Figure 2-S1

Figure 2-S1

Wild-type double transgenic zebrafish at four dpf were microinjected with fluorescently tagged LPS in the tectal brain. To localize the LPS relative to the lymphatic and vasculature structures, two transgenes were used: mrc1a:GFP (Jung et al., 2017) and kdrl:mCherry (Fujita et al., 2011), respectively. Since mrc1a:GFP is known to also be expressed by macrophages (Jung et al., 2017), we generated a new macrophage reporter (mpeg1:BFP) to provide a third channel in blue to distinguish lymphatic from macrophage expressions. Blue arrows point to microglia/macrophage. (a) Three multi-tiled representative frontal plane optical slices from an in vivo confocal z-stack at 1.5 hpi show top, middle, and bottom sections through the head and anterior trunk of the LPS-injected animal. (b) Schematic of the frontal plane optical sections imaged. (c, d) High magnification images corresponding to dotted region in top slice in a). Most superficial layer of the CNS where LPS (red) is concentrated in the hindbrain ventricle and adjacent interstitial parenchymal space (c, asterisk) as well as the most-dorsal junction of the hindbrain-spinal cord (d, asterisk). (e, f) High magnification images of dotted region in middle slice shown in a). LPS is found localized to the lymphatic structures (blec and islv), and abundant in the interstitial space extending from the hindbrain ventricle (asterisk). (g,h) High magnification images corresponding to bottom slice in a). LPS is highly abundant in the spinal canal (sc) as well as tectal and hindbrain lymphatic cells (blec). (i) Top and middle slices of a different LPS-injected animal show the same distribution of LPS at 1.5 hpi. LPS does not appear to localize within the blood vasculature in the CNS or trunk to the same extent as it overlaps with the lymphatic cells. Top slice shows vasculature DLAV surrounded by but not overlap with LPS, arrows. *, interstitial space; DLAV, dorsal longitudinal anastomotic vessels; DLLV, dorsal longitudinal lymphatic vessel; blec, brain lymphatic endothelial cells; islv, intersomitic lymphatic vessels; sc, spinal canal; hv, hindbrain ventricle; hb, hindbrain; lym, lymphatics. Skin is highly auto-fluorescent and labeled by all channels at the border of every tissue. Scale bars all show 50 µm.