Fig. 5: CRISPR-Cas9 knockdown of ESR1 expression in adult ARN^KISS neurons.

a–c Photomicrographs showing EGFP/Cas9 (green), ESR1 (blue), and mCherry (red) expression in the ARN of mice receiving no gRNA injection (a), gRNA-ESR1-3 (b), and gRNA-LacZ (c). Almost all GFP cells co-express ESR1 (blue/light blue nuclei) in a and c compared with only two cells in b. a and b are from opposite sides of the ventricle of mouse 16265 that received a unilateral injection of gRNA-ESR1-3. To facilitate identification of dual-labeled cells, the same photomicrographs are shown to the right with the mCherry channel removed and ESR1 now displayed in magenta. Dual-labeled cells have an asterisk in the nucleus. d Mean ± SEM and individual data points showing numbers of EGFP/Cas9 cells detected per section in the presence of gRNA-LacZ, gRNA-ESR1-3 (3) and gRNA-ESR1-6 (6) in the rostral (rARN), middle (mARN) and caudal (cARN) (N = 6 or 7 per experimental group). e Mean ± SEM and individual data points showing % of kisspeptin (EGFP/Cas9) cells expressing ESR1 in the presence of gRNA-LacZ, gRNA-ESR1-3, and gRNA-ESR1-6 at the three rostro-caudal ARN levels (N = 4–7 per experimental group). f Mean ± SEM and individual data points for all regions of the ARN combined showing % of kisspeptin (EGFP/Cas9) cells expressing ESR1 in the presence of gRNA-LacZ, gRNA-ESR1-3 (gRNA3), and gRNA-ESR1-6 (gRNA6). ***P < 0.0001, **P = 0.002, post hoc Dunnett’s test versus LacZ (N = 6 or 7 per experimental group). g Mean ± SEM and individual data points showing % of tyrosine hydroxylase (TH) cells expressing ESR1 in the presence of gRNA-LacZ, gRNA-ESR1-3 (gRNA3), and gRNA-ESR1-6 (gRNA6) (N = 6 or 8 per experimental group). Source data are provided as a Source Data file.