Figure 7.

Figure 7.

(A) Handmade schematic of CSF distribution in a 30 hpf zebrafish embryo with CSF colored in red. A path ventral to the DV/MV and the RV lets CSF flow and merges into the CC. Dorsal to the CC, a path that is continuous to the RV merges with the CC in the filum terminale. (B1, B2) 2-photon imaging of TexasRed injected in the DV/MV of 30 hpf embryos shows the ventral opening of the primitive lumen of DV/MV and RV in lateral (B1) and coronal (B2) views. The path ventral to RV merges into the CC and has a smaller diameter. (B3, B4) two photon imaging of tight junction (ZO-1) and nuclei (DAPI) labeling of fixed 30 hpf embryos in lateral (B3) and coronal (B4) views highlights the complex circulation system of CSF in primitive lumens. (C1) Experimental measurement of the ventral opening of the RV lumen relative to its arc length. The diameter sharply increases at the beginning of the CC (black dashed line). (C2) Histogram of the ventral opening of the RV lumen showing diameter and length measured in 11 WT 30 hpf embryos (diameter of 7.1 ± 0.7 μm, ratio relative to CC diameter of 0.55 ± 0.12, as discussed in Methods, and 233 ± 24 μm long median ± 0.5 s.d.). (D1) Inverse contrast kymograph of 20 nm beads flowing in the path ventral to RV lumen after injection in the DV/MV. (D2) CSF flow profile inside the path ventral to RV, captured with our automatic kymograph analysis. (E1, E2) 2-photon imaging of TexasRed injected in the DV/MV of a 30 hpf embryo showing the dorsal opening of the CC primitive lumen as it leaves the RV (E1) and as it connects to the main opening of the CC lumen in the filum terminale (E2). The dorsal opening of the CC primitive lumen in its rostral part is also shown in coronal view (E3) and in a fixed 30 hpf embryo after a ZO-1 staining (E4). (F1, F2, F3) 2-photon imaging of TexasRed injected in the DV/MV of a three dpf larva showing the CC lumen in rostral (F1), central (F2), and caudal positions (F3). (F4) 2-photon imaging of TexasRed injected in the DV/MV of a five dpf larva indicates that the caudal end of the RV is at this stage directly connected to the single opening of the CC. Scale bar is 100 μm in (B1), 50 μm in (B3, E1, E2), 30 μm in (B2, B4, F1, F2, F3, G), and 15 μm in (D1, E3, E4). The vertical scale bar in (D1) is 3 s.