Figure 4

Figure 4

terfa knockdown impairs the macrophage response following caudal fin amputation. (A) Experimental design: injection of terfa morpholino (terfa MO) or control morpholino (ctrl MO) at the 1-cell stage in the zebrafish transgenic line Tg(mpeg1:mCherry). Macrophages were isolated from both ctrl morphants and terfa morphants (100 larvae per condition) at 72hpf. The p15/16 expression level was measured and compared between the sorted macrophages and other cells populations (other cells), consisting of all non-macrophage cells from the zebrafish larva using qRT-PCR. (B) Relative mRNA expression levels of cdkn2a/b in the terfa morphants were assessed at 72 hpf by qRT-PCR. Ef1a was used as reference gene (data are the mean ± SEM, n = 6 independent experiments, Mann-Whitney tests were performed, p = 0.0649). (C) Relative mRNA expression level of cdkn2a/b in macrophages at 72hpf. Ef1a was used as reference gene (data are the mean ± SEM). * p < 0.1 (D) Experimental design: injection of terfa morpholino (terfa MO) or control morpholino (ctrl MO) at the 1-cell stage in Tg(mpeg1:mcherry-F/tnfa:eGFP-F) larvae. Caudal fins were amputated at 72 hpf and macrophage recruitment was analyzed from 3 to 72 hpA. (E) Representative images of maximal projections of caudal fin of Tg(mpeg1:mcherry-F/tnfa:eGFP-F) ctrl or terfa morphants at 24 hpA. All macrophages (mCherry-labeled) are in red and tnfa+ macrophages are in green. (F) Total number of macrophages (red) recruited to the wound area in ctrl (dark red) and terfa (pink) morphants at the indicated time points after amputation over 3 days (upper panel). Relative number of tnfa+ macrophages among all macrophages at the wound site in ctrl (dark green) and terfa (light green) morphants at different time points over 3 days. Data are the mean ± SEM, n = 50 larvae at 6, 24 and 48 hpA, n = 20 larvae at 19, 43 and 72 hpA, and n = 10 larvae at 3 hpA; **** p < 0.0001, * p < 0.1 (Tukey's multiple comparisons test).