Inherited deficiency of HCLS1-associated protein X1 (HAX1) in human leads to the development of severe congenital neutropenia (CN), which is characterized by impaired neutrophil development. Patients with HAX1 deficiency are prone to life-threatening infections beginning in their first months of life unless treated by recombinant human granulocyte colony-stimulating factor (rhG-CSF) or bone marrow transplantation. But approximately 10% of these patients do not respond to the rhG-CSF therapy. Therefore, there is an urgent need for new tailored therapeutic strategies for the treatment of this disease. However, there is a lack of an animal model that allows high-throughput compound screening for the HAX1 associated neutropenia. In this study, we sought to determine the role of hax1 in zebrafish hematopoiesis. We used two approaches to interfere with the zebrafish hax1. First, we injected antisense morpholino that efficiently blocked the translation of hax1 mRNA. As a second approach, we used the CRISPR-Cas9 technique to introduce mutations in the zebrafish hax1 gene. Both approaches were used in the in wild-type embryos and transgenic (mpo:gfp) reporter line, where the neutrophils are labeled with GFP. We found that hax1 knockdown reduced the number of neutrophils, without affecting the development of HSPCs and monocytes/macrophages. Compared with their wild-type counterparts, hax1 morphants exhibited reduced expression levels of hcls1 and cebpa. Whereas the expression level of cebpb, a marker of emergency granulopoiesis, was upregulated. Next, we examined whether hax1 knockdown impairs the cellular viability because CN patients who harbor HAX1 mutations exhibit increased apoptosis of myeloid progenitors. Although zebrafish hax1 morphants showed increased cell death throughout the embryo, apoptosis was not triggered in the hematopoietic site. Therefore, our results suggested that reduced neutrophil numbers in zebrafish hax1 morphants is due to decreased production of neutrophils rather than increased cell death. To determine whether our zebrafish model is suitable for discovering human drugs, we treated zebrafish hax1 morphants with human G-CSF and rescued the quantitative reduction of neutrophils. Overall, we have successfully established a novel in vivo model for studying the HAX-1 role in the granulopoiesis, which might open new avenues for developing therapeutic strategies for CN.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
