Objectives: Phospholipase C (PLC) gamma 1 has been shown to mediate signal transduction of tyrosine kinases and affect function of hematopoietic cells. However, its role in hematopoiesis during embryonic development is currently unclear. In this study, we examined this issue using morpholino (MO) gene knockdown in zebrafish embryos
Methods: MO targeting at the exon-1-intron-1 junction of zebrafish PLC-gamma1 was injected into embryos at the one- to four-cell stage (referred herein zPLC-gamma1(MO) embryos). Primitive hematopoiesis was examined quantitatively by flow cytometry in Tg(gata1:GFP) embryos and by real-time quantitative polymerase chain reaction at 18 hours-post-fertilization (hpf), before the onset of circulation. The embryos were also treated with receptor inhibitors of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor at 25, 1, and 30 micromol/L, respectively, from one cell until 48 hpf.
Results: Erythropoiesis was reduced in zPLC-gamma1(MO) embryos, as shown by the reduction in gata1(+) cells (wild-type: 4.32% +/- 0.10% vs zPLC-gamma1(MO): 2.38% +/- 0.11%, p = 0.021) and gata1 and alpha-embryonic hemoglobin expression [0.47 +/- 0.06-fold (p = 0.013) and 0.46 +/- 0.04-fold (p = 0.013)]. Expression of scl, lmo-2 (early hematopoietic progenitors), pu.1, and l-plastin (myelomonocytic lineages) as well as fli1 (vascular progenitors) were unaffected. Fli1(+) cells in Tg(fli1:GFP) embryos were also unaffected by zPLC-gamma1(MO). When embryos were incubated with receptor inhibitors of vascular endothelial growth factor (VEGFRTKI), fibroblast growth factor (SU5402), or platelet-derived growth factor (AG1296), only VEGFRTKI reduced erythropoiesis [VEGFRTKI: 2.10% +/- 0.07% (p = 0.021) vs SU5402: 4.08% +/- 0.12% (p = 0.248) vs AG1296: 4.12% +/- 0.14% (p = 0.149)].
Conclusion: PLC-gamma1 is involved in the regulation of primitive hematopoiesis in zebrafish embryos, which is distinct from its later effect on vascular formation.