The role of phospholipase C gamma 1 in primitive hematopoiesis during zebrafish development
Received 3 November 2006; received in revised form 20 November 2006; accepted 20 November 2006.
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-γ1 was injected into embryos at the one- to four-cell stage (referred herein zPLC-γ1MO 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 μmol/L, respectively, from one cell until 48 hpf.
Results
Erythropoiesis was reduced in zPLC-γ1MO embryos, as shown by the reduction in gata1+ cells (wild-type: 4.32% ± 0.10% vs zPLC-γ1MO: 2.38% ± 0.11%, p = 0.021) and gata1 and α-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-γ1MO. 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-γ1 is involved in the regulation of primitive hematopoiesis in zebrafish embryos, which is distinct from its later effect on vascular formation.
Division of Hematology and Bone Marrow Transplantation, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong, China
Offprint requests to: Anskar Y.H. Leung, M.D., Ph.D., K418, K Block, Department of Medicine, Queen Mary Hospital, Pok Fu Lam Road, Hong Kong, HKSAR, China
ABSTRACT