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First published online December 21, 2007
doi: 10.1242/10.1242/dev.008904

Development 135, 401-410 (2008)
Published by The Company of Biologists 2008
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AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression

Jing-Ruey J. Yeh1,2,3,*, Kathleen M. Munson1,2,3, Yvonne L. Chao1,2, Quinn P. Peterson1,2, Calum A. MacRae1,2 and Randall T. Peterson1,2,3,*

1 Developmental Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.
2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
3 The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.


Figure 1
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  		Fig. 1. Expression of AML1-ETO in zebrafish embryos causes an accumulation of hematopoietic cells. (A) Schematic diagram of the DNA fragment used to generate the Tg(hsp:AML1-ETO) zebrafish line. (B,C) Bright-light images of 1-dpf embryos that have been subjected to heat treatment. The accumulated hematopoietic cells in Tg(hsp:AML1-ETO) embryos are indicated with red arrows (C). The areas in the boxes are shown at a higher magnification in D and E.

 

Figure 2
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  		Fig. 2. AML1-ETO transgenic zebrafish possess functional cardiovascular systems. (A) flk1 in situ hybridization of heat-treated wild-type and Tg(hsp:AML1-ETO) embryos harvested at designated stages as indicated. (B) Bright-light and fluorescent images of fli1-EGFP transgenic embryos in wild-type or Tg(hsp:AML1-ETO) background. These embryos have been subjected to the same heat treatment. The arrowhead indicates the site of the accumulated blood cells. (C) Fluorescent microangiography shows that the vasculature is continuous and the site of the accumulated blood cells is connected to the rest of the circulatory system.

 

Figure 3
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  		Fig. 3. The accumulated hematopoietic cells in Tg(hsp:AML1-ETO) embryos are enriched for immature blast cells. (A-G) Cytology of hematopoietic cells from wild-type (A,D,E) and Tg(hsp:AML1-ETO) (B,C,F,G) embryos at 40 hpf. Low magnification (A,B); high magnification (C-G). Arrowheads, blast-like cells (C); black arrow, a bi-nucleated heterophil/neutrophil (D).

 

Figure 4
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  		Fig. 4. The transcriptional changes in hematopoietic cells of Tg(hsp:AML1-ETO) embryos correlate with the transcriptional changes caused by AML1-ETO expression in human patients and in human cell lines. The embryos used for array #1 were heat shocked two times at 40 and 42°C for one hour each time at 16 and 24 hpf respectively. The blood cells were extracted at 38 hpf. The embryos for arrays #2 and #3 were heat-shocked three times at 40, 42 and 42°C for one hour each time at 16, 24 and 38 hpf respectively. The blood cells were extracted at 40 hpf. The ratios between the signals from Tg(hsp:AML1-ETO) and wild-type samples are shown in the heat map. The corresponding changes in the human studies are indicated in the References column.

 

Figure 5
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  		Fig. 5. AML1-ETO disrupts definitive hematopoiesis. (A,B) In situ hybridization of (A) aml1 and (B) cmyb at 33 hpf indicates that definitive hematopoiesis is blocked in Tg(hsp:AML1-ETO) embryos.

 

Figure 6
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  		Fig. 6. AML1-ETO reprograms hematopoietic cell fate, converting erythropoiesis to granulopoiesis. (A) In situ hybridization of gata1, fluorescent images of zpu.1-EGFP transgenic fish, and in situ hybridization of mpo and l-plastin. AML1-ETO expression results in gata1 downregulation. Subsequently, pu.1 expression is increased. Finally, the accumulated blood cells express the granulocytic cell marker mpo but not the monocytic cell marker l-plastin. All embryos were subjected to the heat treatment and then were collected at designated stages as indicated. (B) Proposed effects of AML1-ETO in hematopoietic progenitor cells. MPC, myeloerythoid progenitor cell; GMP, granulocyte/monocyte progenitor; MEP, megakaryocyte/erythroid progenitor. The red crosses indicate the steps suppressed by AML1-ETO. The parentheses indicate the markers for each cell type or the genes involved in the processes. These data suggest that AML1-ETO reprograms hematopoietic cell fate, resulting in an enrichment of myeloblasts that express mpo (boxed and shaded).

 

Figure 7
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  		Fig. 7. AML1-ETO suppresses the monocytic cell fate. (A) In situ hybridization of l-plastin, mpo and pu.1. Tg(hsp:AML1-ETO) embryos were injected with gata1 morpholino (MO). Half of the injected embryos were heat-treated to induce AML1-ETO expression. AML1-ETO suppresses the expression of pu.1 and l-plastin but promotes the expression of mpo in gata1 morphants. (B) In situ hybridization of mpo. Injections of pu.1 MO decreased the expression of mpo in AML1-ETO-expressing embryos, inicating that Pu.1 is essential for the specification of granulocytes.

 

Figure 8
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  		Fig. 8. AML1-ETO downregulates scl, leading to the early effects of AML1-ETO expression in primitive hematopoietic cells. (A) In situ hybridization of scl. AML1-ETO expression results in scl downregulation specifically in hematopoietic cells. The expression of scl in the head (asterisk), in the endothelial primordium (arrowhead), and in the posterior tail region (arrow) was not affected. (B) In situ hybridization of gata1, mpo and fluorescent images of zpu.1-EGFP transgenic zebrafish. Injections of scl mRNA restored gata1 expression and reversed pu.1 and mpo induction in Tg(hsp:AML1-ETO) embryos.

 

Figure 9
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  		Fig. 9. Trichostatin A suppresses the effects of AML1-ETO in zebrafish embryos. (A-C) DMSO or 0.5 µM TSA was added to Tg(hsp:AML1-ETO) embryos 2 hours before (A,B) or at the end of a 1-hour heat treatment (C) at 38°C at 18 hpf. Tg(hsp:AML1-ETO) embryos that had not been subjected to the heat treatment were used as a control. TSA treatment rescues scl and gata1 downregulation and blocks the accumulation of Mpo+ cells caused by AML1-ETO expression.

 

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