Fig 1: Tubb4aD249N/D249N mice have severely delayed myelination.(A–B) Immunohistochemical assays were performed according to a time course (A) in the Corpus callosum (CC) and Cerebellum (Cb) with the analyzed area in grey. (B) For each assay, the CC and Cb were imaged and quantified in WT, Tubb4aD249N/+, and Tubb4aD249N/D249N mice at P14, P21, and at end-stage (ES;~P35-P40), except for Eriochrome cyanine (Eri-C), PLP and MBP which was also assessed at one year in WT and Tubb4aD249N/+ mice only in CC. (C–D) The CC shows significantly decreased Eriochrome cyanine (Eri-C) staining (blue) in Tubb4aD249N/D249N mice, which worsens over time. (E–F) Tubb4aD249N/D249N mice show significant and worsening decreased of Eri-C staining in the Cb. (G–J) Loss of Eri-C staining (G–H) as well as MBP immunostaining (red) (I–J) in CC seen in 1-year-old Tubb4aD249N/+ mice. (K–N) PLP immunostaining (green) of CC (K–L) and decreased protein levels by western blot in the forebrain from P21 and ES mice (M–N) demonstrate a defect in the normal expression of myelin proteins. (O–R) A similar effect is seen in PLP immunostaining (O–P) and western blot (Q–R) in Cb. (S–Z) MBP protein staining in CC (S–T) and western blot (U–V) in the forebrain, as well as the Cb (W–Z) also showed a significant and progressive decrease in Tubb4aD249N/D249N mice. Statistical tests performed by two-way ANOVA, Tukey post-hoc test or Unpaired t-test. n = 4 mice/group for P14 (except n = 3 for Tubb4aD249N/+ mice) and P21, n = 3 mice/group for ES and 1 year. Data presented as mean and SEM. *p<0.05 and ***p<0.001. Scale bar = 1 mm (C, E, G) or 250 µm (I, K, O, S, W). Western blots, lanes; 1, 2, and 3 represent WT, Tubb4aD249N/+, and Tubb4aD249N/D249N mice, respectively.Figure 2—source data 1.Source files of graphical data for Myelin quantification.Figure 2—source data 2.Source files of graphical data for Western blots quantification of MBP and PLP.