Down-regulation of rpl15 gene expression inhibits development of early hematopoiesis in zebrafish

Abstract

Objective To investigate the effect of down-regulation of ribosomal protein l15 (rpl15) gene on early hematopoietic development in zebrafish. Methods The rpl15 morphilino antisense oligo (MO) was designed and synthesized to down-regulate the expression of rpl15 by microinjection into embryo of zebrafish (0~0.75 hpf) at the unicellular stage. qRT-PCR and Western blotting were used to verify the effectiveness of down-regulating rpl15 gene. The development of the injected (MO) and wild-type (WT) groups were observed separately using a somatic microscope. O-dianisidine staining was used to detect the expression of hemoglobin. qRT-PCR was used to detect the changes in the expression of the red lineage-specific transcription factors hbae3 and hbbe1. Whole embryo in situ hybridization and qRT-PCR were performed to detect the changes in the expression of key transcription factors of early hematopoietic development such as red lineage-specific transcription factor gata1, myeloid transcription factor pu.1, granulocyte marker mpo, targeted he matopoietic marker transcription factors runx1 and c-myb, and primitive group cell hematopoietic markers scl and lmo2. TUNEL assay was used to detect the changes of apoptosis in hematopoietic group. Results Microinjection of rpl15 MO successfully down-regulated the expression of rpl15 in zebrafish, and the MO-injected group exhibited abnormal phenotypes such as body segment curvature, developmental delay, and pericardial edema when compared with the control group. Hemoglobin synthesis was significantly reduced in the MO group than the control group. The results of in situ hybridization indicated that the expression of gata1 was significantly reduced (P < 0.05), but the mRNA levels of pu.1, mpo, runx1, c-myb and lmo2 were not affected, while that of scl was obviously reduced (P < 0.05). TUNEL assay showed no significant change in apoptosis of hematopoietic progenitor cells. Conclusion Down-regulation of rpl15 inhibits the early hematopoietic development of zebrafish, and it may be related to the significantly reduced expression of gata1 and scl.

Keywords

• rpl15
• hematopoietic development
• zebrafish
• morpholino antisense oligo