Fig. 3

Analysis of fluorescent OPC and myelin reporter intensities. The finding that iGluSnFR-expressing zebrafish larvae shows a reduction in dorsal Olig2⁽⁺⁾ cells at 3 dpf (see Figure 2c) could be replicated and extended to ventral cells of the proximal region of the spinal cord by examination of global Olig2:dsRed fluorescence intensities. (a) Low-resolution epi-fluorescence microscopic images of proximal zebrafish spinal cords in lateral view. Olig2:dsRed and GFAP:iGluSnFR double transgenic fish at 3 dpf on the left and only Olig2:dsRed-expressing siblings are depicted on the right (red channel only). Scale bar: 100 μm. (b) Quantification of relative Olig2:dsRed fluorescence intensities as a parameter for cell numbers. Data were analyzed by an unpaired two-tailed t test. *** indicates a p-value < 0.001; t = 9.208. The dot plot graph shows the arithmetic mean ± standard deviation. N = 3, n in iGluSnFR⁽⁺⁾ = 43 (red dots), n in iGluSnFR⁽⁻⁾ = 45 (blue squares). (c) Expression of CldnK is decreased in iGluSnFR⁽⁺⁾ larvae at 5 dpf compared to controls. CldnK linked to red fluorescent expression in Tg(CldnK:mem-TdTomato) zfl is considerably high in control littermate larvae which do not express iGluSnFR (right) compared to Tg(CldnK:mem-TdTomato) Tg(GFAP:iGluSnFR) double transgenic larvae (left) at 5 dpf. (d) As a marker for myelination, the relative expression of ClaudinK:mem-TdTomato fluorescence intensities was measured and showed a considerable decrease in iGluSnFR⁽⁺⁾ (red dots) larvae versus littermate iGluSnFR⁽⁻⁾ controls (blue squares). The fluorescence level (pixel intensity) of the proximal spinal cord of each larva was quantified within the same area and same microscopy settings. Dot plot graphs show averaged values of pixel density areas (mean ± StDev) Data were analyzed by an unpaired two-tailed t test. p-Values are significant ***p < 0.001; t = 4.597; N = 2 independent experiments with a total of 56 larvae (n)