Article Figures & Data
Figures
- Figure 1.
Male pups have higher levels of Foxp2 protein. The relative amount of Foxp2 protein was quantified in tissue collected from the cerebellum, amygdala, cortex, thalamus, hypothalamus and POA at 4 d of age. A, Males had significantly more Foxp2 than females in every brain region except the hypothalamus and POA, two brain regions closely associated with reproduction but not vocal communication. Data are expressed as mean (±SEM), n = 7–8/group, significant sex difference (*p < 0.05, t test). B, In these same animals, basal levels of Foxp1 protein were not sexually dimorphic in any examined brain region. C, Photomicrographs of a PN4 female showing Foxp2 expression in key brain regions known to express Foxp2 as well as the hippocampus which shows no positive staining. Scale bar, 50 μm.
- Figure 2.
Male pup ultrasonic vocalizations are different from female pup vocalization and Foxp2 siRNA reverses this pattern. A, Infusion with siRNA or scrambled control RNA to 4-d-old pups resulted in control treated males making almost twice as many distress USVs per unit time as control treated females. The mean frequency and amplitude of the calls are significantly lower in males compared with their female littermates (two-way ANOVA; p < 0.05). Treatment with siRNA directed against Foxp2 mRNA 2 d before testing significantly reduced the total number of calls in males but increased the number in females. The same pattern was seen for frequency whereas amplitude was modified by siRNA treatment only in females (F test; p < 0.05). Data are expressed as mean (±SEM) of calls, n = 8 /group. Groups with the same symbol are significantly different from each other. B, Typical sonograms of USVs from a control female (top), siRNA Foxp2-treated female, control male and siRNA Foxp2-treated male (bottom). C, Noninjected controls were not different from the scrambled injected controls (t test; p > 0.05, n = 8/group).
- Figure 3.
SiRNA against Foxp2 was effective at reducing protein levels both in vivo and in vitro. A, In vivo infusion of siRNA directed against Foxp2 mRNA on the day of birth and the subsequent day significantly reduced Foxp2 protein levels 24 h later but had no impact on the closely related Foxp1 (F < 1.0, p > 0.05). N = 2–3/group with equal numbers of males and females. B, In vitro transfection of primary cell cultures on DIV 3 with siRNA directed against Foxp2 mRNA reduced Foxp2 protein levels in both the cortex and striatum. C, By postnatal day 4, we again confirmed a sex difference in Foxp2 protein levels, with males having higher levels than females, and the suppression of Foxp2 levels by siRNA was no longer apparent in males (F < 1.0, p > 0.05). However, females treated with Foxp2 siRNA showed an increase in Foxp2 protein compared with scrambled control treated females, suggesting a rebound following inhibition (F test; p < 0.05), n = 8 /group with equal numbers of males and females). Groups with the same symbol (*) are significantly different from each other.
- Figure 4.
Dams retrieve males before females because of their USVs. Four-day-old pups were removed from the nest for 5 min and marked to identify sex, then placed back on the opposite side of the home cage as shown in panel 1. The dam retrieved the pups one by one as shown in panel 2 (A). The rank order sum quantification of pup retrieval based on sex in control dams with litters that received no treatment indicated male pups were more likely to be retrieved before females (Mann–Whitney U = 36.0, p < 0.05, B, based on 6 litters). When litters were treated with either siRNA against Foxp2 or scrambled control (C), control males were again significantly more likely to be retrieved first and females retrieved last. Males treated with siRNA against Foxp2 were significantly less likely to be retrieved compared with control males, whereas females treated with Foxp2 siRNA, which have USVs similar to control males (Fig. 1A) were significantly more likely to be retrieved before control females (Kruskal–Wallis test = 20.5, p < 0.05, groups with the same letter are significantly different from each other (C, based on 5 litters).
- Figure 5.
FOXP2 is differentially expressed in boys' versus girls' cortex. Postmortem cortex from the left hemisphere of Brodmann's area 44 from 4-year-old boys and girls were obtained from the NICHD Brain and Tissue Bank, University of Maryland, Baltimore. The relative amount of FOXP2 and FOXP1 protein was quantified by Western blot. A, Cortex from girls had significantly more FOXP2 than cortex from boys. Data are expressed as mean (±SEM), n = 5/group, significant gender difference (*p < 0.05, t test). B, In these same samples, there was no sex difference in level of FOXP1 protein. GAPDH indicates loading control. C, Regression analysis between protein levels (via Western blot) and mRNA levels for FOXP2 showed a significant positive linear relationship (r = 0.70, p = 0.02, r2 = 0.50).
