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 zDRmo, zChdmo, and zRSmo, respectively. Those coinjected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DRmo and zChd+RSmo. Results zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChdmo embryos. At 48 hours postfertilization, zDRmo embryos showed increased Hb compared with WT or zRSmo embryos (2.36 × 10−2 ± 1.13 × 10−3 vs 1.85 × 10−2 ± 5.60 × 10−4 vs 1.79 × 10−2 ± 1.31 × 10−3 U, p < 0.05). zChd+DRmo embryos also showed increased Hb compared with zChdmo or zChd+RSmo embryos (4.60 × 10−2 ± 2.79 × 10−3 vs 3.17 × 10−2 ± 1.07 × 10−3 vs 3.05 × 10−2 ± 1.25 × 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 zDRmo vs zRSmo (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.