Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene

Objective. We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. Methods. MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those coinjected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR and zChd+RSmo. Results. zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, P < 0.05). zChd+DRmo embryos also showed increased Hb compared with zChd(mo) or zChd+RSmo embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DRmo compared with zChd+RSmo embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DRmo vs zChd+RSmo embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DRmo embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. Conclusion. Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development. (c) 2006 International Society for Experimental Hematology.