Extended Data Fig. 6: Ependymal cell activation is tightly correlated to the PMT wave and can be induced by the neuropeptide melanin concentrating hormone.
a, Dorsal view of a single slice through the optic tectum of Et(EP:mCherry) fish. Inset dashed box indicates location along the anteroposterior axis. Scale bar, 50 μm. b, Slice through the optic tectum of a 7-dpf Et(EP:mCherry);Tg(α-tubulin:gal4;UAS:GFP) triple transgenic fish. Arrows, mature ependymal cells (magenta) co-expressing α-tubulin (green). c, Slice through the optic tectum of a 7-dpf Et(EP:mCherry);Tg(GFAP:gal4;UAS:GFP) triple transgenic fish. Mature ependymal cells largely do not overlap with GFAP-positive cells. d–g, Ventricular cells activated before the PMT wave are EP:mCherry-positive ependymal cells. Confocal z-slice through a 7-dpf zPSG;EP:mCherry double transgenic fish before stimulation (d), ependymal cell activation (e) and PMT wave (f). g, Mean ± s.e.m. ΔF/F trace of five randomly selected double-positive cells showing the transient activation of ependymal cells before the PMT wave. Arrows indicate the time from which images shown in d–f are taken. Scale bar, 50 μm. h–m, MCH peptide induces activation of ependymal cells. Representative maximum projection through SPIM volume acquisition before (h, j) and 5 min after intracerebroventricular injection with carrier (i) or MCH peptide (k). l, Mean normalized fluorescence between carrier (n = 9 fish) and MCH (n = 12 fish) groups from the peritectoventricular zone (dashed lines) before and after injection. m, Box plot of normalized fluorescence percentage change values between carrier- and MCH-injected groups with black circle depicting mean. *P < 0.05; two-sided Student’s t-test. Scale bar, 100 μm. n, Pigment contraction is tightly coupled to discrete activations during PMT-wave dynamics. Representative frames selected from a single z-recording of a pigmented zebrafish during a PMT wave; (1) colour-coded selection of all pigments covering the optic tectum as a representative sample; (2) during darkness and baseline activity, pigments are spread out and cover more pixel area; (3, 4) pigments contract with ependymal cell activation and the PMT wave; (5) when the wave abates, pigments re-spread. Scale bar, 100 μm. o, Quantification of pixel area plotted with representative colour from subpanel (1) in n at the different time points shown in subpanels (1) to (5) in n. *P < 0.05, repeated-measures ANOVA, n = 10 pigments. See Supplementary Information for pigment dynamics description and reproducibility information.
