Ribosomal Protein Gene Knockdown Causes Developmental Defects in Zebrafish

The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6–7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development

Lateral Views of Wild-Type and MO-Injected Embryos

<div><p>The genes targeted by MOs and the observation time are indicated for each image.</p> <p>‘<i>rpl5</i>; MO116’ and ‘<i>rpl5</i>; MO117’ indicate two MOs designed and injected separately for two functional copies of <i>rpl5</i> on the zebrafish genome.</p> <p>‘<i>rpl5</i>; MOmix’ indicates the mixture of MO116 and MO117.</p> <p>Control MOs that included 5 mispaired bases were also used; one example of a control MO injection is shown (<i>rpl38</i>; misMO).</p> <p>Note that compared to the control, the <i>rpl38</i> morphant is shorter and displays a light-colored eye and thin yolk sac extension.</p> <p>Scale bar, 500 µm.</p></div

Specific Morphological Changes in Brain and Body Trunk

<div><p>Examples of morphants displaying characteristic deformations are shown.</p> <p>The targeted genes and the deformed areas of the morphants are indicated.</p> <p>(A, E) Wild type (WT).</p> <p>(B) <i>rps15</i>; enlarged 4th ventricle (white arrow).</p> <p>(C) <i>rps29</i>; enlarged lens (arrowhead) and undulated rhombencephalon (white arrow).</p> <p>(D) <i>rpl28</i>; protruded forehead.</p> <p>(F) <i>rps3a</i>; wider trunk and downward-curving tail.</p> <p>(G) <i>rps29</i>; wavy notochord and extremely bent tail.</p> <p>(H) <i>rpl35a</i>; sharply downward bent tail.</p> <p>Anterior is to the left.</p> <p>Bars: A∼D, 100 µm; E∼H, 200 µm.</p></div