Extended Data Fig. 9: Age- and cell-type-specific sex differences in POA gene expression.

(a) Difference between male and female proportion of adult nearest neighbors at each age. p-value indicates results from two-sided t-test, male versus female distances across all cell types. Boxplots show median, interquartile range, and 1.5 * interquartile range, with cells from n = 2 biological replicates per sex per age. p = 0.0002 (P4) and 3.64×10⁻⁶ (P10). (b) Left: All clusters that show significant sex differences in at least one age group according to male-female PCA distance. Right: number of sexDEGs among these clusters per age. N = 6-7 female and 6 male samples at each age, excepting P65 with 3 female and 5 male samples. N.S. = not significant. (c) At P65, sex differences in excitatory clusters measured by distance between male and female sample centroids in PCA space (normalized to intra-sex distance; left column) or number of sexDEGs detected (middle column). Grayscale matrix on the right indicates sex hormone receptor expression. N.S. = not significant. (d) Esr1 gene expression (upper) and cell-type-specific motif enrichment in accessible chromatin (lower) amongst MPN cell types. (e-f) Top GO terms for sexDEGs found only at specific ages in e-M1 (e) or i-M5 (f). (g) Gene expression values for 115 sexDEGs detected at P4-P10 but not other ages in e-M1, pseudobulked across cells within samples (each column represents a single sample). Control samples from mutant/control experiments included to increase sample number. (h) Violin plots showing expression of Pdzrn4, a sexDEG, in i-M5 neurons at all 8 ages separately in males and females. (i) RNA in situ hybridization confirms stronger expression of Pdzrn4 in males than in females in Esr1+ neurons at E18. (j) Bias-corrected ATAC-footprints centered around the Esr1 motif binding site, quantified separately for male and female i-M5:MPN/StHy^Gal/Fbn2 cells at P0 (left), P10 (middle), or P18 (right). (k) Percentage of cells identified as i-M3:Pe/VMPO^Kiss1/Th in each sex at each age. Each point represents a single sample (3 POAs pooled). Boxplots show median, interquartile range, and 1.5 * interquartile range, with cells from n = 2-4 biological replicates per sex per age (except E16, 1 replicate per sex per age).