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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2021)
The transcription factor Pax6 is prominently expressed in the glutamatergic neurons during cerebellar development. When mutated, the Pax6-null Sey cerebellar granule cells (GC) are disorganized and foliation is disrupted. To elucidate the gene network regulated by Pax6 in cerebellar development, the Sey mutant transcriptome was analyzed and differentially expressed genes were identified. In this thesis, I examine some of these genes and their role in cerebellar development, as well as the relationship of these molecules with Pax6. Wntless (Wls) is up-regulated in the Sey cerebellum. Wls expression is restricted to the interior face of the rhombic lip (iRL) in normal cerebellar development. Whereas in the Sey cerebellum, Wls-expressing cells expand into the EGL, indicating that Pax6 normally suppresses Wls expression. Furthermore, examination of Wls and other rhombic lip (RL) markers in the wildtype embryos demonstrates that the RL is dynamically demarcated into four molecularly distinct compartments. Conversely, Tbr1 and Tbr2 are down-regulated in the Sey cerebellum. These are cell markers of cerebellar nuclei (CN) neurons and unipolar brush cells (UBCs), respectively. The absence of CN neurons and UBCs in the Sey mutant are revealed using standard immunohistochemistry and Nissl staining. Cell death analysis demonstrates that there is enhanced cell death in Sey mutant CN neuron progenitors, GCs and UBCs. Cell proliferation analysis also shows a reduction in the Sey RL progenitor pool during the genesis of UBCs and GCs. To elucidate the requirement of Wls in cerebellar development, I examine the conditional Wls knockout (Wls-cKO) in which Wls is ablated in the RL during mid-gestation. Ectopic Pax6-expressing cells are found in the Wls-cKO RL indicating that Wls normally suppresses Pax6. The Wls-cKO cerebellum displays a smaller vermis and foliation defects. Granule cells are disorganized and UBCs are missing from the mutant cerebellum. The lack of Wls also affected cells of the VZ-lineage such as Purkinje cells and interneurons. This thesis provides novel insights into the molecular network underpinning cerebellar development, in particular the requirement of Pax6 and Wls. This work also reveals the spatial compartments in the developing RL that are defined by Pax6, Wls and other molecular markers.
The cerebellum is critical for motor functions such as coordination, precision and accurate timing of movement; as well as non-motor functions including cognitive and emotional processes. During cerebellar development, genesis and fate decisions of cerebellar neural precursors are controlled by genetic networks consisting of transcription factors and their downstream targets. The objectives of my thesis are: 1) to construct the transcriptional network of the developing cerebellum based on gene expression and gene regulation; 2) to investigate the temporally regulated usage of alternative promoters in cerebellar development; 3) to generate a cerebellar granule cell specific transcriptome dataset to identify genes that are dynamically expressed, or significantly enriched in cerebellar granule cells during development; and 4) to study the roles of a newly discovered, dynamically expressed transcription factor - Kruppel-like Factor 4 (Klf4) in cerebellar granule cell development. Taking advantage of high-throughput next generation sequencing technology, we used HeliScopeCAGE, which combines single molecule fluorescent sequencing technology (Helicos) and Cap Analysis of Gene Expression (CAGE), to generate a new transcriptome time series for cerebellar development. We were able to discover hundreds of gene regulators that are important for cerebellar development through differential expression and motif activity analyses. In addition, I analyzed the temporal shift of usage in alternative promoters of a gene, and found that different forms of gene products have distinct functions during cerebellar development. Furthermore, to study the granule cell-specific transcriptome, I used laser microdissection to isolate granule cells from the cerebellum. Comparison of the granule cell transcriptome with the whole cerebellar transcriptome allowed me to identify genes that are dynamically regulated or significantly enriched in the granule cells. Lastly, I studied a mouse knock-out model of Klf4, a potentially key gene regulator from previous analyses, and found that Klf4 does, in fact, have an important role in early granule cell proliferation. This work also showed that Klf4 is involved in the regulation of other important granule cell transcription factors such as Pax6 in the cerebellum for the first time.
