Analysis of thrombocyte development in CD41-GFP transgenic zebrafish

Thrombocytes are the nucleated equivalent of platelets in non-mammalian vertebrates like the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alphaIIb, GpIIb) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP) tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours post-fertilization (hpf) by 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 non-mobile 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 anti-sense morpholinos for the stem cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The non-mobile 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.