(A) Effect of IOP on Meckel´s cartilage morphology. Left: Representative image of Alcian blue staining of 7 dpf control zebrafish (left) and IOP-treated (right) larvae. Scale bar 100 μm. Right: Graph depicting the quantification of Meckel’s cartilage (n = 3 larvae per group). The difference is best visible in the top middle where the stained area is thinner and more faint in the IOP animal. Scale bar 100 μm. Statistical analysis was performed with a Student’s t test, *p<0.01 (B) Changes in expression of TH-regulated genes in zebrafish larvae exposed to IOP. mRNA quantification of dio2, dio3, tshb, ttr and gh in controls and larvae exposed to IOP 0.5 μM or T3 10 nM for 3 dpf and to IOP 0.5 μM for 7 dpf. Data are represented as individual values in vertical graphs (n = 5 pools from independent experiments of 50–60 larvae per pool and condition). Graphs are showing individual values and mean ± SEM. Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the corresponding control groups. Significant differences are indicated as *p <0.05, **p <0.01, and ***p <0.001.

(A) Side view painting of 3 dpf zebrafish larva showing D + DT + TeO; CeP and SCT location; (B) Schematic drawing of the brain region (forebrain, midbrain and hindbrain) with identification of myelinated longitudinal axons in a violet color. (C) Representative photomicrograph of zebrafish larvae with lateral view and R-C orientation, whole-mount Black-Gold II staining in D + DT + TeO; CeP and SCT at 3 dpf (Unstained CTRL, Solvent Ctrl/BG-II, IOP/BG-II, T3/BG-II and IOP + T3/BGII). In each corner, the amplification of sagittal sections identifying the selected area for analysis and the positive myelin pixels, recognized by the IPP6 program; scale bar: 500 μm and 50 μm. (D) Quantification of myelin density expressed as myelinated area (MA) in pixels² in D + DT + TeO; CeP and SCT in CTRL, IOP, T3 and IOP + T3 larvae (mean ± SEM with n = 5/condition). Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the control group. *p <0.05, **p <0.01, ***p <0.001.

(A) Photomicrograph with lateral view and R-C orientation, whole-mount Black-Gold II staining in D + DT + TeO; CeP and SCT at 7 dpf (Unstained CTRL, Solvent CTRL/BG-II and IOP/BG-II), scale bar: 500 μm and 50 μm. In each corner, the amplification of sagittal sections identifying the selected area for analysis and the positive myelin pixels, recognized by the IPP6 program; (B) Graph showing the quantification of myelin density expressed as myelinated area (MA) in pixels² (individual values and mean ± SEM with n = 5/condition). Statistical analysis was performed with Student’s t test. ***p<0.001, ****p <0.0001.

mRNA quantification of olig2, sox10, mbpa, mpz and plp1b in controls and larvae exposed to IOP 0.5 μM, T3 10 nM or IOP 0.5 μM + T3 10 nM for 3 dpf and to IOP 0.5 μM for 7 dpf. Data are represented as individual values in vertical graphs (n = 5 pools from independent experiments of 50–60 larvae per pool and condition). Graphs are showing individual values and mean ± SEM. Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the corresponding control groups. Significant differences are indicated as *p <0.05, **p <0.01, and ***p <0.001.

(A) Lateral scan of the head using confocal microscopy and generating multiple 3D optical sections; scale bar: 100 μm. (B) Representative photomicrograph of zebrafish larvae with dorsal view and R-C orientation, whole-mount Black-Gold II staining in D, DT and at 3 dpf (wild-type, CTRL, IOP, T3 and IOP + T3). scale bar: 50 μm. (C) Schematic drawing with dorsal view of the head; the asterisks represent signal in the habenula and between the telencephalon (Tel) and tectum opticum (TeO) (red); signal within the TeO (white) and signal in the dorsal Tectum and between TeO and cerebellar plate (CeP) (yellow). The black arrow represents the orientation of the larvae, rostral-caudal (R-C). (D) The images are the representation of the maximum intensity projection of the entire set of Z-stacks: uninjected CTRL (WT), CTRL, larvae treated with IOP 0.5 μM, T3 10 nM or IOP 0.5 μM + T3 10 nM; scale bar: 50 μm. (E) Graphs depicting the volume of the myelinated area in voxels, Data are shown as individual values and mean ± SEM (approximately n = 15 larvae/group). Statistical analysis was performed with one-way ANOVA coupled with Tukey’s multiple comparison test with respect to the control group. **p< 0.01 and ***p< 0.001.

Dorsal view of the head of zebrafish larva at 3 and 7 dpf. (A) Pictures show whole mount NG2 immunoreactivity in control (upper panel) and IOP-treated larvae (lower panel). The latter show significantly decreased immunoreactivity (B) Negative control (without primary antibody) (C) Quantification of total fluorescence intensity of NG2 immunoreactivity. Individual values and the mean ± SEM are shown (n = 5 larvae/group). Statistical analysis was performed using Student’s t test. Significant differences are indicated as **p< 0.01. Scale bar 100 μm.