Developmental analysis of Brn3b+ neurons in the dorsal midbrain using the <i>Brn3b</i>^GFP/+ mouse.

(A) Schematic diagram of a coronal image showing the brain areas (boxed) used for analysis (left) and a sagittal image depicting the level (dashed line) where a coronal section was obtained (right) at P9/P10. (B) Schematic diagram illustrating the level of the sagittal sections as lateral distances from the midsagittal line at E16 and P1/2. (C, D) Double immunostaining with anti-Brn3b (red) and anti-GFP (green) shows no Brn3b expression in the Brn3bGFP/flox:: En1-Cre at P9/P10. Magnified view of the boxed areas showing the loss of GFP+ (i.e., Brn3b+) neurons in the superficial SC (i, ii), indicated by arrows (n = 3 animals/genotype). DAPI (blue). (E, F) Representative images of sections stained for Brn3b (red) and GFP (green) at E16. DAPI (blue). (G) Quantification of GFP+ neurons (iii) and their distribution (iv) showed no obvious differences at E16 (2,306.9 ± 83.7/mm2 for Brn3b GFP/+, 2,319.6 ± 56.6/mm2 for Brn3b GFP/flox:: En1-Cre, n = 3 animals/genotype). Unpaired two-tailed Student’s t test (mean ± SEM; p = 0. 906). (H, I) Representative images of sections stained for Brn3b (red) and GFP (green) at P1/2. DAPI (blue). (J) Quantification of GFP+ neurons (v) and their distribution (vi) revealed that Brn3b loss decreases the number of neurons in the superficial and intermediate layers (613.6 ± 37.8/mm2 for Brn3b GFP/+, 431.3 ± 39.6/mm2 for Brn3b GFP/flox:: En1-Cre, n = 3 animals/genotype). Superficial and intermediate layers were defined as located within 350 μm from the pia. Unpaired two-tailed Student’s t test (mean ± SEM; p = 0.029 [*] for the cell number and p = 0.013 [*] for the distribution). Magnified view of the boxed area (vii). No clear difference was detected in the deep SC/PAG (1,857.1 ± 65.4/mm2 for Brn3b GFP/+, 1,863.0 ± 101.0/mm2 for Brn3b GFP/flox:: En1-Cre, n = 3 animals/genotype). Unpaired two-tailed Student’s t test (mean ± SEM; p = 0. 964). The dashed lines delineate the pial surface (C–F and H–I). DAPI (blue). Scale bars: 200 μm (C–D) and 50 μm (E–F and H–I). The data underlying this figure can be found in S1 Data. (TIF)