spry4 Is Required for Normal Hematopoietic Development in Zebrafish Embryos

<div><p>(A) The observed frequency of hematopoietic defects in <i>gata1:</i>DsRed Tg zebrafish embryos are indicated for two independent <i>spry4</i>-targeted MOs (MO1 and MO2; black bars), a four-base mismatched control MO (MM MO; no bar indicates a 0% frequency), a low-dose injection of MO1 and MO2 individually and in combination (gray bars), and MO2 coinjected with a human <i>SPRY1</i> DNA expression vector or a GFP control (white bars) (error bars = standard deviation of the mean, *<i>p</i> < 0.05, **<i>p</i> ≤ 0.01).</p> <p>(B) Representative pictures of the phenotypes seen with <i>spry4</i> MO1 at 48 h using <i>fli1:</i>EGFP/<i>gata1:</i>DsRed double Tg zebrafish embryos that have EGFP⁺ vascular endothelial cells and DsRed⁺ erythroid cells. The embryos display a more drastic reduction in DsRed⁺ blood cells with some blood pooling and pericardial edema at higher MO doses (++MO) without major vasculature defects. The <i>gata1:</i>DsRed and <i>fli1:</i>EGFP Tg images are representative of three experiments of <i>n</i> > 40 embryos each.</p> <p>(C) Q-RT-PCR analysis of <i>hbae1</i> and <i>lcp1</i> transcripts in <i>spry4</i> MO1, control mismatch MO and a <i>gata1</i> MO-injected zebrafish at 48 hpf (*<i>p</i> < 0.05).</p> <p>(D) Injection of 30 pg of human <i>SPRY1</i> DNA expression vector resulted in an expansion of DsRed⁺ hematopoietic cells in the posterior ICM in greater than 50% of successfully injected embryos at 32 hpf (three experiments of <i>n</i> = 30), and a representative bright-field image is pictured with the fluorescence micrograph overlaid (left). Q-RT-PCR analysis of <i>hbae1</i> and <i>lcp1</i> transcripts in <i>SPRY1</i> overexpressing embryos relative to uninjected clutchmate controls (three experiments of <i>n</i> = 5 embryos) (right).</p>