Abstract
In this study, to investigate the secondary function of Rpl10a in zebrafish development, morpholino antisense oligonucleotides (MOs) were used to knock down the zebrafish ribosomal protein L10a (rpl10a). At 25 hpf (hours post-fertilization), embryos injected with the rpl10a MO showed an abnormal morphology, including short bodies, curved tails, and small yolk sac extensions. We observed pigment reductions, edema, larger yolk sacs, smaller eyes and smaller yolk sac extensions at 50 hpf. In addition, reductions in the expression of primordial germ cell (PGC) marker genes (nanos1 and vasa) were observed in rpl10a knockdown embryos. A rescue experiment using a rpl10a mRNA co-injection showed the recovery of the morphology and red blood cell production similar to wild-type. Moreover, the CRISPR-Cas9 system was used to edit the sequence of rpl10a exon 5, resulting in a homozygous 5-bp deletion in the zebrafish genome. The mutant embryos displayed a morphology similar to that of the knockdown animals. Furthermore, the loss of rpl10a function led to reduced expression of gata1, hbae3, and hbbe1 (erythroid synthesis) and increased tp53 expression. Overall, the results suggested that Rpl10a deficiency caused delays in embryonic development, as well as apoptosis and anemia, in zebrafish.
Highlights
Ribosomal proteins (RPs) function in protein synthesis
The zebrafish rpl10a gene is located on chromosome 8 and contains six exons, resulting in a 648 bp cDNA sequence
To investigate the effect of Ribosomal protein L10a (Rpl10a) deficiency, we knocked down the rpl10a gene using morpholino antisense oligonucleotides (MOs) injection to inhibit protein translation
Summary
Ribosomal proteins (RPs) function in protein synthesis. Many lines of evidence suggest other roles for RPs, including DNA repair, apoptosis, translational regulation, developmental control, and red blood cell development[1]. Rpl10a has secondary functions in embryogenesis and ovary development, the effects of Rpl10a on primordial germ cells, or PGCs, during early embryogenesis have not been reported. In Drosophila, PGCs deficient in Nanos activity showed abnormal development, shifts to gonad failure, a reduction in egg number, and morphological abnormalities[18]. These results in fruit flies were similar to results from zebrafish[17]. Histology was used to support the morphological defects and the effects of rpl10a knockdown on PGC marker genes, including nanos[1] and vasa gene expression, which were investigated using qRT-PCR and whole-mount in situ hybridization
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