Transcription factor analysis of zebrafish macrophages via ATAC-seq and RNA-seq. (A) Motif enrichment analysis in zebrafish macrophages at 3 days post fertilization (dpf). The analysis reveals a strong enrichment of Ets family motifs, with 9 of the top 10 motifs belonging to the Ets family. (B) Expression levels of Ets family members in zebrafish macrophages, as determined via RNA-seq. Bar plots represent the mean ± standard error of the mean (S.E.M.).

Morpholino knockdown-mediated screening of Ets family members in zebrafish macrophage development. (A) Representative confocal images showing macrophages in the head region of Tg(mpeg1:eGFP) zebrafish larvae at 3 dpf following morpholino-mediated knockdown of Ets family members. (B) Quantification of macrophage numbers in the head regions of the transgenic zebrafish shown in (A). Data were analyzed using one-way ANOVA followed by Dunnett’s test, with n ≥ 20, and shown as mean ± S.E.M. (C) Representative confocal images showing macrophages in caudal hematopoietic tissue (CHT) at 3 dpf following morpholino-mediated knockdown of Ets family members. (D) Quantification of macrophage numbers in the CHT of the transgenic zebrafish shown in (C). Data were analyzed using one-way ANOVA followed by Dunnett’s test, with n ≥ 15, and shown as mean ± S.E.M. Statistical significance is indicated as ns (p ≥ 0.05), **** p < 0.0001. Scale bar: 50 μm.

Reduction in macrophages in elf1 crispants. (A) Representative confocal images showing macrophages in the head region in transgenic zebrafish at 3 dpf following CRISPR-mediated mosaic mutants. Scale bar: 50 μm. (B) Quantification of macrophage numbers within the brain regions of the transgenic zebrafish shown in (A). Unpaired, two-tailed Student’s t-test, with n ≥ 20, and results are shown as mean ± S.E.M. (C) Representative confocal images of macrophages in whole zebrafish larvae at 1, 2, and 3 dpf. Scale bar: 200 μm; imaging depth approximately 200 μm. (D–F) Quantification of macrophage numbers within specific regions of the transgenic zebrafish shown in (C), including the posterior blood island (PBI) and caudal hematopoietic tissue (CHT). Unpaired, two-tailed Student’s t-test for n ≥ 12, 18, and 9, respectively; results are shown as mean ± S.E.M. Statistical significance indicated as ** p < 0.01 and **** p < 0.0001.

Macrophage behavior following tail amputation in elf1 crispants. (A) Time-lapse images showing macrophage infiltration in controls and elf1 crispants at 3 dpf. Imaging of the caudal fin region was initiated two hours post-amputation. The white dashed line marks the position of transection. Scale bar: 50 μm. (B) Quantification of macrophage numbers within the damage regions shown in (A). Data were analyzed using two-way ANOVA followed by Sidak’s tests, with n ≥ 7, and shown as mean ± S.E.M. Statistical significance indicated as * p < 0.05 and ** p < 0.01.

Overexpression of a dominant-negative form of Elf1 (DN-Elf1) in macrophages disrupts their response to injury. (A) Time-lapse imaging of macrophage infiltration at 3 dpf in control zebrafish and zebrafish expressing DN-Elf1 specifically in macrophages. The background transgenic line used was Tg(mpeg1:Gal4FF/UAS:eGFPcaax). Imaging of the caudal fin region was initiated two hours post-amputation. The white dashed line marks the position of transection. Scale bar: 50 μm. (B) Quantification of macrophage numbers within the damage shown in (B). Data were analyzed using two-way ANOVA followed by Sidak’s tests, with n ≥ 7, and shown as mean ± S.E.M. Statistical significance indicated as ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Macrophage behavior following tail amputation in cxcr4b crispants. (A) Expression levels of cxcr4b in zebrafish macrophages, as determined via RNA-seq. Bar plots represent the mean ± S.E.M. (B) Relative expression of cxcr4b in macrophages of elf1 crispants compared to the control group at 3 dpf. Unpaired, two-tailed Student’s t-test results for n = 4 are shown as mean ± S.E.M. Statistical significance indicated as **** p < 0.0001. (C) Time-lapse images showing macrophage infiltration in controls and cxcr4b crispants at 3 dpf. Imaging of the caudal fin region was initiated two hours post-amputation. The white dashed line marks the position of transection. Scale bar: 50 μm. (D) Quantification of macrophage numbers within the damage regions shown in (C). Data were analyzed using two-way ANOVA followed by Sidak’s tests, with n ≥ 10, and shown as mean ± S.E.M. Statistical significance indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Ectopic expression of cxcr4b partially rescues macrophage function in elf1 crispants. (A) Time-lapse images of macrophage infiltration in controls and cxcr4b ectopic expression in elf1 crispant zebrafish larvae at 3 dpf. Imaging of the caudal fin region was initiated two hours post-amputation. The white dashed line marks the position of transection. Scale bar: 50 μm. (B) Quantification of macrophage numbers within the damage regions shown in (A). Data were analyzed using two-way ANOVA followed by Sidak’s tests, with n ≥ 18, and shown as mean ± S.E.M. Statistical significance indicated as ** p < 0.01, *** p < 0.001, and **** p < 0.0001.