Abstract
Neutrophils play an essential role in the innate immune defense system in vertebrates. During hematopoiesis, the full function of neutrophils involves maturation of granules and related enzymes. Yet, transcription regulators that promote neutrophil maturation remain largely undefined. Here, two hematopoiesis-defective zebrafish mutants, runx1w84x and c-mybhkz3, were used to investigate the in vivo roles of Runx1 in cooperation with c-Myb in regulating neutrophil maturation. Loss of runx1 impairs primitive neutrophil development. Additional regulation of c-myb+/− and c-myb−/− induces a more severe phenotypes suggesting a synergistic genetic interaction between c-myb and runx1 in neutrophil maturation. Further studies revealed that the two transcription factors act cooperatively to control neutrophil maturation processes via transactivating a series of neutrophil maturation-related genes. These data reveal the in vivo roles of Runx1 in regulating primitive neutrophil maturation while also indicating a novel genetic and molecular orchestration of Runx1 and c-Myb in myeloid cell development. The study will provide new evidence on the regulation of neutrophil maturation during hematopoiesis.
Highlights
The function of mature neutrophils requires the development of characteristic neutrophil granules along with stored enzymes
The signal intensity for Sudan Black B (SB) staining of the neutrophils was lower in mutants compared with that in siblings (Fig. 1, A, B and D), suggesting that Runx1 is involved in neutrophil maturation
By utilizing the zebrafish model, the in vivo roles of Runx1 were elucidated in neutrophil maturation
Summary
Mature neutrophils are characterized by abundant granules in the cytoplasm, which can be stained by Sudan Black B (SB) [14, 24]. Chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were performed to ascertain whether the two transcription factors can directly bind to the putative sites in vivo To address this question, embryos were injected with Myc-tagged c-myb or runx plasmids to overexpress c-Myb and Runx for ChIP-PCR analysis in zebrafish. The results showed that all putative binding sites of the target genes were coprecipitated using an anti-MYC antibody (Fig. 4B) This result demonstrates that these neutrophil-specific genes are all directly regulated by c-Myb and Runx respectively. From the genetic epistasis and biochemical analysis, it can be concluded that Runx functions as a positive regulator for neutrophil maturation in early development It interacts and cooperates with c-Myb to transactivate a panel of neutrophil maturation-related genes (Fig. 5)
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