Insights into spinal cord biology and its contributions to adolescent idiopathic scoliosis: From GWAS to animal models

The aim of this thesis was to investigate the relationship between the spinal cord, adolescent idiopathic scoliosis (AIS), biological sex and a transcription factor, LBX1. AIS is a multifactorial gene of unknown aetiology affecting 3-5% of the population. The physical manifestations of the disease are primarily a 3-dimensional rotational curvature. However, it also compromises pulmonary and cardiac function. Treatment is typically invasive and of those ultimately requiring surgical intervention; 90% of these cases with progressive AIS are female, exhibiting a severe sex bias. However, the etiology of AIS remains elusive. Advances in genetic screening have enabled clinicians to undertake powerful populationbased studies known as genome wide association studies (GWAS). From these GWAS, several genes have been identified as associated with AIS. The most promising candidates is a SNP, located near the LBX1 gene. LBX1 is a transcription factor with known roles in patterning the spinal cord during embryonic development. In addition to GWAS, recent studies in mice have demonstrated that the proprioceptive system, our body’s sense of position in space, controls spinal alignment and that disruption to this system in mice results in scoliosis. The links between AIS, sex, LBX1 and the adult spinal cord has not been investigated in mouse models. We firstly investigated the developing neural tube in mice, examining global gene expression pre- and post-puberty and between males and females using RNA-sequencing. While there were marked differences in the expression profile between the pre-puberty and post-puberty samples, the differences between the sexes were not as many as expected. In the pre-puberty spinal cords, we saw a number of developmentally linked genes upregulated including those involved in myelination and response to stimulus, suggesting and confirming the notion of the spinal cord continuing its development/maturation. Following puberty, the genes overrepresented were largely involved in processes related to neuronal signalling, that is; vesicle transport, exocytosis, viii neuron projection development, among other suggesting the primary role for the spinal cord post-puberty was to relay information. Lbx1 function has been studied extensively in developmental contexts and have been shown to be critical in the appropriate patterning of the spinal cord, as such we wanted to determine: 1) if it had continued expression in the adult spinal cord, 2) the role of Lbx1 in the spinal cord and, 3) where it might be expressed within adult the spinal cord and whether this would provide insight into its role postnatally. Firstly, immunofluorescence and in situ hybridisation we performed to determine the spatial expression patter of Lbx1. Lbx1 was found to be expressed throughout dorsal horn interneuron populations dI5 and dI4 dorsally, as well as dI1 and the motor neurons of the ventral horn. Subsequently, Chromatin Immunoprecipitation with high throughput sequencing on Lbx1 in the adult murine spinal cord was carried out. A total of ~3200 putative target genes of Lbx1, were identified, many with roles related to neuronal signalling, synapse formation and neuronal projection development. To better understand the role of Lbx1 in the adult murine spinal cord, colocalization with markers for various neuronal subtypes was performed. Lbx1 expression overlapped largely with proprioceptive and nociceptive neuronal populations. Lastly, we wanted to determine whether the LBX1 associated SNP (rs11190870) resulted in changes to Lbx1 expression in a mouse model. The SNP of interest was located within a putative regulatory module in humans and a highly conserved, homologous region was identified in mice. To investigate the role of this regulatory region, which we termed the AIS-CRM, we employed CRISPR-Cas9 gene editing to delete a 189 bp region, removing the putative AIS-CRM. Initial investigations were largely observational, looking for gross morphological defects and behavioural changes in these mice carrying this deletion (AISCis Regulatory Model Δ). The AIS-CRMΔ mice performed more poorly on simple proprioceptive tasks than their wild type (WT) counterparts, pre-puberty and up to 4 months of age, after which the proprioceptive performance was similar between the AISCRMΔ and WT mice. Following this finding of slightly altered proprioceptive function, the vertebral column was examined more closely by means of microCT. Three-dimensional analysis of the vertebral column showed an increase in vertebral rotation in the AIS-CRM ix mice but also a larger range of variability between all samples suggesting difficulty in maintain a stable spine. Having linked the proprioceptive defect and the vertebral instability, we wanted to determine what may be driving these physical differences. We examined the genetic expression of Lbx1 transcripts in the neural tube and spinal cord. Initially we examined Lbx1 expression at E12.5 is period of cell fate specification, and the pre-pubertal spinal cord (PN28) to determine whether changes in Lbx1 were occurring embryonically or prior to the adolescent transition through puberty. Lbx1 expression peaks at E12.5, is higher in male NT than females and is also expressed 5-fold higher in AIS-CRMΔ mice relative to their WT counterparts. Pre-pubertal Lbx1 expression is similar between the sexes and displays no difference between WT and AIS-CRMΔ samples. RT-qPCR analysis revealed that at E10.5, while Lbx1 mRNA expression is not significantly different between the sexes, there is a ~9-fold increase in the AIS-CRMΔ compared to the WT. At E15.5 there is not statistically significant difference between sexes or genotypes, a trend also seen in the adult samples. This suggests that migration of neurons (E10.5) and their cell fate acquisition (E12.5) are possibly the timepoints affected by the deletion of the Lbx1 CRM. To investigate any spatial changes to gene expression, in situ hybridisation analysis was performed. The spatial expression of Lbx1 changes in the developing neural tube at E12.5 with staining expanding from the lateral and medial regions of the neural tube to the ventro-lateral region. The expression pattern between adults is similar, however, staining in the AIS-CRMΔ spinal cord appears to be more widespread. This thesis presents the first functional investigation into the possible link between genome, Lbx1 and AIS development due to altered spinal cord development in the embryo, resulting in lasting effects which manifest physically in the form of a proprioceptive deficit and vertebral rotation