Journal: bioRxiv

Article Title: Brain-synthesized estrogens regulate cortical migration in a sexually divergent manner

doi: 10.1101/654731

Figure Lengend Snippet: (A) Western blot of lysates taken from developing cortex demonstrating aromatase expression at different stages of development. (B) Representative RT-PCR analysis for sry gene used to determine sex of P0 animals used in subsequent analysis. (C) Representative confocal image of aromatase staining in P0 cortex of female and male. Aromatase positive cells can be observed in the SVZ/VZ, IZ and CP in both female and male developing cortex. Yellow dotted boxes indicate high magnification images shown in D. (D) High magnification images from C. Red arrows indicate aromatase positive immunoreactivity within cell body of developing neurons in either female or male P0 cortex. Yellow arrow heads indicate aromatase expression within processes of developing neurons in P0 cortex of both sexes. (E) Quantification of relative aromatase expression throughout P0 cortex of female and male. This revealed that males have a significantly higher expression of aromatase at this stage of development within the cortex. Conditions were compared by Student’s t test; **P<0.001; n=13-15 sections from 8 pups/sex. Data are presented as Box and Whisker plots with error bars showing minimum and maximum data points; each data point represents an individual section. Scale bars = 100 μm (C) and 20 μm (D).

Article Snippet: A myc-DDK-tagged aromatase construct (pCMV6-myc-DDK-aromatase) was purchased from Origene (Rockville; Cat. No. MR224509); the pCAG-eGFP has previously been described ( Srivastava et al. , 2012b ).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Whisker Assay

Journal: bioRxiv

Article Title: Brain-synthesized estrogens regulate cortical migration in a sexually divergent manner

doi: 10.1101/654731

Figure Lengend Snippet: (A) Western blot of cell lysates taken from hEK293 cells expressing myc-aromatase in the presence or absence of shRNA against Cyp19a1 (shRNAa-d) or a control construct (shRNA_scram). (B) Quantification of myc-aromatase expression reveals that shRNA_c (aka shRNA_arom) produced ∼60% knockdown of exogenous aromatase. Conditions were compared by one-way ANOVA; F(5,12)=7.077, p=0.003, Tukey Post Hoc, *, p < 0.05; n = 3 independent cultures. Data are presented as Box and Whisker plots with error bars showing minimum and maximum data points; each data point represents an individual experiment. (C and D) Representative confocal images of female (C) or male (D) P0 cortex in utero electroporated at E14.5 with either control (shRNA_scram) or shRNA_arom and co-stained for aromatase and GFP. GFP was used to identify cells expressing control or Cyp19a1 shRNA. (E) Quantification of aromatae expression in GFP+ cells in control or shRNA_arom conditions in female or male P0 cortex. This revealed that shRNA-arom reduced endogenous aromatase expression by ∼ 50% in both sexes. Data are represented as a percentage of control condition. Conditions were compared by two-way ANOVA; F(1,18)=95.59, p<0.0001, Bonferroni Post Hoc, ***, p < 0.001; n=8-12 sections from 4-6 pups/sex. Each data point represents an individual section. Scale bars = 20 μm.

Article Snippet: A myc-DDK-tagged aromatase construct (pCMV6-myc-DDK-aromatase) was purchased from Origene (Rockville; Cat. No. MR224509); the pCAG-eGFP has previously been described ( Srivastava et al. , 2012b ).

Techniques: Western Blot, Expressing, shRNA, Construct, Produced, Whisker Assay, In Utero, Staining

Journal: bioRxiv

Article Title: Brain-synthesized estrogens regulate cortical migration in a sexually divergent manner

doi: 10.1101/654731

Figure Lengend Snippet: (A) Representative confocal images of female P0 cortex in utero electroporated at E14.5 with either control (shRNA_scram) or shRNA_arom and stained for GFP. (B) Quantification of GFP+ cells distribution throughout the developing female cortex. In conditions where Cyp19a1 had been knockdown (shRNA_arom), a significant more GFP+ cells were observed in the lowest bin, equating to the SVZ/VZ of the P0 cortex. Concurrently, a decrease in the number of GFP+ cells was measured in the upper most bins, equating to the upper portion of the CP. Conditions were compared by two-way ANOVA; F(9,140)=5.873, p<0.0001, Bonferroni Post Hoc, **, p<0.01, ***, p < 0.001; n=10-12 sections from 6-8 pups/condition/sex. (C) Representative confocal images of male P0 cortex in utero electroporated at E14.5 with either control (shRNA_scram) or shRNA_arom and stained for GFP. (D) Quantification of GFP+ cells distribution throughout the developing male cortex. Loss of aromatase induced by Cyp19a1 knockdown (shRNA_arom) resulted in a significant increase of GFP+ cells in the upper portion of the CP. Congruent with this, a decrease in GFP+ cells was observed in the SVZ/VZ of the P0 male cortex. Conditions were compared by two-way ANOVA; F(9,160)=10.08, p<0.0001, Bonferroni Post Hoc, *, p<0.05, ***, p < 0.001; n=7-11 sections from 5-7 pups/condition/sex. Scale bars = 50 μm.

Article Snippet: A myc-DDK-tagged aromatase construct (pCMV6-myc-DDK-aromatase) was purchased from Origene (Rockville; Cat. No. MR224509); the pCAG-eGFP has previously been described ( Srivastava et al. , 2012b ).

Techniques: In Utero, shRNA, Staining