The Human AF9 Homologue in Zebrafish Is Involved in Primitive Hematopoietic Development

Introduction: Human AF9 (alias MLLT3) gene was initially identified as one of the most common translocation partners of MLL associated with acute myeloid leukemia. More recently it has been demonstrated that AF9 can be a positive regulator of early erythroid/megakaryocytic lineage decisions. However, many biological functions of AF9 and its role in leukemia remain unclear. Here, we aimed to identify the zebrafish homologue of human AF9 and to analyze its function using the zebrafish model system. Methods and Results: A single zebrafish af9 gene was identified by homology searching at zebrafish genome database (www.ncbi.nlm.nih.gov/). The putative af9 protein showed 60% identity and 73% similarity to human counterpart. These results from the perspective of phylogenetic and synteny analyses indicated that zebrafish af9 was the likely homologue of human AF9. We used whole-mount in situ hybridization to analyzed its spatial and temporal expression patterns during embryonic development. Af9 was expressed specifically in the posterior-lateral mesoderm at the 5-somite stage and in the intermediate cell mass (ICM, site of primitive hematopoiesis) between 18 to 24 hours post fertilization (hpf). In order to characterize the role of af9 in early hematopoietic development, we performed over-expression and loss-of- function experiments in zebrafish embryos, and then analyzed various hematopoietic markers by reverse transcriptase quantitative PCR (RT-qPCR). Inducing ectopic af9 expression by mRNA injection we found an increased expression of gata1 and hbae1 (erythroid markers) and reduced expression of runx1, scl, lmo2, c-myb (stem cell/precursor associated markers) as well as pu.1 and l-plastin (myeloid markers) at 22 hpf-old embryos. Conversely af9 loss-of-function obtained using anti-sense morpholinos showed decreased expression of gata1 and hbae1 mRNA, while pu.1 and l-plastin increased their expression in morphant embryos at 22 hpf. The same morphants did not show any appreciable change in stem cell/precursor marker expression. Conclusion: The finding that gata1 expression is controlled by af9 suggests that this gene might promote the erythroid development in zebrafish primitive hematopoiesis. As the genetic programs of AF9 are evolutionary conserved between human and zebrafish, these findings provide new insights and opening further perspectives in leukemia research and hematopoiesis studies.