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 co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo).


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+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) 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+DR(mo) compared with zChd+RS(mo) 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+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene.


CONCLUSIONS
Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.
Kwan
Tommy T
TT
Department of Medicine, University of Hong Kong, Hong Kong.
Liang
Raymond
R
Verfaillie
Catherine M
CM
Ekker
Stephen C
SC
Chan
Li C
LC
Lin
Shuo
S
Leung
Anskar Y H
AY
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D004195:Disease Models, Animal; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006023:Glycoproteins / Q000008:administration & dosage; D006410:Hematopoiesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006454:Hemoglobins / Q000096:biosynthesis / Q000187:drug effects; D036341:Intercellular Signaling Peptides and Proteins / Q000008:administration & dosage; D009025:Morpholines / Q000008:administration & dosage; D018124:Receptors, Tumor Necrosis Factor / Q000008:administration & dosage* / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D006454:Hemoglobins; D036341:Intercellular Signaling Peptides and Proteins; D009025:Morpholines; D018124:Receptors, Tumor Necrosis Factor; C414429:hematopoietic death receptor; C054234:chordin; D053148:Caspase 3; D020169:Caspases