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L. Doll et al.
model has been established and, therefore, the experi- mental model system is based on in vitro neutrophil dif- ferentiation of induced pluripotent stem cells from patients with HAX1-associated CN.19,20 Mice lacking Hax1 display normal granulopoiesis and their predominant phenotype is post-natal lethality due to neuronal apopto- sis.15 Hence, there is an urgent need for an in vivo model to study the role of HAX1 in granulopoiesis and leukemoge- nesis. Here, we chose to use zebrafish (Danio rerio) as a vertebrate model because cellular and molecular mecha- nisms underlying granulopoiesis are largely conserved between zebrafish and humans,21-25 and 82% of disease- causing human genes have an orthologue in zebrafish.26,27 For example, zebrafish harboring mutations in g-csfr,23,24 cxcr4,28 and g6pc329 display impaired neutrophil develop- ment, mimicking CN patients who lack the correspon- ding orthologue gene. Thus far, it was unknown whether hax1 is involved in zebrafish granulopoiesis. Here, we show that this gene is expressed in zebrafish hematopoi- etic cells and is indispensable for neutrophil development, which makes zebrafish a suitable model for studying HAX1-associated neutropenia.
Methods
Zebrafish
Zebrafish lines were maintained according to standard proto- cols and handled in accordance with European Union animal protection directive 2010/63/EU and approved by the local gov- ernment (Tierschutzgesetz §11, Abs. 1, Nr. 1, husbandry permit 35/9185.46/Uni TÜ). All experiments described in the present study were conducted on embryos younger than 5 days post-fer- tilization (dpf). In this study, we used the wild-type TE strain of zebrafish. The transgenic (tg) reporter lines tg(mpo:gfp) and tg(lyz:dsRED) have been described previously.30,31
Injection of morpholino, single-guide RNA and messenger RNA
Three morpholinos (GeneTools) targeting the hax1 gene were used in this study (Online Supplementary Table S1). For the rescue experiment, full-length complementary DNA (cDNA) of zebrafish hax1 was isolated and cloned into the pMC vector.32 Capped messenger RNA (mRNA) was synthesized using the mMESSAGE mMACHINE SP6 kit (Ambion). Morpholino and 5- 10 ng/mL of hax1 mRNA were co-injected into one-cell stage zebrafish embryos. To perform transient CRISPR-Cas9 targeting of the hax1 gene, single guide RNA (sgRNA) target site (5’- GGGTTTTTCGGGATTCCCGG-3’) was predicted and evaluat- ed for off-target site by using the CCTop web tool.33 sgRNA (15 ng/mL) and 150 ng/mL of Cas9 mRNA (a kind gift from J. Wittbrodt, Heidelberg University) were co-injected into one-cell stage transgenic tg(mpo:gfp) embryos.
Heat-inducible g-csfa construct
The full-length cDNA of zebrafish g-csfa (or csf3a) was isolat-
ed and cloned into the pTGH2 plasmid containing a bi-direction- al heat-inducible promoter34 flanked by Tol2 binding sites. The resulting plasmids were then injected at a dose of 20 ng/mL with 10 ng/μL mRNA of Tol2 transposase into one-cell-stage embryos. Injected embryos at 1 dpf were heat treated at 39°C for 1 h. Green fluorescent protein-positive (GFP+) embryos were select- ed for subsequent whole mount in situ hybridization (WISH) analysis.
Whole mount in situ hybridization
RNA in situ hybridization of zebrafish embryos was performed
as described previously35 using digoxigenin-labeled RNA antisense probes, which are listed in Online Supplementary Table S2.
Statistical analysis
GraphPad Prism software (version 8) was used to produce graphs and for the statistical analysis. In this study, an unpaired, two-tailed Wilcoxon-Mann-Whitney test was used to compare the means of different data sets.
Additional details of the material and methods are available in the Online Supplementary Material and Methods.
Results
Zebrafish hax1 is expressed in hematopoietic cells
An in silico analysis was carried out to characterize zebrafish hax1. A high degree of synteny was found between human and zebrafish hax1 with the upab2l and pygo2 genes upstream, and aqp10b and cks1b genes down- stream in the corresponding genomic loci (Figure 1A). Multiple alignment and phylogenetic analysis revealed strong amino acid similarities over the entire coding region of Hax1 protein between zebrafish and its ortho- logues in other vertebrates (Figure 1B, Online Supplementary Figure S1). To elucidate whether hax1 is expressed in zebrafish hematopoietic cells, we first searched for hax1 in two single-cell RNA-sequencing data- bases36,37 of adult zebrafish whole kidney marrow (akin to mammalian bone marrow). In both databases, hax1 was found to be expressed in neutrophils, macrophages, and erythrocytes, albeit at low levels (data not shown). WISH was performed to determine the hax1 spatial expression patterns during embryonic development, revealing that hax1 is ubiquitously expressed throughout gastrulation and early embryonic segmentation (Figure 1C). At later stages, the hax1 transcript was detected in the posterior intermediate cell mass and caudal hematopoietic tissue (Figure 1C-F, black arrows), which are the sites of primi- tive and definitive hematopoiesis, respectively, in zebrafish embryos.21 The expression of hax1 in hematopoietic cells was also confirmed by double fluores- cent in situ hybridization analysis, revealing co-localiza- tion of hax1 and cmyb, a marker of hematopoietic stem cells (Figure 1D). Notably, hax1 transcript was also detect-
ed in the brain at 4 dpf (Figure 1G, arrow).
Loss-of-function of Hax1 leads to reduced neutrophil numbers in zebrafish embryos
To determine whether hax1 has a role in zebrafish hematopoiesis, a morpholino (MO)-mediated gene knock- down strategy was used. Specifically, three MO targeting the hax1 gene were used (Online Supplementary Figure S2A). One MO was designed against the ATG start codon site (hereafter named atg-MO), whereas the other two blocked the correct splicing of either exon 1 (hereafter named e1-MO) or exon 2 (hereafter named e2-MO), lead- ing to a truncated Hax1 protein (Online Supplementary Figure S2B-E). Quantitative analysis showed that wild- type hax1 transcript levels decreased to 90% and 70% in the embryos injected with e1-MO and e2-MO, respective- ly (Figure 2A, Online Supplementary Figure S2F and G). Notably, injection of MO did not result in embryonic mal-
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haematologica | 2021; 106(5)