Fig 1: TTR protein is undetectable in the SVZ in vivo. (a) TTR staining using the monoclonal Rabbit-anti-mouse TTR antibody on coronal sections from the P4 WT brain shows strong specific signal in the choroid plexus (red box), as expected. Inserts (magnification of white box) show TTR presence in the cellular cytoplasm, while no TTR is detected in the negative control. Scale bars: 50 µm, insert: 10 µm. (b) No TTR signal was detectable in the dorsal, lateral or ventral SVZ of coronal brain sections of neither male, nor male adult WT mice (n = 3 per group). Scale bars: 50 µm. NC: negative control.
Fig 2: Ttr KO results in decreased neuroblast and increased OPC generation in the lateroventral SVZ. (a) Schematic representation of the region of interest (the lateroventral SVZ, red box). (b) DCX+ neuroblasts (red) and OLIG2+ OPCs (green) are observed across the dorso-ventral axis of the lateroventral SVZ. Inserts show DCX+ and OLIG2+ cells in detail (white boxes). Scale bars: 10 µm. (c) Statistical analysis revealed a significant decrease in DCX+ cells in Ttr KO mice as compared to WTs, contrasting a significant increase in OLIG2+ cells (n = 6–8, two-tailed Student’s T test). (d) The ratio of DCX+ neuroblasts vs. OLIG2+ OPCs in the lateroventral SVZ is reduced in both male and female Ttr KO mice as compared to WTs (n = 3–4, one-way ANOVA, followed by Tukey Post-hoc test). (e) Ttr KO mice display a significant reduced number of DLX2-DCXhigh-expressing mature neuroblasts (green bars), suggesting hampered neuronal differentiation (n = 6–8, two-tailed Student’s T tests). Bars represent mean ± SD. St: striatum; v: ventricle. *P < 0.05, **P < 0.01.
Fig 3: Ttr KO results in increased pOPC numbers in the corpus callosum of male mice only. (a) Schematic representation of the region of interest (corpus callosum, red box). (b) OLIG2+ oligodendroglial cells (green) are abundantly present in the corpus callosum of WTs. The majority of them are mature oligodendrocytes also expressing CC-1 (red). Scale bars: 50 µm. (c) Statistical analysis revealed that OLIG2+ cell number per mm² in WTs is higher in females, and that Ttr KO results in a significantly increased pOPC number in males only, reaching WT female values (n = 6, two-way ANOVA, followed by Tukey Post-hoc test). (d) Detailed pictures showing mature oligodendrocytes co-expressing OLIG2 (green) in the nucleus and CC-1 (red) in the cytoplasm (arrowheads), while immature pOPCs only express OLIG2 (arrow). (e) The proportion of OLIG2+ cells co-expressing CC-1 does not differ statistically between all groups (n = 4–5, two-way ANOVA). Bars represent mean ± SD. CC: corpus callosum; v: ventricle. *P < 0.05.
Fig 4: Detection of Ttr mRNA expression in SVZ cells. (a) Ttr mRNA expression levels in FACS-sorted SVZ cells. High Ttr levels were found in quiescent and activated NSCs (CD133+), after which they dropped in TAPs (EGFR+) and NPCs (EGFR+ CD24+). Cells committed to the neuroblast lineage (CD24+) showed increased Ttr expression levels (n = 3–5, Kruskal Wallis ANOVA followed by Mann-Whitney U tests). (b) Ttr mRNA expression levels were measured in magnetic bead-sorted neuronal (PSA-NCAM+) and oligodendroglial (AN2+) cells after 1 and 5 days of in vitro differentiation. Ttr mRNA levels were significantly higher in neuronal cells after 1 day of in vitro differentiation (n = 3–4, two-tailed Student’s T tests). (c) Visualisation of Ttr mRNA transcripts in choroid plexus epithelial cells (red box on the schematic section) using RNAscope. Ttr mRNA transcripts (green dots, magnification of the white box) are abundantly present in the cytoplasm surrounding the nuclei (blue). Scale bar left picture: 50 µm, inserts: 10 µm. (d) Representative pictures of the lateral and ventral SVZ of WT mice (n = 4) showing few cells with a high number of Ttr mRNA transcripts (white arrows, magnification of white boxe), and some cells with a few transcripts (arrowheads, magnification of white boxes). Scale bar overview pictures: 50 µm, inserts: 10 µm. (e) No transcripts were detected in Ttr KO mice (n = 3). Scatter dot plots represent mean ± SEM. NPC: neural precursor cell; ns: not significant; NSC: neural stem cell; TAP: transient amplifying progenitor. *P < 0.05. **P < 0.01.
Fig 5: Ttr KO results in decreased neuroblast numbers in the male dorsal SVZ. (a) Schematic representation of the region of interest (the dorsal SVZ, red box). (b) DCX+ neuroblasts (red) densely populate the dorsal SVZ in males and females, together with several OLIG2+ OPCs (green). The DCX+ cell density in Ttr KO mice is clearly lower, while OPC numbers are similar. Scale bars: 20 µm. (c) Statistical analysis revealed a significantly reduced DCX+ cell number in the dorsal SVZ of male Ttr KO mice as compared to WTs (n = 5–6, two-way ANOVA, followed by Tukey Post-hoc test). (d) The ratio of DCX+ neuroblasts versus OLIG2+ OPCs in the dorsal SVZ is reduced in males only (n = 5–6, two-way ANOVA, followed by Tukey Post-hoc test). (e) Ttr KO mice display a significant reduced number of DLX2-DCXhigh-expressing mature neuroblasts, and a borderline-significant increase in DLX2lowDCXlow-expressing immature neuroblasts, suggesting hampered neuronal differentiation (n = 6–8, two-tailed Student’s T tests). Bars represent mean ± SD. CC: corpus callosum; dSVZ: dorsal subventricular zone; v: ventricle. *P < 0.05, ***P < 0.001.
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