Significant statistical differences are indicated by * for p < 0.05 and **** for p < 0.0001.

Trace and density maps. Example of traces for all genotype larvae, obtained from a recording session of 3 min (left). Coloured density maps represent the proportion of larvae at each position in the well (middle). Black and white density maps represent the proportion of larvae at each position in the well (right). Top to bottom: WT, mef2ca^b1086/b1086, mef2ca^b1086/+, mef2cb^fh288/fh288 and mef2cb^fh288/+.

Analysis of trajectories for wild-type (N = 46), mef2ca^b1086/b1086 (N = 19), mef2ca^b1086/+ (N = 46), mef2cb^fh288/fh288 (N = 53) and mef2cb^fh288/+ (N = 42) larvae. (A) The arena-surface division into inner and outer regions used for data analysis. (B) Time percentage spent in an outside disk. (C) Total distance covered by each individual in a group during the recording time of 3 min. (D) Maximum velocity median of every group during the recording time of 3 min. (E) Mean velocity during movement bouts. (F) Median percentage of time in activity. Significant statistical differences are indicated by * for p < 0.05 and ** for p < 0.01.