Fig. 1

Knockout of Atf4a leads to growth impairment in zebrafish. (A) Schematic of the zebrafish atf4a gene structure. The CRISPR/Cas9 target sequence is located in the second exon. The PAM sequence is shown in green. Sequencing chromatograms of wt and atf4a mutant alleles are shown below, followed by the corresponding wt and predicted mutant Atf4a protein sequences. (B) Relative expression levels of atf4a and atf4b mRNA in wt, atf4aΔ2/Δ2, atf4aΔ4/Δ4zebrafish at 72 hpf. mRNA levels were normalized to β-actin mRNA. (C–E) Relative expression levels of slc7a11, asns and shmt2 mRNA in wt, atf4a−/− and atf4a−/− microinjected with atf4a mRNA at 72 hpf. mRNA levels were normalized to β-actin mRNA. (F–H) Relative expression levels of slc7a11, asns and shmt2 mRNA in wt, atf4aΔ2/Δ2 and atf4aΔ4/Δ4 treated with or without thapsigargin for 6 h at 72 hpf. mRNA levels were normalized to β-actin mRNA. (I) Body length of wt, atf4aΔ2/Δ2 and atf4aΔ4/Δ4 at 24 hpf and 72 hpf. (J) The protein level of p-AktSer473, Akt, p-S6kThr389 and S6k in zebrafish embryos of wt and atf4a−/− at 24 hpf, 48 hpf and 72 hpf. β-actin was used as a loading control. (K) Body length of wt, atf4a−/− and atf4a−/− microinjected with atf4a mRNA at 72 hpf. (L) The protein level of p-AktSer473 and Akt in zebrafish embryos of wt, atf4a−/− and atf4a−/− microinjected with atf4a mRNA. β-actin was used as a loading control. (M) The graph shows the ratio of p-AKTSer473 to AKT. All data are presented as mean ± SEM from three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. atf4a: Activating transcription factor 4a; atf4b: Activating transcription factor 4b; slc7a11: Solute carrier family 7 member 11; asns: Asparagine synthetase; shmt2: Serine hydroxymethyltransferase 2, β-actin: Beta-actin.