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Embryonic zebrafish have long been used for lineage tracing studies. In zebrafish embryos, the cell fate identities can be determined by whole-mount in situ hybridization, or by visualization of live embryos if using fluorescent reporter lines. We use embryonic zebrafish to study the effects of a leukemic oncogene AML1-ETO on modulating hematopoietic cell fate. Induced expression of AML1-ETO is able to efficiently reprogram hematopoietic progenitor cells from erythroid to myeloid cell fate. Using the zebrafish model of AML1-ETO, we performed a chemical screen to identify small molecules that suppress the cell fate switch in the presence of AML1-ETO. The methods discussed herein may be broadly applicable for identifying small molecules that modulate other cell fate decisions.
Many leukemic oncogenes, including AML1-ETO, contribute to leukemogenesis by modulating hematopoietic stem/progenitor cell differentiation. Embryonic zebrafish is a powerful model to study hematopoietic cell fate. Within the first day post-fertilization, zebrafish embryos develop two localized pools of hematopoietic progenitor cells (HPCs). The anterior blood island expresses pu.1, and will give rise to the myeloid cells (1). On the other hand, the posterior blood island expresses gata1, and will differentiate into the erythroid cells (1). These synchronized pools of HPCs are useful for studying the signaling pathways that underlie or affect hematopoietic cell fate determination.
We have shown that expressing AML1-ETO, an oncogene frequently associated with acute myelogenous leukemia, leads to a rapid and efficient cell fate switch in the posterior blood island of zebrafish embryos (Fig. 1). This fate switch is characterized by downregulation of gata1, suggesting suppression of erythropoiesis, and upregulation of mpo, indicating conversion into the granuocytic cell fate (2). Furthermore, to identify candidate small molecules that can reverse AML1-ETO’s effects and the mechanisms by which AML1-ETO reprograms hematopoietic cell fate, we conducted a chemical suppressor screen using zebrafish embryos. From this screen we have identified several classes of compounds that restored gata1 expression in the presence of AML1-ETO (3). The chemical suppressors of AML1-ETO identified from the in vivo zebrafish screen may provide not only new insights into AML1-ETO-mediated hematopoietic differentiation but also new means to block AML1-ETO’s effects in the clinical settings.
For the chemical suppressor screen of AML1-ETO we used a transgenic zebrafish line, Tg(hsp:AML1-ETO), in which AML1-ETO expression is controlled by a zebrafish heat shock hsp70 promoter. Thus, AML1-ETO expression can be induced by incubating zebrafish embryos at 37–42°C as compared to the regular embryo culture temperature at 24–28.5°C. Tg(hsp:AML1-ETO) embryos were arrayed into 96-well screening plates. Compounds from the chemical library were also added to the screening plates, and the plates were subjected to the heat treatment to induce AML1-ETO expression. Subsequently, the embryos were fixed and processed for whole-mount in situ hybridization of gata1 through both manual and automated procedures.
Conceivably, the present method could also be adapted for investigation of other cell fate decisions by using other zebrafish lines and cell markers. The combination of facile detection of cell fates and high-throughput in vivo small molecule screening surely will make the embryonic zebrafish a unique model system to study reprogramming/cell fate modulation.
The authors would like to thank Dr. Randall T. Peterson for his advice and support during the development of this project. J.-R. J. Yeh is supported by a Career Development Award (AG031300) from the National Institute of Aging. This work was supported by RO1 CA118498 and the Claflin Distinguished Scholar Award.