Expression of genes in the 16p11.2 locus during human fetal cortical neurogenesis
Morson, Sarah Elisabeth
The process of the brain developing from a single fertilized egg to the most sophisticated known organ requires precise spatial and temporal control to produce the necessary correct brain size and architecture. A particular region of interest is the cerebral cortex, responsible for higher functions such as language, reasoning and conscious thought. Its expansion in size and complexity from smaller mammals, such as mice, to humans is thought to contribute to our higher functions. However, a caveat of this increased complexity is the increased challenge of generating such a complex structure, and the potential for subtle changes during neurodevelopment to manifest in neurodevelopmental disorders such as Autism Spectrum Disorders (ASD). ASD is a spectrum disorder diagnosed early in childhood based on a range of diagnostic criteria. It is frequently characterised by impaired social interaction, repetitive behaviour and delayed development. While ASD patients share some symptoms, the genetic underpinnings of ASD are highly heterogeneous, with mutations to many single genes or larger genetic regions implicated as ASD risk factors. The 593kbp 16p11.2 locus encompasses 29 protein coding genes and its copy number variation (CNV) by heterozygous microduplication or microdeletion is implicated in around 1% of ASD cases, with many patients born with macrocephaly (deletion) or microcephaly (duplication), potentially indicating a possible problem with generating the correct number of brain cells during development. This suggests the hypothesis that some of the 16p11.2 region genes are involved in neural proliferation in early corticogenesis, and changes to the levels of these genes may affect proliferation, contributing to the 16p11.2 patient phenotype. This hypothesis is supported by a 16p11.2 deletion mouse model which exhibits ASD-like symptoms and altered proliferation in the cortex during embryonic development. Given that the 16p11.2 CNV’s 1% autism incidence makes it the most frequent aetiology of ASD, this region is a promising area of study to understand how genetic dysregulation during critical prenatal cortical neurogenesis can contribute to the ASD phenotype. Despite its strong association with ASD, very little is known about the majority of the 16p11.2 genes, especially regarding brain development. In this thesis, focussing on the developing human neocortex, we aimed to identify which, if any, of these 29 genes were expressed in progenitor cells and describe their expression pattern during critical stages of cortical neurogenesis. We first used a bioinformatics approach to identify 16p11.2 genes expressed in progenitors to narrow down from the 29 genes in the region. We analysed a publicly available single-cell RNA sequencing (scRNA-seq) dataset from the proliferative zones, the ventricular zone and subventricular zone, of the 16-18 gestational week (GW) human fetal cortex. We identified six genes as being highly expressed in the cortical progenitor cells, and two as being significantly higher expressed in progenitors compared to post mitotic cells: KIF22 and ALDOA. We described their protein expression pattern in vivo at key stages of human fetal cortex development. We showed KIF22 protein to be expressed in the germinative zones, and its expression to be restricted to proliferating cells, suggesting a role for this protein in proliferation. We showed KIF22 protein levels to vary with the cell cycle, increasing from G1 through S and G2 phases to peak in mitosis. This suggests that changing the KIF22 protein level, as in the microduplication or microdeletion patients, will affect cell cycle and proliferation manifesting in changes to cortical size and architecture contributing to the 16p11.2 phenotype. ALDOA protein was shown to be present throughout the cortex, although higher in the proliferating regions. We demonstrated that ALDOA is predominantly localised to cytoplasm, and its protein levels or sub-cellular localisation do not change in proliferating or non-proliferating cells. ALDOA has a critical role in energy metabolism, and we can hypothesise that due to its expression throughout the cortex any changes to the ALDOA protein by the 16p11.2 CNV will induce a wide range of effects on brain development In conclusion we have identified two genes highly expressed in progenitors and expressed at much lower levels in post-mitotic cells from the 16p11.2 locus. These genes provide interesting targets for future studies to elucidate the mechanism by which they mediate proliferation and the effects of manipulating their protein levels. This is outwith the scope of this PhD thesis however a range of new techniques are emerging such as cerebral organoids, which can be easily manipulated. These will be a powerful tool to address the hypotheses produced by the descriptive work of this PhD thesis.