Fig 2: CP macrophage distribution: Epithelium is labeled with Notch2, macrophages with Iba1. (A) LHS. Arrows point to epiplexus cells. Scale bar: 400 µm. (B) RSS: an epiplexus cell is seen at the contact point with brainstem. Scale bar: 100 µm.

Fig 3: Microvascular network and frond structure. (A) SCAN within a single villus in the loose part of LHS of a female rat. The anastomotic pattern of the small capillary network (arrow) is evident. Collagen IV labeling. (B) Same section as in (A), additionally labeled for CP epithelium (Notch2, cyan) and arterioles (SMA, green). One flat villus is en face in this section (asterisk), and its whole lateral surface is visible. The distribution of large capillaries at the periphery and small capillary (SCAN) in the center is evident. This SCAN attaches to its parent arteriole following a loose spiral course (see Supplementary Video 6). Scale bar: 200 µm. (C) Terminal region of corkscrew-like villi from the loose part of CSS. Arterioles are seen in the middle, associated with spiraling capillaries. Asterisks mark the direction of the parent artery. Green, SMA; red, ColIV; cyan, Notch-2. Scale bar: 100 µm. (D) Single optical slice with a magnification of a portion of the top villus in (C), showing that the arteriole is associated with capillaries running within the same villus. Scale bar: 50 µm. Green, SMA; red, ColIV; blue, Notch-2. (E) Z-stack maximal projection from 50 optical sections (3 µm steps) displaying a SCAN associated with its arteriole and LTC. Scale bar: 100 µm. (F) Three-dimensional reconstruction of the LHS surface of a female rat facing the DCN. Top: Notch-2 (cyan) + ColIV (red) + SMA (green) labeling. Bottom: colIV labeling (red). Notice the passage from leaflike villi (asterisk) to digitiform villi (arrow) to flat surface (arrowhead) passing from the caudal to the rostral part of LHS. Scale bar: 500 µm. (G) Schema of hCP vascular network. Arteries (ART) divide into arterioles, which give rise to SCANs (magenta) through divergent connections. At their other end, SCANs contact LTCs (green) which course back to reach veins (VE). Since LTCs are very long, a single capillary may fill an entire frond, and SCANs are attached via multiple outlets to a single LTC. Probably due to growth with spatial constraints, both SCANs and LTCs may display a tortuous pathway; differential growth of SCANs and LTCs (and possibly pre-SCAN vessels) may produce all microvascular patterns observed.

Fig 4: Overall shape of the hindbrain CP. (A) Schema of the hindbrain CP with relative hindbrain surrounding areas. RSS, rostral sagittal segment; CSS, caudal sagittal segment; MHS, medial horizontal segment; LHS, lateral horizontal segment. (B) Overall CP shape obtained from segmentation of the Notch2 signal Z-stack maximal projection of the light-sheet 3D dataset of a female rat cleared brain. Notice the presence of an incisure between MHS and LHS (asterisks). Scale bar: 1 mm.