Analysis of thrombocyte development in CD41-GFP transgenic zebrafish

Thrombocytes are the nucleated equivalent of platelets in nonmarnmalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoterandhavegeneratedtransgeniczebra- fish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP(+) cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP(+) cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.