Dox-induced expression of Il-1? in the CNS promotes dose-dependent mortality and neuroinflammation in the transgenic CNS/Il-1? model (A) Design of DNA constructs used to make the zebrafish transgenic lines Tg(gfap:rtTA, cmlc2:EGFP) and Tg(TRE:GSP-il1bmat, cmlc2:mCherry). The combination of these transgenic lines is designated as ?CNS/Il-1?? to indicate doxycycline (Dox) inducible expression of Il-1? in the CNS. (B) Experimental timeline for all experiments. This graphic represents the timing of Dox induction and in vivo imaging. Dox (0?10 ?g/mL) was added at approximately 6 hpf for all experiments. Survival was monitored daily through 7 dpf. Confocal imaging was performed at 4 dpf for neuroinflammation (Figures 1D and 1F) and at 2 dpf for imaging brain vasculature (Figures 2, 3, 4, 5, and 6). (C) Kaplan-Meier analysis monitoring survival probability. CNS/Il-1? embryos were treated with Dox at 0, 1, 2.5, 5, or 10 ?g/mL at 6 hpf, then monitored for survival every day until 7 dpf (?p < 0.05; ??p < 0.01; ???p < 0.001; not all comparisons are shown). (D) Representative confocal microscopy images showing neutrophils (mpx:EGFP) and blood vessels (kdrl:mCherry) in the head. CNS/Il-1? embryos were untreated (No Dox) or treated (Dox; 1.0 ?g/mL) at 6 hpf and then imaged at 4 dpf (dorsal view; anterior left). Scale bar is 100 ?m. (E) Quantification of neutrophils (mpx:EGFP) in the heads of untreated (No Dox) or treated (Dox; 1.0 ?g/mL) CNS/Il-1? larvae at 4 dpf (n=4). (F) Representative confocal microscopy images showing microglia/macrophages (mpeg1:mCherry) and blood vessels (kdrl:EGFP) in the head for context. CNS/Il-1? embryos at 6 hpf were either untreated (No Dox) or treated (Dox; 1.0 ?g/mL), then imaged at 4 dpf (dorsal view; anterior left). Scale bar is 100 ?m. (G) Quantification of microglia/macrophages (mpeg1:mCherry) in the heads of untreated (No Dox) or treated (Dox; 1.0 ?g/mL) CNS/Il-1? larvae at 4 dpf (n=5). Error bars in E and G represent means ± SEM (?p < 0.05; ???p < 0.001).

Il-1? expression disrupts CNS angiogenesis in the transgenic CNS/Il-1? model (A) Representative confocal microscopy images showing dose-dependent effects of Dox on brain vascular development. CNS/Il-1?, kdrl:EGFP embryos were untreated (No Dox) or treated with Dox (0.1, 1.0, or 10.0 ?g/mL) at 6 hpf and then imaged at 52 hpf (dorsal view; anterior left). Scale bars are 100 ?m. (B) Quantification of the number of CtAs in CNS/Il-1? embryos treated with Dox (0, 0.1, 1.0, or 10.0 ?g/mL) (n = 5). (C) Representative confocal microscopy images showing whole embryo vasculature in CNS/Il-1?, kdrl:EGFP embryos at 2 dpf (lateral view; dorsal top; anterior left). CNS/Il-1? embryos were either untreated (No Dox) or treated (Dox; 10.0 ?g/mL) at 6 hpf. Note the loss of CNS vasculature with Dox treatment but no effect on trunk vasculature with Dox treatment. Scale bar is 100 ?m. (D) Quantification of the number of intersegmental vessels (ISVs) at 52 hpf in CNS/Il-1? embryos either untreated (No Dox) or treated (10.0 ?g/mL Dox) (n = 4). Error bars in B and D represent means ± SEM (??p < 0.01; ???p < 0.001; no label = not significant).

CRISPR/Cas9 il1r1 mutants rescue Il-1?-induced mortality and CNS angiogenesis (A) Schematic of the zebrafish il1r1 gene showing the locations targeted by the CRISPR/Cas9 RNP complexes (cr1 and cr2) (top) and the resulting deletion of intron 8 (bottom). The exon and intron sizes in base pairs (bp) and the forward and reverse primers (F and R) used for genotyping are shown (not to scale). (B) PCR showing the genotyping of CRISPR/Cas9 deletion of il1r1. CNS/Il-1? embryos from heterozygous il1r1+/? adults were treated with Dox (10.0 ?g/mL), selected as sick or healthy, and then genotyped by PCR. Note that all healthy embryos have the il1r1?/? deletion and all sick embryos carry a wild-type il1r1 allele. (C) Kaplan-Meier analysis monitoring survival probability of il1r1 mutants. CNS/Il-1? embryos either il1r1+/+ or il1r1?/? were untreated (No Dox) or treated (Dox; 10.0 ?g/mL) at 6 hpf and then monitored for survival every day until 7 dpf (???p < 0.001; not all comparisons are shown). (D) Representative confocal microscopy images showing rescue of CNS angiogenesis in Dox-induced il1r1 deletion mutants. CNS/Il-1?, kdrl:EGFP embryos either il1r1+/+ or il1r1?/? were untreated (No Dox) or treated (Dox 10 ?g/mL) at 6 hpf and then imaged at 54 hpf (dorsal view; anterior left). Scale bars are 100 ?m. (E) Quantification of the number of CtAs in wild-type il1r1 (+/+) and mutant il1r1 (?/?) embryos either treated with no Dox (?) or treated with 10.0 ?g/mL Dox (+). Error bars represent means ± SEM (?p < 0.05; ??p < 0.01; ???p < 0.001; no label = not significant). (F) Still frames from time-lapse confocal imaging of CNS/Il-1?, kdrl:EGFP embryos either il1r1+/+ or il1r1?/? without (No Dox) or with (10.0 ?g/mL Dox) treatment (lateral view; dorsal top; anterior left). Shown here are snapshots at 4-h intervals over 12 h of acquisition beginning at the onset of CNS angiogenesis (30 hpf). Stereotypical tip cells are indicated at 34 hpf (yellow arrowheads). See Videos S1, S2, S3, and S4 for more detail.

