PSA-NCAM⁺ migrating neuron, GFAP⁺ glial fiber, and calretinin⁺ neuron differentiation defects in the mutant RMS. (A–F) Immunostaining of PSA-NCAM on sagittal sections (A and D) and counterstaining with DAPI (B and E) revealed a thin RMS in the control brain (A) compared with an enlarged RMS in the mutant (D). (C and F) Higher magnification of PSA-NCAM and DAPI staining in the regions outlined in B and E, respectively. Note the existence of PSA-NCAM⁺ neurons migrating in organized chains in the control brain (C), whereas in the region of accumulation of neurons in the mutant, the PSA-NCAM⁺ cells are more numerous and appear less organized (F). (G and I) Low-magnification images of GFAP immunostaining on sagittal sections in control and mutant brains. The RMS is outlined with dashed lines. A meshwork of GFAP⁺ fibers associated with the stream was observed in the control RMS (G), whereas in the mutant, the GFAP⁺ glial fibers cover a larger region where the accumulation of neurons is observed (I). Arrowheads indicate regions of dramatically enlarged GFAP staining. (H and J) Higher magnification of GFAP staining in the regions outlined in G and I, respectively. Note the existence of disorganized glial fibers in the mutant (J) compared with the control (H). (K–N) Calretinin immunostaining counterstained with DAPI in the RMS on sagittal brain sections from control (K) and mutant (L) brains. Note that, in the control brain, few calretinin⁺ cells were observed, whereas in the mutant brain, numerous calretinin⁺ cells were present in the RMS2 region. (M) An enlargement of the region outlined in L. Calretinin⁺ cells present complex morphologies. (N) Calretinin⁺ cells were quantified at four different levels of the RMS. Note the increase in the number (Nb) of calretinin⁺ neurons in mutant RMS2. ***, P < 0.001. n = 5. Data are represented as mean ± SEM. CC, corpus callosum; LV, lateral ventricle. Bars: (A) 140 µm; (C) 24 µm; (G) 210 µm; (H) 45 µm; (I) 210 µm; (J) 45 µm; (K) 60 µm; (M) 12 µm.