Knockdown of gro3 in cooperation with AML1-ETO expression induces abnormal hematopoiesis and absence of circulating blood in zebrafish embryos.Gro3 MO (morpholino oligonucleotide)-injected or uninjected wild-type and Tg(hsp:AML1-ETO) zebrafish embryos were all subjected to the same heat induction. (A) Inspection of uninjected (Control) and gro3 MO injected Tg(hsp:AML1-ETO) zebrafish embryos showed the absence of circulating cells and accumulation of hematopoietic cells in the ICM and the ventral tail region (red arrows). These cells had a blast-like morphology with a large size and relatively little cytoplasm. (B) Percentages of embryos exhibiting loss-of-circulating cells were scored between 30 and 40 hpf. Control and MO indicate uninjected and morpholino oligonucleotide-injected embryos, respectively. AML1-ETO and WT indicate Tg(hsp:AML1-ETO) and wild-type embryos, respectively. n equals the number of embryos scored. P value was obtained using Student t test. The inset gels show the knockdown by RT-PCR of gro3 transcripts with 2 gro3 specific morpholino oligonucleotides. (C) Lack of circulating cells in AML1-ETO–expressing embryos is not caused by a vascular obstruction as evidenced by microangiography. The fluorescein-coupled latex beads injected into the inflow tract of the atrium was able to perfuse the whole vascular system of the Tg(hsp:AML1-ETO) embryos. It revealed a normal vascular pattern but reduced intersomitic vessels in gro3 morphant embryos. (D) Cytologic analysis of hematopoietic cells collected from the circulating blood in 3 uninjected Tg(hsp:AML1-ETO) embryos and the accumulating blood in 3 injected embryos. Whereas the uninjected embryos contain mostly red blood cells, the injected embryos contain abundant immature blast-like cells. Black arrows indicate red blood cells.