(A) Schematic representation of the exon–intron organisation of eef1a1l1 (red), eef1a1a (black), eef1a1b (blue) and eef1a2 (yellow) structures obtained from the Ensembl database. Length (in base pairs) of exons and introns, which are not drawn to scale, are indicated above and below respectively. (B) Percentage identity matrix for zebrafish Eef1as at the nucleotide and amino acid sequence level (in brackets) calculated using Clustal Omega. (C) Phylogenetic relationship among the zebrafish Eef1as and other vertebrate eEF1As using the maximum likelihood method.

The human eEF1A: heEF1A1 and heEF1A2, mouse eEF1A: meEF1A1 and meEF1A2. Identical and similar amino acid residues are indicated by black and grey backgrounds. Red asterisks (*) indicate some of the residues that are mutated in human eEF1A2, which are completely conserved in the four zebrafish eEF1A isoforms [22–27].

(A) Expression of eef1a1l1 in different early embryonic stages detected by RT-PCR. The other eef1a genes were undetected at these stages (data not shown). (B) Expression of eef1a genes in 24, 48 and 72 hpf developmental stages using RT-PCR. NTC, no template control. (C) Expression of eef1a genes in different tissues of adult zebrafish detected by RT-PCR. (D) Expression levels of eef1a genes in brain, muscle and liver tissues, and (E) comparison of the relative levels of their transcripts in these tissues. Expression values were normalised to those of ATPsynth, NADH and 16S. Results are means ± SD, n=3. *P<0.05; **P<0.01; ***P<0.0001. For comparison, data were presented as the gene expression ratio of the target mRNA to the geometric mean of reference genes for each tissue. (F) Western blot showing Eef1a2 expression in brain, liver and muscle zebrafish tissues using eEF1A2-Abcam antibody (1:1000). Control is muscle tissue from mouse. (G) Validation of eEF1A2-Abcam antibody (ab82912) specificity using lysates isolated from individually transfected HEK293T cells with GFP-tagged Eef1a constructs. Abbreviation: Zf, zebrafish.

(A) Schematic showing outcross mating of founder (F0) fish and wild-type fish showing recovered F1 sequences (number of F1 fish for each allele are indicated in brackets, target sequences (yellow highlight) and PAM site (purple) with red showing inserted bases) and the predicted effect of Ins4 and Del2 mutant allele with aberrant residues shown in red (right). (B) No overt difference in homozygous Del2 (6 months) and homozygous Ins4 (8 months) adult fish from wild-type (6 months) adult fish. (C) The position of the three different primer sets; eef1a2S (blue triangle), eef1a2P (black triangle) and 3′eef1a2 (red triangle) is illustrated in relation to the gRNA target site (PAM site sequence in red). Expression levels of eef1a2 in homozygote Del2 (top panel) and homozygote Ins4 (bottom panel) fish using eef1a2P and 3′eef1a2 primer sets. Results were normalised to ATPsynth, NADH and 16S. (means ± S.E.M; n=3), *P<0.05. (D) Anti-GFAP antibody stained transverse sections of spinal cords of homozygous Del2 and Ins4 adult fish showed no sign of neurodegeneration. Negative control of a no primary (No Pri) was included which showed no staining. Scale bar = 100 µm. (E) Expression levels of eef1a1l1 (left), eef1a1a (middle) and eef1a1b (right) mRNA in homozygous Del2 (top panel) and Ins4 (bottom panel) adult brain. Data were normalised to ATPsynth, NADH and 16S and were presented as means ± S.E.M.; n=3.

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