Abstract
Pervasive sex differences between male and female brain development have been implicated in neurodevelopmental disorders featuring a distinct sex bias, such as autism. To further our understanding of sex differences we hypothesised that sex-differential microRNA expression occurs in the developing brain. MicroRNAs are powerful regulators of gene expression whose function is essential for normal brain development, and are thought to mediate development of sex differences.
Small RNA sequencing was performed with RNA isolated from E15.5 mouse brains (n=3 per sex), representing early brain differentiation. Comparison of microRNA expression identified 12 microRNAs that were differentially expressed between sexes (p<0.05). Differences in expression were validated using real time quantitative RT-qPCR for miR-10, and miR-205 and miR-5099.
miR-10 is encoded within the Hox gene complex, a cluster of genes conferring positional identity to structures along the anterior-posterior axis of the developing embryo. As miR-10 is thought to be controlled in the same spatially co-linear manner as the Hox genes, we anticipated that it has a distinctive expression domain. Using in situ hybridisation with a locked nucleic acid (LNA)-probe targeting miR-10, strong staining for miR-10 expression was found in the hindbrain and anterior spinal cord of embryos at E15.5.
Not only did we investigate known microRNAs, we also analysed novel microRNAs aligning to the mouse Anti-Müllerian Hormone AMH gene. Previous experiments identified a non-coding RNA (ncRNA) transcribed at this locus, bearing hallmarks of a primary microRNA. Subsequent preliminary small RNA sequencing at E12.5 (n=1 per sex) showed microRNA profiles at the AMH locus unique to each sex. Using RT-qPCR validation, female-specific expression of a novel microRNA aligning to exon 3 of AMH was determined. Interestingly, an increase in microRNA expression occurred in mouse lines where the exons 1 and 2 of the AMH gene were deleted. This observation lead to identification of a putative repressor element within exon 2, thought to negatively regulate expression of ncAMH.
Our findings indicate that sex-differential expression of microRNAs occurs in the developing mouse brain during a key stage of brain patterning. The identified microRNAs are known to influence development and neurological functions, and therefore their expression in a sex-differential manner may contribute to sex differences established in the developing brain.