Neural stem cells and their precursors, collectively referred to as neural progenitor cells (NPCs), are present in discrete regions of the mature brain, namely the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ), and rostral migratory stream (RMS). These NPCs divide and give rise to new neurons in a process called adult neurogenesis. Genetic influence is a major determinant of adult neurogenesis. However, the genetic architecture underlying NPC proliferation and differentiation is poorly understood. My thesis aims to gain insights into the genes regulating NPC proliferation using a phenotypic-driven, genome-wide approach. I first examined nine inbred mouse strains housed in the same condition and across different ages from 60 days (P60) to 2 years. Wide inter-strain differences and negative impact of age on the number of NPCs were observed in the RMS. Genetic background had a significant effect on NPC proliferation and it also differentially influenced the effect of age on this process. The most dramatic inter-strain difference was detected at P60. Heritability estimated ~50% of the differences in NPC numbers were attributed to the genetic variation among the strains. I used quantitative trait locus (QTL) mapping to survey the entire genome for chromosomal segments referred to as QTLs that contribute to the phenotypic differences. Two panels of recombinant inbred strains, AXB/BXAs and BXDs, were employed for QTL mapping. Genetic variation in QTLs on chromosome (Chr) 6 and 11 were significantly associated with the differences in NPC numbers in the RMS. Additional analyses revealed potential interaction of Chr 6 QTL with other loci. These QTLs are hypothesized to harbor genes important for NPC proliferation and downstream experimentation is required to validate the function of these genes. As proof of concept, a candidate gene called Galanin receptor 2 (Galr2) in the Chr 11 QTL was demonstrated to be a pro-proliferative regulator of NPCs using in vitro techniques manipulating Galr2 expression and Galr2 knockout mice. In summary, I identified novel QTLs underlying NPC proliferation and these loci serve as starting points to identify genes (e.g. Galr2) critical to this process.
Master's Student Supervision (2010 - 2020)
The cerebellum is an important part of the central nervous system (CNS). During early embryonic development, the neuroepithelium of the cerebellar primordium consists of two primary progenitor zones – the rhombic lip (RL) and the ventricular zone (VZ). All glutamatergic cells like granule cells arise from the RL while the GABAergic cells like Purkinje cells arise from the VZ. Each of the progenitor zones gives rise to multiple cell types in a distinct spatiotemporal sequence, but it is not clear what are the underlying genetics that control this sequence. Compartmentation of these progenitor zones has been an emerging field in this line of investigation. Using fluorescent RNA in situ hybridisation, I have characterised the Msx genes, a family of transcription factors downstream of BMP signaling, to show how they spatiotemporally pattern the cerebellar neuroepithelium. Msx1 is compartmentalised within the RL to likely maintain a progenitor pool, while Msx3 is compartmentalised within the VZ to likely be involved in the VZ progenitor fate specification. As external signaling molecules, the BMPs have been implicated in the specification of cerebellar cell types but their downstream molecular cascades are unknown. The results of this study present the Msx genes as strong candidates facilitating this BMP signaling in cerebellum development. In the second part of the study, I have utilised a time-course transcriptome to identify a catalog of brain specific long non-coding RNAs (lncRNAs) expressed significantly in the developing cerebellum. This class of non-coding RNAs is largely heterogenous and uncharacterised in their function. Recent studies, however, have implicated lncRNAs in the genetic regulation of CNS development. The top candidate lncRNA of the catalog, 6330403K07Rik, has been analysed for its spatiotemporal expression in the developing cerebellum. 6330403K07Rik has strong expression in the RL and nuclear transitory zone at E11.5 and in the glutamatergic cerebellar nuclear neurons at E18.5. This two-part study is aimed to further the genetic resolution of cerebellar development through gene expression studies. Developmental defects in the cerebellum are implicated in neurodevelopmental disorders such as Autism Spectrum Disorder, Schizophrenia and ADHD, and understanding the genetics of cerebellar development is important to developing therapeutic interventions.
