The zebrafish ChP show evolutionarily conserved tissue characteristics (A) Diagram of the fChP. Lateral (top) and dorsal (bottom) views. (B) Gt(foxj1b:GFP) labels fChP epithelial cells. Max projection (B1) and single plane (B2) of the fChP showing the folded epithelium (left). Schemes representing the location of (B1)–(E3) (right). (B3) fChP injected with 70 kDa rhodamine B isothiocyanate (RITC)-dextran (magenta) in CSF. (B4) fChP labeled by acetylated-tubulin antibody marking cilia (white arrows) and axons (yellow arrows). (B5 and B6) fChP labeled with 4C4 antibody (arrows: macrophage on basal [yellow] or apical [white] side). (C) Number of 4C4-positive cells in the apical and basal sides of the fChP. (D) Tg(kdrl:GFP) labels endothelial cells in fChP. Max projection (D1) and single plane (D2–D4) of the fChP immunolabeled with 4C4 (arrows: contact with endothelial cells) (D3) and SV2 antibodies (arrows: contact with endothelial [white] or epithelial [yellow] cells) (D4). (E) Hybridization chain reaction (HCR) of fChP for clu (yellow, ChP marker) and plvapa (magenta). Max projection. (E1–E3) Merged and individual channels. (F) Diagram of the hChP. Lateral (top) and dorsal (bottom) views. (G) Gt(foxj1b:GFP) labels hChP. Max projection (G1) and single plane (G2) of the hChP showing the sheet-like epithelium (left). Schemes representing the location of (G1)–(J3) (right). (G3–G5) hChP labeled with 4C4 antibody (arrows: macrophage on basal [yellow] or apical [white] side). (G6 and G7) hChP labeled with acetylated-tubulin antibody (yellow arrows: axons, white arrows: cilia). (H) Number of 4C4-positive cells in the apical and basal sides of the hChP. (I) Tg(kdrl:GFP)-expressing endothelial cells in hChP. Max projection (I1 and I3) and single plane (I2 and I4) of the hChP (white arrow: endothelial cells at the basal side) immunolabeled with acetylated-tubulin (arrows: contact with endothelial [white] or epithelial [yellow] cells) (I3) and SV2 antibodies (arrows: contact with endothelial [white] or epithelial [yellow] cells) (I4). (J) HCR of the hChP with clu (yellow) and plvapa (magenta). (J1–J3) Merged and individual channels. (K) Diagram of the fChP used for transmission electron microscopy. (L) Images of the fChP at low (L1 and L2) and high (L1a and L2a) magnification. (L3–L5) Images of junctions (L3), basal membrane (L4), and motile cilia and microvilli (L5). DAPI labels nuclei. Yellow dotted lines mark either the ChP or ventricles. A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left; Hb, habenula; Tel, telencephalon. Scale bars: (A1–J3) 50 μm, (L1–L2a) 1 μm, and (L3–L5) 200 nm. Wilcoxon signed-rank test (C) (n = 7) (H) (n = 5). ∗p < 0.05. See also Figures S1–S4.

Expression of transporter genes is evolutionarily conserved across vertebrates (A) Weighted Venn diagram showing protein-coding genes expressed in the ChP that have an ortholog in human, rat, mouse, and zebrafish. (B) Weighted Venn diagram showing transporter genes expressed in the ChP that have an ortholog in all four species. (C) Expression (rank and TPM in parentheses) of transporter genes involved in CSF secretion. Darker colors indicate higher ranks. TPMs of duplicated genes were summed into a single value. (D and E) HCR of the fChP for igfbp2a (yellow, ChP marker) (D2 and E2), aqp1a.1 (magenta) (D3), slc12a2 (cyan) (D4), and atp1a1b (magenta) (E3). Single-plane images. (D1 and E1) Merged view. (D2–D4, E2, and E3) Individual channels. (F) Staining of the fChP in Gt(foxj1b:GFP) for ATP1A1. Single plane. (F1–F3) Merged and individual channels. Yellow dotted lines mark the dien-/mesencephalic ventricle (D/MV). DAPI labels nuclei. Scale bars: (D–F) 50 μm. See also Figure S5.

