Glial expression increases during zebrafish brain development with distinct lineages. (A) RT-PCR panel of different genes commonly expressed in glia, amplified in 0–7 dpf zebrafish cDNA samples. A water lane was used as a non-template control. (B,C) Among the amplicons from panel (A), olig2 and gfap label distinct populations at 1 dpf (B) and 4 dpf (C) of brain development. E, eye. Br, brain. Scale bar = 50 μm.

Zebrafish migratory olig2+ glia expand during CNS angiogenesis and co-opt the developing vascular network. (A–F) Daily live confocal images of the Tg(olig2:dsRed) line (red) crossed to the Tg(fli1a:eGFP)^y1 line (green) from 1 to 6 dpf. Red olig2+ glia move out from the ventral brain and migrate along green blood vessels during brain development. Scale bar = 50 μm. (G) A representative maximum intensity projection of a single olig2+ cell (red) sitting on a vessel (green) in the developing zebrafish forebrain (left panel). The orthogonal horizontal (XZ) and vertical (YZ) views (middle and right panel) from the crosshairs where the olig2+ cell sits on a vessel. The orthogonal views are displayed above their corresponding fluorescence line profiles whereby an arrow denotes an olig2+ -vessel interaction and an asterisk shows signal from a nearby vessel without an olig2+ cell. Scale bar = 10 μm. (H)olig2+-vessel interactions quantified from 3 to 6 dpf. A one-way ANOVA with Tukey’s Multiple comparisons test was performed. *p ≤ 0.05, **p ≤ 0.005, ****p ≤ 0.0001. Error bars represent standard error of the mean. n = 7–11 per group.

Wnt inhibition decreases olig2+ cell vessel migration and brain endothelial cell signaling. (A)Tg(olig2:dsRed;fli1a:eGFP) animals were treated for 72 h from 3 to 6 dpf with DMSO vehicle, the Wnt inhibitor, XAV939, or the Vegf inhibitor, AV951. olig2+ cell-vessel interactions were quantified after 72 h of each treatment. A one-way ANOVA with Tukey’s Multiple comparisons test was performed (****p < 0.0001). Error bars represent standard error of the mean. n = 7–8 animals per group. (B) Quantification of individual cells from animals treated in panel (A). A one-way ANOVA with Tukey’s Multiple comparisons test was performed (**p < 0.005). Error bars represent standard error of the mean. n = 6–7 animals per group. (C) Quantification of the total number of olig2+ cell-vessel interactions divided by total number of olig2+ cells per condition. A one-way ANOVA with Tukey’s Multiple comparisons test was performed (*p < 0.005). Error bars represent standard error of the mean. n = 10 animals per group. (D) Representative live confocal images of Tg(glut1b:mCherry) animals treated with either DMSO vehicle control or XAV939 from 3 to 6 dpf. The right panels are the zoomed in areas from the corresponding white boxes in the left panels. Scale bar = 20 μm. (E) Quantification of glut1b:mCherry fluorescence after 72 h of XAV939 treatment. A two-tailed, unpaired t-test was performed (***p < 0.001). Error bars represent standard error of the mean. n = 9–11 animals per group.

Zebrafish gfap+ glia outline the developing brain vasculature. (A) The front-facing view of a volumetric 3D reconstruction showing the interaction (white arrow) of a gfap cell process (green) with a brain vessel (red). (B) A lateral-view of the same image in panel (A). (C,D) White and black images of each channel in panel (A). (E,F) White and black images of each channel in panel (B). (G) A view of the image in panel (A) showing the interaction of the green fluorescent signal with the red fluorescent signal (white) at the crosshairs. (H) Orthogonal views and line profiles for the vertical (YZ) and horizontal (XZ) crosshairs intersecting (white arrow) where the gfap cell meets the vessel in (G). n = 5 animals, both hemispheres, 20 vessels total. Scale bar = 15 μm. (I,J) White and black images of each channel in panel (G).

Ablation of gfap glia impairs CNS angiogenesis via a reduction in Vegfa. (A) Live confocal images of 3 dpf animals in the following treatment groups; gfap:nfsB-mCherry+ treated with DMSO treated (left), gfap:nfsB-mCherry- treated with MTZ (middle), and gfap:nfsB-mCherry+ treated with MTZ (right). MTZ treatment in the transgene containing group reduces vessel outgrowth in the brain. n = 6–11 animals per group. (B) RT-PCR of Vegfa isoforms during 0–4 dpf development. (C) WMIHC of 1 dpf Tg(gfap:GFP) (green) stained for Vegfa protein (red). Vegfa is widely expressed during gfap cell development. E, eye. Br, brain. (D) Western blot analysis of Vegfa protein levels in the same treatment groups in (A). A two-way ANOVA with Tukey’s Multiple comparisons test was performed (***p < 0.0005). Error bars represent standard error of the mean. n = 5 biological replicates/groups per condition. (E) Volumetric analysis of vessels from non-transgenic control siblings treated with MTZ (left) show a reduction in vessel volume in transgenic animals treated with MTZ (right). A one-tailed, unpaired t-test was performed (*p < 0.05). Error bars represent standard error of the mean. n = 8–9 animals per group.

Mature human astrocytes contact developing zebrafish brain vessels. (A–E) Immunocytochemistry of GFP-expressing human astrocytes (green), with DAPI counterstained nuclei (blue) (A), the common marker GFAP (B), and mature markers ALDH1L1 (C), Kir4.1 (D), and GLT-1 (E) (red). Scale bar = 30 μm. (F) 48 h post-implantation, human astrocytes (green) reach out to the surrounding brain vasculature (red) of a 6 dpf Tg(kdrl:mCherry) zebrafish larvae. (G) Green channel highlighting the astrocyte’s morphology. (H) A zoomed in view from the white dotted box in panel (F) showing the astrocyte reaching out to a vessel. n = 5 animals. Scale bar = 10 μm. (I,J) Quantification of human glia-zebrafish vessel interactions. While there was a decrease in human glia present in the brain from 24 to 48 h post-intracranial injection (I), the amount of glial-vessel contacts did not subsequently decrease (J). n = 4 animals in (I) and n = 3–7 animals in (J). A paired, two-tailed t-test was performed (*p < 0.05) in (I) and an unpaired, two-tailed t-test was performed in (J).