Page 95 - 2021_05-Haematologica-web

Zebrafish Hax1-associated neutropenia
Granulocyte colony-stimulating factor rescues reduced neutrophil numbers in the hax1 morphants
Next, two approaches were used to evaluate the expression of hcls1, cebpa and cebpb, which are activated by G-CSF. First, total RNA was isolated from morphants and uninjected embryos at 2 dpf. Quantitative reverse transcriptase polymerase chain reaction analysis was per- formed to determine the levels of expression of hcls1, cebpa and cebpb. This revealed a downregulation of the expression levels of hcls1 and cebpa, while the expression
of cebpb was upregulated in the hax1 morphants (Figure 6A). As a second approach, cells expressing cebpa or cebpb were stained using WISH. Similarly, the number of cebpa+ cells was reduced (Figure 6B), while the number of cebpb+ cells was increased (Figure 6C) in the hax1 morphants. These findings raised the question of whether overex- pression of g-csf is sufficient to rescue the reduced num- ber of neutrophils in hax1 morphants. To address this question, a heat-inducible promoter34 was used to ectopi- cally express zebrafish g-csfa at 1 dpf (Figure 6D).
A
B
C
Figure 3. Migration and phagocytosis of neu- trophils in the hax1 morphants. (A) Experimental design. (B) Injection of Alexa-594 conjugated Staphylococcus aureus debris into the noto- chord of tg(mpo:gfp) embryos at 2 days post-fer- tilization (dpf). Arrows indicate the injected site. Dashed lines indicate the position of the noto- chord. (C) Still photographs from a time-lapse recording illustrating the migration and phago- cytic activity of neutrophils (arrows) in the hax1 morphants. Numbers indicate time in minutes. Scale bars, 40 mm (B) and 10 mm (C). GFP: green fluorescent protein; nc: notochord.
haematologica | 2021; 106(5)
1315