Fabp7b-expressing cells secrete proteins into the CSF and brain ventricles (A) fabp7b RNA expression in the larval brain. (B) Schematic of the Gal4 integration in Gt(fabp7b:2A-Gal4vp16). (C) Dorsal view of Gt(fabp7b:2A-Gal4vp16);Tg(uas:NTR-mCherry);Tg(elavl3:GCaMP6s). (D) Diagrams of the brain and ventricles at 17 dpf. The dotted boxes indicate the location of the images of ventricles in (E2)–(E5). (E) Images of Gt(fabp7b:2A-Gal4vp16);Tg(uas:NTR-mCherry);Tg(uas:starmaker-GFP). Dotted lines mark the brain and eyes. (E1) Reconstructed 3D image. (E2–E5) Dorsal and sagittal views of the anterior and posterior parts of the brain as indicated in (D). TV, telencephalic ventricle; D/MV, dien-/mesencephalic ventricle; RV, rhombencephalic ventricle. A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left. Scale bars: 50 μm. See also Figures S6, S7, and S10.

Fabp7b-expressing cells are required for the maintenance of the ventricular volume (A) Experimental scheme used for measuring ventricular size and blood-CSF barrier integrity after ablation. (B) Diagram of the juvenile brain with insets indicating the location of (C1)–(C6). (C) Max projection images of the TC, fChP, and hChP in control (C1, C2, C4, and C5) and ablated fish (C3 and C6). (D) Quantification of the TC, fChP, and hChP areas in control (DMSO and CtrlMTZ) and ablated fish (MTZ). n =10. The TC and ChP sizes are shown as normalized GFP+ ratio (mean ± standard deviation). p values from post hoc analysis: TC area: DMSO vs. MTZ, ∗∗p = 0.0055; CtrlMTZ vs. MTZ, ∗∗∗p = 0.0005. fChP area: DMSO vs. MTZ, ∗∗∗p = 0.0002; CtrlMTZ vs. MTZ, ∗∗∗p = 0.0007. hChP area: DMSO vs. MTZ, ∗∗p = 0.0012; CtrlMTZ vs. MTZ, ∗∗∗p = 0.0001. (E) Diagrams of the juvenile brain (top) and ventricles (bottom). Projections of 6 horizontal slices in (F1)–(F3), 5 sagittal slices in (F4)–(F6), and 5 sagittal slices in (F7)–(F9). (F) Horizontal (F1–F3) and sagittal (F4–F6 and F7–F9) views of the ventricles following 10 kDa Alexa 647 injection in control (F1, F2, F4, F5, F7, and F8) and ablated fish (F3, F6, and F9). Ventricles are highlighted with dotted yellow lines. (G) Quantification of the anterior (ant.) TV, posterior (post.) TV, and ant. D/MV and RV areas in the control (DMSO and CtrlMTZ) and ablated fish (MTZ). n = 10. p values from post hoc analysis: ant. TV area: DMSO vs. MTZ, ∗∗p = 0.0077; CtrlMTZ vs. MTZ, ∗∗∗p = 0.0008. post. TV and ant. D/MV areas: DMSO vs. MTZ, ∗∗∗p = 0.0009; CtrlMTZ vs. MTZ, ∗∗∗p = 0.0003. RV area: DMSO vs. MTZ, ∗p = 0.0124; CtrlMTZ vs. MTZ, ∗∗p = 0.0015. (H) Diagrams of the brains in the control and ablated fish. Kruskal-Wallis test. The p values on the graph are the results from Kruskal-Wallis test. ∗∗p < 0.01, ∗∗∗p < 0.001. TV, telencephalic ventricle; D/MV, dien-/mesencephalic ventricle; RV, rhombencephalic ventricle. A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left. Scale bars: 50 μm. See also Figures S8–S10.

The brain barrier is maintained in the ablated zebrafish (A) Representative images showing how fluorescent intensities of blood plasma, brain parenchyma, and ventricle were measured in the rhombencephalon in control (A1) and ablated fish (A2). (B) Fluorescent intensities in the ventricles in control (DMSO, CtrlMTZ) and ablated fish (MTZ). Statistical analysis: n = 10 except for D/MV ablated group, where n = 4 due to the collapse of the ventricle. Kruskal-Wallis test, p > 0.05. (C) Diagrams showing the location of (A)–(F). (D) Cldn5 immunostainings of TC, fChP, and hChP in control (D1, D2, D4, and D5) and ablated fish (D3 and D6). Max projections. n > 4. (E and F) Transmission electron microscope images of the fChP in control (E) and ablated fish (F). (E1 and F1) Low- (E2–E4 and F2–F6) and high-magnification (E2, F2, and F6) images of junctions, microvilli (E3 and F3), basal membranes (E5 and F5), and motile cilia (E4 and F4). TV, telencephalic ventricle; D/MV, dien-/mesencephalic ventricle; RV, rhombencephalic ventricle; A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left. Scale bars: (D) 50 μm, (E1, E3, and F1) 1 μm, and (E2–E5 and F2–F6) 200 nm. See also Figures S11 and S12.