Abstract
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,
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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
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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.