Additional file 1 of Species-specific blood–brain barrier permeability in amphibians

Additional file 1: Figure S1 Quantifications of the isolectin+ staining and the tracer retention in the Amphibian CNS.Dot-plot illustrating the proportion of isolectin covered area in the Xenopus, axolotland pre-and post-metamorphic Pleurodeles brain. Data are shown as mean ± SEM and statistical analysis is based on Kruskall-Wallis testand Dunn’s multiple comparisons test.Dot-plot depicting the Manders’ coefficients of all the analyzed tracers in Xenopus and axolotl. The data were color-coded according to the tracers. Each data point represents one animal. Data are shown as mean ± SEM and statistical analysis is based on simple linear regression. Abbreviations: n.s: not significant; Pre: pre-metamorphic; Post: post-metamorphic. Figure S2 Permeability analysis of the 70 kDa tracer using a long survival protocol.Scheme of the experimental design of the 60 min survival protocol.Images depicting the retention pattern of the 70 kDa tracer in the axolotl and the Xenopus hindbrain. Low retention areas are indicated with orange arrowheads. Insets indicate the area of the brain. All images are full z-projections of a confocal stack. Scale bars = 100 µm.Dot-plot analyzing the Misolectin coefficients of the 70 kDa tracer in the axolotl and Xenopus CNS after 20and 60mins survival. Each data point represents one animal. Data are shown as mean ± SEM. Abbreviations: Ax: axolotl; Xe: Xenopus. Figure S3 Details of the EM images from the axolotl and Xenopus brain.Macropinocytosis, plane spacing 80 nm; red asterisk: structure engulfing some EC lumen; white asterisk: macropinosome with flaky material.Micropinocytosis, plane spacing 40 nm; arrow: micropinocytosis in statu nascendi; white arrowhead: small vesicles.Extracellular vesicles containing flaky electron dense material in the Xenopus luminal space.Micropinosomes in Xenopus ECs. Plane spacing in C and D 35 nm. Scale bars in A, B = 500 nm; in C, D = 1 µm. Figure S4 Comparative analysis of the permeability threshold in the Xenopus and axolotl BBB.Dot-plots showing the Misolectin coefficients of all injected tracers following the 20 min survival protocol in Xenopusand axolotl. The data were color-coded according to the molecular size. Each data point represents one animal. Data are shown as mean ± SEM and statistical analysis is based on One-way ANOVA.Experimental workflow to analyze the 1 kDa leakage observed in the axolotl CNS.Confocal image of a transversal section of the axolotl telencephalon injected with 1 kDa tracer and labeled with DAPI.Images depicting the retention pattern of the 1 kDa tracer in Xenopus at stage 49and stage 66. Inset indicate the area of the brain. The images are full z-projections of a confocal stack. Scale bars in D, D’, D’’ = 50 µm; in E, E’, F, F’’ = 100 µm. Figure S5 Comparative analysis of the permeability threshold of the injected tracers in all analyzed species.Dot-plots depicting the Manders’ coefficients of the Xenopus, Axolotl and Pleurodeles BBB. In A, data was colored-coded by tracer, 1 kDa. The yellow area was further analyzed in B.The Manders’ coefficients of the 1 kDa injected Xenopus, axolotl, pre-and post-metamorphicPleurodeles were plotted. Each data point represents one animal.