FIGURE 3.

Vitamin B12 treatment restores normal expression of cortical markers in Tbx1 heterozygous embryos. Panels show coronal sections of embryonic brains at E13.5. (A,a) Immunostaining for TBR1 (A–A”’) revealed the precocious presence of terminally differentiated neurons in the SVZ of Tbx1^lacZ/+ embryos (A’). White brackets define the regions containing TBR1-positive cells in (A,A’). Quantitative analysis confirmed the altered distribution and frequency of mature cortical neurons in the ML-cortex, of Tbx1^lacZ/+ embryos, including the SVZ and CP (a), which was rescued by B12 treatment (A”’,a). (B,b) Immunostaining for phospho-histone 3 (PH3) revealed increased mitotic activity in the proliferative zones (VZ, SVZ) of the ML-cortex of vehicle-treated Tbx1^lacZ/+ embryos (B’,b,b’) compared to vehicle-treated WT embryos (B,b), which was rescued by B12 treatment (B”’,b,b’). (C,c) Immunostaining for KI67. White asterisks identify KI67-negative regions. Quantitative analysis (c) revealed the reduced thickness of the KI67- expressing region (yellow brackets) relative to the total ventricular-to-pial thickness (white brackets) in vehicle-treated Tbx1^lacZ/+ embryos (C’,c) compared to vehicle-treated WT embryos (C), which was rescued by B12 treatment (C”’,c). The cartoon (c’) indicates the position of the ML-cortex in coronal sections, and the position of the counting boxes for cell counts. Scale bars, 100 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, error bars indicate mean ± SD, red horizontal bars with # not significant, n = 5–6 per genotype. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate; ML-Cx, medio-lateral cortex; D, dorsal; M, medial; ML, medio-lateral; L, lateral.