Fig. 3

RagA Promotes CNS Myelination by Repressing Tfeb

(A) Volcano plot depicting the genes differentially expressed (p < 0.01) in rraga^−/− mutants relative to wild-type animals. Among the significantly downregulated genes (< 2.5×) are mpz and plp1b (purple), which at this stage are characteristic of CNS myelin. Expression levels of 17 previously defined targets of TFEB (red) are upregulated (> 2.5) in rraga^−/− mutants. Inset graph depicts previously known TFEB targets (red) upregulated in rraga^−/− mutants. See also Tables S1, S2, and S3.

(B) Diagrammatic representation of the hypothesis that RagA promotes myelination by repressing Tfeb, which inhibits CNS myelination.

(C) Diagrammatic representation of Tfeb protein and of predicted truncated proteins encoded by tfeb^st120 and tfeb^st121 mutant alleles.

(D) Expression of mbp mRNA, as detected by whole mount in situ hybridization, is reduced in CNS of rraga^−/− mutants, but is restored in rraga^−/−;tfeb^−/− double mutants. rraga^−/−;tfeb^−/− double mutants and tfeb^−/− mutants are indistinguishable from wild-type larvae. See also Figure S5. Scale bar, 50 μm.

(E) TEM images of transverse sections of the ventral spinal cord (CNS) at 9 dpf show that myelination is restored in rraga^−/−;tfeb^−/− double mutants. Red arrows indicate unmyelinated axons, and yellow arrows indicate myelinated axons. Scale bar, 1μm.

(F) Quantification of the number of myelinated axons per hemi ventral spinal cord at 9 dpf is shown on the right; graph depicts average values and standard deviation; individual measurements are also shown. 3 individuals of each genotype were analyzed. (Statistical analysis: pairwise comparisons using one-way ANOVA; Tukey post hoc test significant interaction ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001).