Figure 2.
csf1a, but not csf1b, and il34 drive the expansion of embryonic macrophages in vivo. (A-B) csf1a, csf1b, and il34 ligands were overexpressed by mRNA microinjection in 1-cell stage transgenic wild type (WT) embryos. Control embryos were injected with phosphate-buffered saline (PBS). Fluorescence images were acquired at 72 hpf and the area of fluorescent cells was calculated in the caudal hematopoietic tissue (CHT) (area inside of the red box) by FIJI. Results were normalized to injected controls. Scatter plots on the right represent quantification of fluorescent cells in CHT. Each dot in the scatter plot represents 1 larva. (A) Tg(fms:GAL4;UAS:mCherry); control n = 28, + csf1a n = 35, + csf1b n = 35, + il34 n = 28. (B) Tg(mpeg1:EGFP); control n = 41, + csf1a n = 45, + csf1b n = 37, + il34 n = 34. (C) WISH of 48 hpf embryos showing the expression of lcp1 in WT, 2 csf1ra mutants: csf1raV614M (panther) and csf1raΔ5bp, and in csf1ains2bp, csf1bΔ2bp ligand mutants. WT n = 15 (L = 1, M = 9, H = 5); csf1raV614M n = 15 (L = 4, M = 7, H = 4); csf1raΔ5bp n = 15 (L = 12, M = 2, H = 1); csf1ains2bp n = 14 (L = 7, M = 6, H = 1); csf1bΔ2bp n = 16 (L = 4, M = 9, H = 3). (D) lcp1 WISH of 36hpf WT embryos injected with PBS (control), and csf1ains2bp mutant embryos injected with PBS (control), csf1a mRNA or recombinant zebrafish Csf1a protein. WT, control n = 17 (L = 0, M = 14, H = 3); csf1ains2bp, control n = 45 (L = 32, M = 13, H = 0), + csf1a n = 32 (L = 15, M = 17, H = 0), + Csf1a n = 35 (L = 14, M = 21, H = 0). (C-D) Violin plot graphs show the level of lcp1 expression in the CHT region of individual embryos (L = low, M = medium, H = high) with median represented by a black line. (A-D) The level of statistical significance was determined by Mann-Whitney U test. *P < .04, **P < .007, ***P < .0001. All fluorescent images were acquired on Zeiss AxioZoom.V16 with Zeiss Axiocam 506 mono camera and ZEN Blue software. Bright field images of WISH were acquired on Zeiss AxioZoom.V16 with Zeiss Axiocam 105 color camera and processed using the Extended Depth of Focus module in the ZEN Blue software. FIJI and Adobe Photoshop were used for image processing.

csf1a, but not csf1b, and il34 drive the expansion of embryonic macrophages in vivo. (A-B) csf1a, csf1b, and il34 ligands were overexpressed by mRNA microinjection in 1-cell stage transgenic wild type (WT) embryos. Control embryos were injected with phosphate-buffered saline (PBS). Fluorescence images were acquired at 72 hpf and the area of fluorescent cells was calculated in the caudal hematopoietic tissue (CHT) (area inside of the red box) by FIJI. Results were normalized to injected controls. Scatter plots on the right represent quantification of fluorescent cells in CHT. Each dot in the scatter plot represents 1 larva. (A) Tg(fms:GAL4;UAS:mCherry); control n = 28, + csf1a n = 35, + csf1b n = 35, + il34 n = 28. (B) Tg(mpeg1:EGFP); control n = 41, + csf1a n = 45, + csf1b n = 37, + il34 n = 34. (C) WISH of 48 hpf embryos showing the expression of lcp1 in WT, 2 csf1ra mutants: csf1raV614M (panther) and csf1raΔ5bp, and in csf1ains2bp, csf1bΔ2bp ligand mutants. WT n = 15 (L = 1, M = 9, H = 5); csf1raV614M n = 15 (L = 4, M = 7, H = 4); csf1raΔ5bp n = 15 (L = 12, M = 2, H = 1); csf1ains2bp n = 14 (L = 7, M = 6, H = 1); csf1bΔ2bp n = 16 (L = 4, M = 9, H = 3). (D) lcp1 WISH of 36hpf WT embryos injected with PBS (control), and csf1ains2bp mutant embryos injected with PBS (control), csf1a mRNA or recombinant zebrafish Csf1a protein. WT, control n = 17 (L = 0, M = 14, H = 3); csf1ains2bp, control n = 45 (L = 32, M = 13, H = 0), + csf1a n = 32 (L = 15, M = 17, H = 0), + Csf1a n = 35 (L = 14, M = 21, H = 0). (C-D) Violin plot graphs show the level of lcp1 expression in the CHT region of individual embryos (L = low, M = medium, H = high) with median represented by a black line. (A-D) The level of statistical significance was determined by Mann-Whitney U test. *P < .04, **P < .007, ***P < .0001. All fluorescent images were acquired on Zeiss AxioZoom.V16 with Zeiss Axiocam 506 mono camera and ZEN Blue software. Bright field images of WISH were acquired on Zeiss AxioZoom.V16 with Zeiss Axiocam 105 color camera and processed using the Extended Depth of Focus module in the ZEN Blue software. FIJI and Adobe Photoshop were used for image processing.

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