中性粒细胞发生是一个高度有序且被精密调控的过程，造血相关转录因子在其中起着关键作用。造血相关转录因子通过与其辅因子相互作用或转录因子之间相互作用形成复杂的调控网络，调控网络的异常可导致白血病的发生。目前参与该过程的转录因子有几十种，它们的结构与功能被广泛研究，但对转录因子之间调控关系的研究则相对缺乏。人PU.1和cMYB参与中性粒细胞发生的多个阶段且它们的异常通常与血液疾病相关，但目前对于在体情况下两者之间是否存在调控关系以及如何相互作用尚不明确。本研究利用cMyb过表达(cmyb^hyper)和Pu.1缺陷(pu.1^G242D/G242D)的斑马鱼模型，通过整体原位杂交、qRT-PCR、荧光报告系统以及拯救实验检测中性粒细胞发育过程中Pu.1和cMyb的相互作用关系。结果显示，在pu.1^G242D/G242D突变体中，中性粒细胞的cmyb表达量显著升高，而在cmyb^hyper中，中性粒细胞的pu.1表达量则无明显变化。进一步在 pu.1^G242D/G242D 突变体中注射MO (morpholino)来降低cmyb的表达，并使用SB以及BrdU染色检测中性粒细胞的数量和增殖情况，发现cmyb的敲低可以拯救突变体中中性粒细胞异常增生的表型。这些结果表明，在中性粒细胞发育过程中Pu.1可以负调控cMyb的表达量。最后，本研究通过构建多位点突变质粒和荧光报告系统，证实了Pu.1能够直接结合在cmyb启动子+72 bp位点，从而负调控其表达。综上所述，本研究明确了Pu.1可以通过调控cmyb的表达参与中性粒细胞的发育，为理解两者之间调控关系及其在疾病中的作用提供了新的线索。

Granulopoiesis is a highly ordered and precisely regulated process in which hematopoietic-related transcription factors play crucial roles. These transcription factors form complex regulatory networks through interactions with their co-factors or with each other, and anomalies in these networks can lead to the onset of leukemia. While the structures and functions of dozens of transcription factors involved in this process have been extensively studied, research on the regulatory relationships between these factors remains relatively limited. PU.1 and cMYB participate in multiple stages of neutrophil development, and their abnormalities are often associated with hematologic disorders. However, the regulatory relationship between these factors in vivo and their mode of interaction remain unclear. In this study, zebrafish models with cMyb overexpression (cmyb^hyper) and Pu.1 deficiency (pu.1^G242D/G242D) were utilized to systematically investigate the interaction between Pu.1 and cMyb during granulopoiesis through whole-mount in situ hybridization, qRT-PCR, fluorescence reporting systems, and rescue experiments. The results showed a significant increase in cmyb expression in neutrophils of the pu.1^G242D/G242D mutant, while there was no apparent change in pu.1 expression in cmyb^hyper. Further experiments involving injection of morpholino (MO) to decrease cmyb expression in pu.1^G242D/G242D mutants, followed by SB and BrdU staining to assess neutrophil quantity and proliferation, revealed that reducing cmyb expression could rescue the abnormal proliferation phenotype of neutrophils in the pu.1^G242D/G242D mutant. These findings suggest that Pu.1 negatively regulates the expression of cMyb during neutrophil development. Finally, through the construction of multi-site mutation plasmids and a fluorescent reporter system, confirmed that Pu.1 directly binds to the +72 bp site in the cmyb promoter, exerting negative regulation on its expression. In conclusion, this study delineates that Pu.1 participates in neutrophil development by regulating cmyb expression. This provides new insights into the regulatory relationship between these two factors and their roles in diseases.