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J. Lian et al.
translation,15,16 protein degradation,17,18 and microRNA pro- cessing.19 The accumulation of excess heme and its precur- sors in tissues can cause oxidative damage via the genera- tion of reactive oxygen species.11 Thus, cellular heme homeostasis must be tightly controlled.
The 5-aminolevulinate synthase 1 (ALAS1) is a mito- chondrial enzyme that catalyzes the condensation of glycine and succinyl-CoA, forming 5-aminolevulinic acid. ALAS1 is the first and rate-limiting enzyme of the heme biosynthetic pathway, which is conserved from lower to higher organisms.20 ALAS1 (also called hepatic ALAS or non-specific ALAS) is ubiquitously expressed throughout the body, whereas another isoform, ALAS2 (also called ALAS-E), is predominantly expressed in erythroid cells, to meet the need of the large amounts of heme required for hemoglobin synthesis.11 It has been reported that human ALAS2 mutations cause X-linked sideroblastic anemia;21 ALAS2-deficient mice and zebrafish also display severe anemia,22,23 revealing the major contribution of ALAS2 to erythroid heme biosynthesis, and hence how it is essential to erythroid differentiation. In contrast to ALAS2, there are no reported human diseases directly caused by muta- tions in ALAS1. In mice, Alas1-null embryos are lethal by embryonic day 8.5 (E8.5),24 thus, the in vivo physiological role of ALAS1 is unclear. Using GFP knock-in mice (Alas1+/GFP), ALAS1 was found to be highly expressed in the liver, exocrine, endocrine glands, and myeloid cells, where large amounts of heme are required to meet the needs of tissue-specific hemoproteins, such as MPO, NADPH oxi- dase, and CYP450.24 Notably, Alas1 is also expressed high- er in neutrophils than macrophages,24 suggesting cell-spe- cific roles in neutrophils. However, the function of ALAS1 in neutrophils is still unknown.
Taking advantage of their transparent body, we can observe neutrophil morphology to trace neutrophil behav- iors in live zebrafish. Zebrafish is an ideal model for study- ing neutrophil biology.25,26 Here we report a novel role of Alas1 in regulating neutrophil development using a neu- trophil lineage-deficient mutant zebrafish line (previously named smu350) that was isolated from our ENU mutant collection.27 We demonstrated that alas1 was the causative gene for the mutant and revealed that alas1 was essential for neutrophil development and physiological function.
Methods
Fish maintenance
Zebrafish were raised and maintained under standard condi-
tions.28 Embryos were maintained in egg water containing 0.2 mM
N-phenylthiourea (Sigma-Aldrich, St. Louis, MO, USA) to prevent
pigment formation. All work involving zebrafish was approved by
Southern Medical University Animal Ethics Committee. The fol-
lowing strains were used: AB, Tg(lyz:DsRed),29 Tg(gata1:DsRed), vltm651/+ and alas1smu350/+.
Treatment with succinylacetone
Succinylacetone (SA) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in egg water. Zebrafish embryos were placed in culture dishes containing 1 mM SA at 5 hours (h) post fertilization (hpf) till the desired stage.
Bacterial infection
The eGFP-labeled E. coli strain XL10 was cultured in LB broth with 50 mg mL-1 ampicillin at 37°C until reaching an optical den-
sity at 600 nm between 0.5-0.8. Bacteria were washed with sterile phosphate buffered saline (PBS) three times, harvested by centrifu- gation at 5000 x g for 5 minutes (min) and resuspended in sterile PBS. The working concentration of E. coli was 2x109 mL-1, and approximately 0.5 nL of bacterial suspension was subcutaneously injected over a somite into 3-day-post-fertilization (dpf) embryos with 0.02% tricaine using a PLI-100A Pico-Injector (Warner Instruments, Hamden, CT, USA) as previously described.26,30 For bacterial colony forming assays, every injected embryo was washed with sterile PBS three times, and then homogenized in 200 mL of sterile PBS at the desired time points. Then, 10 mL of homogenate was plated on LB medium with ampicillin and cul- tured at 37°C overnight. The results are the average of two sepa- rate experiments.
Statistical analysis
Data were recorded and analyzed using GraphPad Prism 7 and IBM SPSS v.23. Two-tailed Student t-test and Mann-Whitney U test were used for comparisons between parametric and non-para- metric data, respectively. One-way analysis of variance (with Bonferroni or Dunnett T3 post-test adjustment) was used for para- metric data to make multi-comparisons. Differences were consid- ered significant at P<0.05. Data are expressed as the mean±Standard Deviation (SD).
Results
Neutrophil deficiency in smu350 mutant zebrafish
To identify new regulators of neutrophil development, we conducted a genetic screen for neutrophil-deficient zebrafish mutants using Sudan black B (SB) staining. From this screen, we isolated the neutrophil-deficient smu350 mutant, which lacked the SB signal as early as 36 hpf (Figure 1A). The early loss of the SB signal in smu350 mutants suggested defects in embryonic neutrophils, as SB+ cells represent embryonic neutrophils that are initiat- ing from embryonic myelopoietic tissue.31 Because myeloid progenitors that are derived from rostral blood islands will progress to neutrophils during embryonic myelopoiesis, we first determined if there were defects in the formation of myeloid progenitors. The results showed that pu.1 expression at 22 hpf was normal (Figure 1B), sug- gesting the presence of myeloid progenitors in smu350 mutants. Therefore, we speculated that the loss of the SB signal was due to defects in neutrophil maturation. To test this possibility, we detected the transcript and protein activity of the myeloid-specific peroxidase (Mpx), which is an abundant granule protein in neutrophils.2 Whole- mount in situ hybridization (WISH) showed that mpx mRNA expression was intact (Figure 1C), suggesting the presence of neutrophils in smu350 mutants. To further examine Mpx enzyme activity, diaminobenzidine (DAB, a peroxidase substrate) staining was performed. The results showed that while the signal in the yolk sac (representing hemoglobin peroxidase activity32) was present, signals rep- resenting neutrophil peroxidase activity were absent in smu350 mutants (Figure 1D), suggesting that Mpx lost its catalytic activity. As neutrophil granules are abundant with Mpx, we directly monitored neutrophil granule mor- phology via video-enhanced differential interference con- trast (VE-DIC) analyses of live embryos. The results showed that neutrophils from siblings (alas1smu350/+ and alas1+/+ embryos from a heterozygous alas1smu350/+ in-cross) had abundant visible and highly mobilized granules, while neutrophils from smu350 mutants lacked such granules
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haematologica | 2018; 103(11)