DEV008904 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. The loss-of-circulation phenotype in Tg(hsp:AML1-ETO) embryos is dependent on the induced expression of AML1-ETO. The control Tg(hsp:AML1-ETO) embryos and Tg(hsp:AML1-ETO) embryos that had been injected with antisense morpholino oligonucleotides complementary to the translation start site of the human AML1-ETO mRNA (hAML1-MO) were subjected to the same heat treatment. At 36-40 hpf, the percentages of the embryos exhibiting a complete loss-of-circulation phenotype were scored. Injection of hAML1-MO restored circulation in heat-treated Tg(hsp:AML1-ETO) animals. Total numbers of embryos scored for each condition are indicated as n.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Time-course of AML1-ETO expression and the loss-of-circulation phenotype. Tg(hsp:AML1-ETO) embryos were incubated at 38°C at various stages during development as indicated for 1 hour. The percentages of embryos without circulation were scored between 32-40 hpf. Total numbers of embryos scored for each condition are indicated as n. While inducing AML1-ETO expression before 19.5 hpf can elicit the AML1-ETO phenotype with complete penetrance, inducing AML1-ETO expression after 21 hpf greatly diminishes its effect.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Transient expression of AML1-ETO results in accumulation of immature blast cells that fail to mature. (A,B) Cytology of hematopoietic cells from wild-type (A) and Tg(hsp:AML1-ETO) (B) embryos at 5 dpf. All of the embryos have been subjected to 1-hour 38°C incubation at 16 hpf. All of the wild-type fish exhibit abundant circulating blood cells at the time of harvest. Only some of the Tg(hsp:AML1-ETO) embryos possess circulating blood cells at the time of harvest. Pools of accumulating blood cells can still be seen in many of the Tg(hsp:AML1-ETO) embryos. (A) The wild-type sample was extracted from the circulating blood cells. It contains mostly mature erythrocytes, while immature blast cells (arrowhead) are scarcely seen. (B) The Tg(hsp:AML1-ETO) sample was extracted from the non-circulating blood cells. It contains a mixture of erythroid cells (arrows) and immature blast cells (arrowheads).

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. The Mpo+ cells accumulated in Tg(hsp:AML1-ETO) embryos are unlikely to be immature erythroid progenitors. (A) Bright field and (B) fluorescent image of double in situ hybridization of Mpo (purple color, no fluorescence) and βe3 globin (red color, red fluorescence) at 31 hpf. (C) Combined image of both bright field and fluorescence image. The photos show the side view of the embryo close to the end of yolk extension (marked in blue in C). Two filled arrows point to the Mpo+ cells stained purple but have no red fluorecence, suggesting that these cells do not express βe3 globin. By contrast, the cell that expresses βe3 globin does not express Mpo (open arrow).

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. AML1-ETO expression results in cebpa downregulation in hamatopoietic cells. In situ hybridization of cebpa indicates that in AML1-ETO transgenic embryos, hematopoietic expression of cebpa is blocked (red arrows), whereas intestinal expression of cebpa is unaffected (black arrows). Both embryos have been subjected to the same heat treatment.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. AML1-ETO suppresses the erythroid and monocytic cell fates, and promotes the granulocytic cell fate in chordin morphants. (A) In situ hybridization of gata1, mpo and l-plastin in wild-type and AML1-ETO transgenic embryos. All of the embryos have been injected with 50 mM chordin antisense morpholino oligonucleotides (Open Biosystems) and subjected to the same heat treatment. The expanded pool of blood cells in the chordin morphants expresses gata1 and l-plastin (A, upper panels), whereas induced expression of AML1-ETO downregulates both gata1 and l-plastin but upregulates mpo (A, lower panels). The embryos were at 22 hpf for gata1 staining and 31 hpf for mpo and l-plastin staining. (B,C) Cross sections of chordin morphants stained with l-plastin probes. While l-plastin+ cells are often seen in the wild-type embryos, these cells are not seen in the AML1-ETO transgenic embryos.

• Supplemental Table S1 - Adobe PDF