Abstract
Sex differences pose a challenge and an opportunity in biomedical research. Existing data is primarily derived from studies that fail to include female subjects and/or fail to stratify results by biological sex. This limitation hampers our understanding of the many disorders that demonstrate a sex difference in phenotype, prevalence, severity, and response to treatment. Addressing this flaw by studying the basis of sex differences will reveal new information pertinent to sex-biased disorders and help ameliorate gaps in our understanding of female biology.
Understanding how sex chromosomes and hormones affect disease-causing mechanisms will shed light on the mechanisms underlying largely idiopathic sex-biased neurodevelopmental disorders such as ADHD, schizophrenia and autism. Gene expression is a crucial conduit for the influence of sex on developmental processes, therefore this investigation has focused on sex differences in gene expression and regulation of gene expression. Mounting interest in microRNAs (miRNAs), small, non-coding RNAs that fine-tune gene expression, for their contribution to normal and pathological neurodevelopment, prompted us to test how miRNA expression also differs between the sexes in the developing brain.
Here I report the first comprehensive investigation of sex-biased gene and miRNA expression in the developing mouse brain. Using the mouse brain at age embryonic day 15.5 (E15.5), I pursued four lines of investigation: (1) characterising sex differences in gene and miRNA expression; (2) determining the mechanisms that contribute to sex-biased miRNA expression; (3) investigating whether anti-Müllerian hormone (AMH) contributes to sex-biased gene expression; (4) identifying novel miRNA in the developing mouse brain. Together, these experiments highlight sex-biased RNA biology underlying mammalian brain development.
Firstly, I utilized high-throughput sequencing approaches to identify genes and miRNAs that showed significantly different expression between male and female mouse brains at E15.5. Robust sex differences were identified for a portion of genes and miRNAs, confirming the influence of biological sex on RNA in this context. Many miRNAs showing the greatest differences between male and female have established roles in neurodevelopment, implying that sex-biased expression could drive sex differences in developmental processes. In addition to highlighting sex differences for individual miRNAs, gene ontology analysis suggests a number of broad categories in which sex-biased RNAs might be acting to establish sex differences in the embryonic mouse brain.
The first results chapter yields a number of genes, miRNAs, and pathways of interest that may contribute to sex differences in brain development. However, it is also important to understand how and why these differences arise in addition to understanding their consequences. Therefore, the second aspect of my investigation aimed to determine the mechanisms that cause the observed pattern of sex-biased miRNA expression. Using RNA-seq and small RNA-seq data generated in the first results chapter, in conjunction with miRNA databases, I tested six possible mechanisms. Evidence showed that many different mechanisms, including hormonal, genetic and regulatory, all contribute to sex-biased miRNA expression. A complex combination of causes reinforces the notion that miRNA expression in the developing brain has been sculpted by evolution to drive sex differences.
The hormonal mechanisms in the second part of my project focused on gonadal sex steroids as they are established regulators of sexual differentiation. But the sexually dimorphic gonads generate other factors, such as AMH, that may be potent regulators of sex differences in development. Thus, the third section of my project aimed to test whether this hormone contributes to sex differences in gene expression in the E15.5 mouse brain. Limited sample numbers hampered the ability to confirm this hypothesis. Preliminary findings suggest that AMH influences a large portion of the transcriptome in the embryonic male brain, but that compensatory mechanisms appear to prevent considerable sex differences in gene expression due to AMH.
Experiments with an AMH targeted deletion mouse line also resulted in the serendipitous discovery of a novel, non-coding transcript at the mouse Amh locus. By testing the hypothesis that this transcript is a precursor for a novel miRNA, I amassed several lines of evidence for miRAmh, a rodent-specific miRNA encoded within Amh intron 3 that is usually repressed in the embryonic mouse brain. Therefore, I have demonstrated dual roles for the mouse Amh locus in the developing brain.
The fourth and final aspect of my project was prompted by the discovery of miRAmh. Many advances in small RNA-seq technology and bioinformatics pipelines have been made since the most recent publication to discover novel miRNAs in the E15.5 mouse brain. Therefore, my high-quality, small RNA-seq datasets generated in this tissue could be mined using an unbiased miRNA discovery strategy to build on those previous findings. Fifty putative novel miRNAs were identified, from which ten were selected for further validation. A combination of conservation analysis, spatiotemporal expression, and functional prediction were employed to determine the authenticity of novel miRNA candidates. These findings demonstrated that miRNAs tend to remain undiscovered if they are derived from other non-coding RNA (ncRNA), or if study design fails to incorporate both sexes.
Work presented in this thesis builds a strong case for sex differences at the level of RNA in the developing mammalian brain. Not only has this pattern of sex-biased expression been carefully established via several mechanisms, but those functions of sex-biased genes, miRNAs and novel miRNAs provide a plausible basis for functional sex differences in the brain to emerge. Furthering our understanding of sex-biased RNA networks will help pinpoint the changes underpinning sex-biased neurodevelopmental disorders.