Understanding the Genetics of the Outer Subventricular Zone as a Primer for Gyrification

<p dir="ltr">The mechanisms that regulate gyrification in some species but not in others have been the subject of investigation for decades. To date, numerous theories and perspectives have been proposed to explain how the cerebral cortex acquires its characteristic folds. It is now generally accepted that cortical gyrification is driven by tightly regulatory mechanisms governing neural progenitor cell proliferation, differentiation, and neuronal migration. Although these developmental processes are broadly conserved between gyrencephalic and lissencephalic species, spatiotemporal differences in gene expression and the regulation of these processes result in striking differences to the topographical architecture of the cerebral cortex. </p><p dir="ltr">The outer subventricular zone (oSVZ) is an expanded germinal layer in the brain that increases significantly in size at the onset of cortical folding. This layer is a hallmark of gyrencephalic development, rich in two populations of basal progenitor cells: basal radial glial cells (bRGCs) and basal intermediate progenitor cells (bIPCs). In the gyrencephalic brain, these basal progenitors have greater proliferative capacity. Through successive proliferative mitoses, they generate vast numbers of post-mitotic neurons that populate the cortical plate (CP) and promote cortical expansion. In contrast, the oSVZ is comparatively thinner in lissencephalic species, with sparse bRGCs and limited capacity for proliferative divisions. Understanding these mechanisms and differences is critical for elucidating how gyrification is regulated. Beyond identifying differential gene expression patterns, analysing the expression dynamics of key candidate genes offers a powerful approach to uncovering their functional roles during cortical development.</p><p dir="ltr">The overall aim of this thesis was to determine the genetic and cellular mechanisms that drive cortical folding, with a particular focus on the role of bRGCs and their transcriptional regulation within the oSVZ. This thesis therefore sought to identify differentially expressed genes (DEGs) in the oSVZ correlating to the coronal gyrus (CG) and suprasylvian sulcus (SSS) at the onset and middle of gyrification, and to select candidate DEGs for follow up investigation, including a functional genetic manipulation in ferret neural progenitor cells.</p><p dir="ltr">To investigate the genetic drivers of cortical folding, it is essential to determine differences in gene expression in the oSVZ niche, which includes bRGCs, and to understand these differences throughout cortical development and folding using clear and reproducible methods. In Chapter 2, SPIA paired-end RNA sequencing (RNA-Seq) was performed in the oSVZ beneath the CG and SSS at the onset (P5) and middle (P15) or cortical folding in the ferret. The data revealed several DEGs between the CG and SSS regions at the onset of folding. Additionally, several DEGs were identified in each region from the onset to the middle of cortical folding. Gene ontology and KEGG pathway analyses using DEG lists successfully identified novel biological processes and pathways enriched between the CG and SSS at the onset, and within each region during gyrification. Furthermore, candidate genes identified through literature review were cross-referenced with the RNA-Seq data to determine their expression patterns in the analysed samples and ages. Consistent with the literature, several of these genes were also differentially expressed between the CG and SSS oSVZ at the onset of gyrification. </p><p dir="ltr">Building on the results of Chapter 2, ten genes were selected for further mRNA and protein level validation: (1) SOX9, (2) FOXM1, (3) FOXC1, (4) GLI1, (5) PI4KB, (6) TUBA4A, (7) AJAP1, (8) PRUNE1, (9) SEMA3D, and (10) STAT3. In Chapter 3, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed on remaining P5 and P15 RNA obtained in Chapter 2 to validate the RNA-Seq results for a subset of candidate genes. To further expand on these findings, immunofluorescence staining was performed on a subset of these targets (SOX9, FOXM1, FOXC1, GLI1, and pSTAT3) in the ferret at neurodevelopmental stages encompassing oSVZ genesis (E34), expansion (E38-P2), gyrification onset (P5), middle (P15), and advanced stages of gyrification (P25). The data revealed that most expression differences observed in Chapter 2 were consistent with the RT-qPCR results. Additionally, the data revealed that SOX9 and pSTAT3 were differentially expressed in the oSVZ as early as E34, potentially priming the cortex for gyral and sulcal formation. The results also indicated that FOXM1 was significantly upregulated in the CG oSVZ compared to the SSS at the onset of cortical folding. Based on these findings, FOXM1 was selected as a prime candidate for further experimentation. </p><p dir="ltr">In Chapter 4, a bioinformatic investigation into FOXM1 focussed on a unique transcript variant comprising an exon proposed to regulate primate cortical folding. The aim was to determine whether this unique exon sequence was found in FOXM1 transcripts of other species, including both gyrencephalic and lissencephalic species, and to assess the conservation of the remaining coding sequence of FOXM1. Using protein, nucleotide, and translated nucleotide BLAST across three databases, it was found that human FOXM1 exon 9 was not present in all other primates, questioning its necessity for gyrification. Furthermore, a multiple sequence alignment of FOXM1 found that the remaining gene sequence was 99% conserved across multiple species, irrespective of gyrencephaly. </p><p dir="ltr">Lastly, Chapter 5 aimed to generate and characterise a population of ferret neural progenitor cells for genetic manipulation and to perform a functional assessment of FOXM1 in these cells. A previous study showed that the inclusion of exon 9 in the lissencephalic mouse brain led to the formation of cortical folds, suggesting that FOXM1 containing exon 9 may act as a primate-specific cortical folding gene. However, given the findings in Chapter 4 - that exon 9 is not present in other gyrencephalic species – and contrasting literature suggesting that this transcript variant is transcriptionally inactive, further investigation was needed. This study found that exon 9 may influence mRNA and protein-level expression of key neurodevelopmental markers in ferret neural progenitor cells suggesting it may have a regulatory role in the processes involved in cortical folding. </p><p dir="ltr">Overall, this thesis identified novel DEGs and pathways within the oSVZ that are associated with the location and development of the CG and SSS in the gyrencephalic ferret brain. Additionally, the functional investigation of the candidate gene FOXM1 highlighted the complexity and nuances of gene regulation during cortical development. Collectively, the findings support the notion that region-dependant differences in gene expression are fundamental to the development of the cerebral cortex and have generated a valuable database of DEGs that may act as early determinants or priming factors in the formation of gyri and sulci.</p>