Il-1? disrupts glut1b:mCherry expression in brain endothelial cells during CNS angiogenesis (A) Representative confocal microscopy images showing Dox-dependent effects on glut1b:mCherry expression. CNS/Il-1?, kdrl:EGFP, glut1b:mCherry embryos were untreated (No Dox) or treated with Dox (0.1, 1.0, or 10.0 ?g/mL) at 6 hpf and then imaged at 52 hpf (dorsal view; anterior left). Scale bars are 100 ?m. (B) Representative confocal microscopy images showing control kdrl:EGFP, glut1b:mCherry embryos (no CNS/Il-1?) at 2 dpf. Embryos were either untreated (No Dox) or treated with 10.0 ?g/mL (Dox) at 6 hpf. Scale bars are 100 ?m. (C) Quantification of the average glut1b:mCherry fluorescence intensity in the hindbrain vasculature of CNS/Il-1?, kdrl:EGFP, glut1b:mCherry embryos at 2 dpf (n = 5). Note the dose-dependent decrease in glut1b:mCherry signal indicating a defect in barriergenesis. (D and E) Quantification of the number of CtAs (D) and average glut1b:mCherry fluorescence intensity (E) in the hindbrain vasculature of control kdrl:EGFP, glut1b:mCherry embryos (no CNS/Il-1?) without (No Dox) or with (10.0 ?g/mL Dox) treatment (n = 5). Note that Dox alone has no impact on barriergenesis. Error bars in C, D, and E represent means ± SEM (?p < 0.05; ??p < 0.01; ???p < 0.001; ns or no label = not significant).

il1r1 crispants rescue glut1b:mCherry expression in brain endothelial cells during CNS angiogenesis (A) Representative confocal microscopy images showing rescue of glut1b:mCherry expression in il1r1 crispants. CNS/Il-1?, kdrl:EGFP, glut1b:mCherry embryos were injected with CRISPR/Cas9 RNP complexes (cr1 and cr2) at the one-cell stage. Control embryos and il1r1 crispants were either untreated (No Dox) or treated (10.0 ?g/mL Dox) at 6 hpf and then imaged at 52 hpf (dorsal view; anterior left). Scale bars are 100 ?m. (B and C) Quantification of the number of CtAs (B) and average glut1b:mCherry fluorescence intensity (C) in the hindbrain vasculature of control (CRISPR ?) and il1r1 crispants (CRISPR +) either untreated (Dox ?) or treated with 10.0 ?g/mL Dox (Dox +) (n = 5 for each condition). Error bars in B and C represent means ± SEM (?p < 0.05; ??p < 0.01; ???p < 0.001; no label = not significant).

Il-1? reduces Wnt/?-catenin transcriptional activation in brain endothelial cells (A) Representative confocal microscopy images showing Wnt/?-catenin transcriptional activity. CNS/Il-1?, kdrl:EGFP, TCF:mCherry embryos were untreated (No Dox) or treated with Dox (0.1, 1.0, or 10.0 ?g/mL) at 6 hpf and then imaged at 52 hpf (dorsal view; anterior left). Scale bars are 100 ?m. (B) Quantification of TCF:mCherry-positive endothelial cells in the hindbrain vasculature (n = 7). Values are the number of TCF:mCherry-positive cells in kdrl:EGFP-positive blood vessels divided by the length of the hindbrain vasculature in each embryo. All values were normalized to the average of the No Dox group. (C) Representative confocal microscopy images showing control kdrl:EGFP, TCF:mCherry embryos (no CNS/Il-1?) at 2 dpf (dorsal view; anterior left). Embryos were either untreated (No Dox) or treated with 10.0 ?g/mL (Dox) at 6 hpf. (D) Quantification of TCF:mCherry-positive endothelial cells in the hindbrain vasculature (n = 7). Values are the number of TCF:mCherry-positive cells in kdrl:EGFP-positive blood vessels divided by the length of the hindbrain vasculature in each embryo. All values were normalized to the average of the No Dox group. Scale bars for A and C are 100 ?m. Error bars in B and D represent means ± SEM (?p < 0.05; ??p < 0.01; ???p < 0.001; ns or not labeled = not significant).