Brn3b expression in the dorsal midbrain, thalamus, and retina.

(A–C) Schematic diagrams of coronal images showing the brain area (boxed) used for analysis (left) and sagittal images depicting the level (dashed line) where the coronal section was obtained (right) at P1 (A) and P9-P12 (B and C). (D–G) Brn3b expression in Brn3bflox/+:: En1-Cre mouse, visualized by immunostaining at P1 (D) and P10 (F) and by AP labeling at P1 (E) and P12 (G). A magnified view of the boxed areas (i–iv) showed that Brn3b+ neurons are located close to the pia at P1 and become confined to the SO layer at P10/P12 (n = 3 mice/age/visualization method). Due to the poor quality of AP antibody staining and the required tissue clearing for AP staining, immunostaining and AP labeling were conducted using different sections/animals. (H, I) Brn3b immunostaining in WT mouse at P9. Brn3b expression is missing in the upper layer of superficial SC (SGS: H), consistent with observation in the Brn3bflox/+:: En1-Cre mice. No Brn3b signals were detected in LP (I). Immunostaining with anti-Brn3b was conducted at P9 because of very weak signals produced by Brn3b antibody beyond P12 (n = 3 mice). To demarcate dorsal lateral geniculate nucleus (dLGN) and LP in the thalamus, calbindin antibody (green) was used (Grubb and colleagues [57]). (J) No AP signals were detected in the retina of Brn3bflox/+:: En1-Cre mouse (n = 3 animals). Dashed lines indicate either the pia surface or boundary of dLGN and LP (D–I). Blue (DAPI). Scale bar: 200 μm. (TIF)