Objective: To explore the relationship between cerebellar pathology and changes in neuronal activity in mouse models of autism-like phenotypes.Methods: We used the rotarod test as a measure of sensorimotor function in our mice and as a means to trigger neuronal activation. Following behavioural testing we obtained brain tissue from our ASD-like mouse models and used histology and microscopy to examine the expression of cFos (a reporter of neuronal activity) and several other structural and functional markers to evaluate cerebellar pathology. Finally, we looked at differences in the morphology, distribution and number of cerebellar glia in our ASD-like mouse models to determine if reactive gliosis contributes to further cerebellar pathology in adult mice.Results: Compared to wildtype littermates, Lc/+ mutant mice performed significantly worse on the rotarod assay of sensorimotor function (p
With the improvement in neuroimaging technology and the increase in survival of preterm infants, the detection of abnormalities in the cerebellum has become increasingly common. Human and animal studies suggest that the preterm cerebellum is particularly vulnerable to damage because of its dramatic growth and complex developmental processes. Cerebellar hemorrhage (CBH) is the most frequently detected cerebellar lesion in premature infants and often leads to long-term neurological sequela, such as motor, affective and cognitive dysfunction. However, how CBH affects the development and function of the cerebellum remains largely unknown. Therefore, our study focuses on developing a mouse model of CBH to determine the anatomical, behavioural, and molecular phenotypes resulting from the hemorrhage in the developing cerebellum. To induce hemorrhage in the fourth ventricle and germinal matrix of the cerebellum, we injected bacterial collagenase, which breaks down surrounding blood vessel walls, into the fourth ventricle of postnatal (P) day two mice. Controls were injected with saline. We then performed various anatomical, behavioural and molecular assessments to detect any changes in the morphology and function of the cerebellum and to unravel the mechanisms of injury and neuroprotection activated as a result of CBH. In our model, we found a delay in cerebellar development, reduction in granule cell and interneuron density, and persistent neurobehavioural abnormalities similar to the abnormalities found in premature infants with CBH. Furthermore, we found a significant upregulation of neurotrophic factor expression and of genes in the sonic hedgehog pathway, which indicate the activation of endogenous neuroprotective mechanisms. Thus, our study provides a novel preclinical model of CBH that can be used to understand the pathophysiology of the disease and for the development and evaluation of preventive therapies and post-hemorrhagic treatments.
Approximately 11% of pregnant woman in Canada continue to use tobacco during pregnancy. Maternal tobacco use increases the risk of complications in pregnancy and also the risk of adverse foetal outcomes. Although tobacco smoke contains over 4000 compounds, studies have established nicotine as the principal component of tobacco smoke that leads to the majority of negative reproductive outcomes associated with maternal tobacco use. It appears the neuroteratogenicity of nicotine is mediated by complex gene-environment interactions. Genetic background contributes to individual differences in nicotine-related phenotypes. The aim of the current study was to investigate the interaction between pre- and perinatal nicotine exposure and genetic background on histological and behavioural measures using DBA/2J (D2) and C57BL/6J (B6) inbred mice. Alterations in neuronal cell populations, regional brain volumes, and behaviour - open field (OF) activity, novel object recognition (NOR), elevated plus maze (EPM), and passive avoidance (PA) - were evaluated on postnatal day (PN) 24 and PN75, following early exposure to nicotine solution (200 μg/ml)starting from 30 days before pregnancy up to pups weaning. Data revealed no difference between treatment groups of dams in gestational weight gain or pup mortality. Histological data showed that early nicotine exposure resulted in decreased striatal volume among preadolescent females and reduced neuronal number within the striatum of preadolescent B6 mice. In the hippocampus the effects of early nicotine exposure appeared more subtle, in which only the granule cell layer of the dentate gyrus in D2 preadolescents was afflicted. Behavioural data showed that early nicotine exposure promoted hyperactivity in D2 female mice and disrupted NOR and PA memory. Specifically, NOR deficits were significant amongst adult male mice whereas PA deficits were unconditional. These data suggest that pre- and perinatal nicotine affects regional brain morphology and leads to neurobehavioural alterations. The observed treatment interactions suggest that genetic background, developmental stage, and sex interact with nicotine to influence nicotine-related phenotypes.