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Fig. 2
Knockdown of beclin 1 suppresses the Spns1 deficiency in zebrafish.
(A) Schematic representation of the zebrafish beclin 1 (zbeclin 1) gene, its mRNA and protein products. A splice-blocking beclin 1 MO was designed to overlap the intron-exon boundary at the 52-splice junction of exon 4 in the zebrafish beclin 1 gene. To detect aberrantly spliced RNA products, two forward primers were designed for exon 3 (EX3 primer) and exon 4 (EX4 primer), and one reverse primer was designed for exon 7 (EX7 primer) within the beclin 1 gene. The zebrafish beclin 1 gene has a total of 11 exons having three unique domains [BH3 domain, coiled-coil (CCD) domain, and evolutionarily conserved (ECD) domain], and the beclin 1 MO was anticipated to disrupt the BH3 domain encoded by exon 4 and exon 5. (B) Splicing detection of zbeclin 1 mRNA by RT-PCR. Amplified PCR fragments show the intact sizes of the two amplicons for EX3-EX7 and EX4-EX7 following control (water) injection or only spns1 MO injection. Either beclin 1 MO (12 ng/embryo) injection or coinjection of spns1 MO (4 ng/embryo) and beclin 1 MO (12 ng/embryo) generated a skipping of exon 4 (beclin 1Δexon4). This was detected by the presence of an altered EX3-EX7 amplicon and undetectable EX4-EX7 product. The deletion of exon 4 was confirmed by sequencing. Injected embryos were harvested for total RNA isolation at 54 hpf. (C and D) Rescue of the spns1 morphant by beclin 1 knockdown. (C) The yolk opaqueness phenotype appearance in control-injected (water), spns1 MO-injected, and spns1 and beclin 1 MOs-coinjected embryos was followed through 72 hpf. At 24 hpf, opaqueness commenced from the yolk extension region, which had almost disappeared or was severely damaged (more than 95% of spns1 MO-injected animals) with an extension of opacity to the entire yolk at 48 hpf. By 72 hpf, yolk opaqueness became highly dense throughout most of the spns1 MO-injected embryos, which usually died within another 24 h. Scale bar, 250 μm. (D) Clarification of the yolk opaqueness phenotype in spns1 morphants at 72 hpf. As described in (C), more than 95% of the spns1 MO-injected embryos showed a ‘mostly opaque’ yolk at 48 hpf, and such embryos subsequently died. Animals showing a ‘partially opaque’ yolk could sometimes be recovered and subsequently survived 96 h and beyond. beclin 1 MO coinjection dramatically increased (more than 10 times) the animal numbers with the partial yolk opaque phenotype. (E) Survival curve for spns1 morphant and spns1;beclin 1-double morphant larvae (log rank test: χ2 = 162.5 on one degree of freedom; p<0.0001).