Lamina formation in the developing cortex requires precise generation, migration and differentiation of cortical neurons. Cortical projection neurons originate from progenitors of the embryonic dorsal telencephalon. The transcription factor Pax6 is expressed in apical progenitors (APs) throughout corticogenesis in a rostro-lateralhigh to caudo-mediallow gradient. The current studies focus on elucidating the spatial and temporal role of Pax6 in cortical development. I first analysed the cortex of PAX77 transgenic mice that overexpress Pax6 in its normal domains of expression. I show that Pax6 overexpression acts cell-autonomously to reduce the proliferation of late cortical progenitors specifically, resulting in the formation of thinner superficial layers in the PAX77 cortex. Increased levels of Pax6 lengthen the cell cycle of APs and drive the system towards neurogenesis. These effects are specific to late stages of corticogenesis, when superficial layer neurons are normally generated, in cortical regions that express Pax6 at the highest levels. The number of superficial layer neurons is reduced in postnatal PAX77 mice, while radial migration and lamina specification of cortical neurons are not affected by Pax6 overexpression. Then, Pax6 was conditionally inactivated in cortical progenitors at mid- or late-stages of corticogenesis by using a tamoxifen-inducible Emx1-CreER line. I report a novel requirement of Pax6 for continuous suppression of ventral fates and concurrent maintenance of an appropriate dorsal identity in cortical progenitors. Pax6 ablation at either mid- or late-stages of corticogenesis increases the proliferation of late cortical progenitors at all levels across the rostral-caudal axis. In the absence of Pax6 from mid-corticogenesis, late-born neurons are severely under-represented and misspecified in superficial layers of the mutant cortex. Notably, Pax6 inactivation during late corticogenesis also affects superficial laminar fate; although the numbers of late-born cortical neurons are not severely affected in superficial layers of the mutant cortex, substantial numbers of late-born cells fail to migrate to appropriate laminar positions and accumulate in the ventricular zone (VZ) of the postnatal mutant cortex. Collectively, these gain- and loss-of-function studies suggest that disruption of Pax6 levels during different developmental time points leads ultimately to impaired formation of superficial cortical layers but through different cellular and molecular mechanisms.

Pax6 is a highly conserved developmental regulator with a complex temporal, spatial and quantitative expression pattern, that is crucial for correct development of the central nervous system, the eye, and pancreas. Accordingly, the Pax6 gene resides in a complex genomic locus containing a large array of long-range tissue-specific cis-regulatory elements primarily identified through multispecies sequence conservation and reporter studies. I have set out to understand how the chromatin architecture of the locus contributes to the mechanism and specificity of cis-regulatory interactions. As well as addressing whether the DNA looping model for regulatory interactions applies to the mouse Pax6 locus, I will identify which elements facilitate such interactions and if they vary between cell types. Utilising ChIP-array technology the distribution and variability of key regulatory histone modifications and factors were assessed in a set of Pax6 expressing and non-expressing mouse cell lines, acting as models for different regulatory states of the locus. Work in other loci suggests a key role for CTCF and cohesin (subunit Rad21) in chromatin organisation and long distance regulatory interactions. ChIP-chip for CTCF/Rad21 across the Pax6 locus identified numerous sites within the gene and at distal regulatory locations. The majority of these sites are cell type invariable. The active enhancer modification H3K27ac identified both known and several novel putative enhancer elements distributed through the locus that are highly cell type specific. A subset of CTCF/Rad21 sites also acquire the active enhancer modification H3K27ac in a cell type dependent manor, suggesting that CTCF/Rad21 may facilitate looping to the target gene from these sites. Using reporter based assays, putative regulatory elements marked by the looping factors and active histone modifications showed a diverse range of functional activities. Unexpectedly only 3 of the 7 CTCF sites tested showed classical insulator activity in an enhancer blocking reporter assay. Surprisingly the strongest insulator tested resided within intron 7 the Pax6 gene. Other CTCF/Rad21 sites were neutral or enhancers in the insulator assay. This reveals the disparity between predicting regulatory properties using ChIP binding profiles alone and the actual outcome of functional reporter experiments. A novel element, CTCF6 showed a ChIP signature of CTCF/Rad21/H3K27ac in all Pax6 expressing tissues, and functioned as a strong enhancer in transient transfection and stable LacZ reporter assays. CTCF6 recapitulated a broad range of Pax6 expression patterns, at multiple embryonic stages, including the brain, neural tube and pancreas. A second novel element, E-120 identified in the pancreatic derived cell line, drove stable embryonic reporter expression in the embryonic pancreas and sub set of brain regions. Together this has expanded the repertoire and size of Pax6’s regulatory landscape particular in the upstream region. Chromatin conformation capture (3C) was used to characterise the dynamic chromatin architecture of the locus and identify the interaction profiles from three CTCF/Rad21 binding regulatory locations within the Pax6 locus. This revealed a core set of regulatory interactions with the Pax6 gene, while individual elements showed a more variable set of cell type specific interactions. The CTCF6 enhancer showed highly cell type specific promoter interactions throughout the Pax6 gene, indicative of enhancer-promoter looping not detected in the non-expressing cells. While the downstream site CTCF5 at the edge of a cluster of regulatory elements known as the DRR (differentially regulated region), interacted with both the gene and an upstream element CTCF7 300 kb away only in the Pax6 expressing locus. Together these results reveal Pax6 has a chromatin hub structure with regulatory loops from upstream and downstream bringing distant yet variable active elements in to the vicinity of the Pax6 promoters where they can act. This work has revealed new roles for CTCF/cohesin sites in transcriptional regulation of Pax6 and how the cis-regulatory activity and structure of the locus varies across different cell types.

During development the nuclei of the thalamus form reciprocal connections with specific regions within the cortex. These connections give rise to the thalamocortical tract. The processes by which axons of the thalamocortical tract are guided to their target regions are poorly understood. It has been shown that diffusible or membrane bound factors can have a chemoattractive or chemorepulsive effect on the tip or growth cone of the axon. Thalamocortical axons may also be guided along ‘pioneer’ axon populations that form a scaffold along which axons may grow. The transcription factor Pax6 has been shown to have a role in a variety of developmental processes such as neuronal patterning, proliferation, migration and axon guidance. It is known that Pax6 is involved in the development of the thalamocortical tract but its exact role is unknown. To explore the role that Pax6 plays in the development of the thalamocortical tract I have used two different mouse models, the small eye (Pax6Sey/Sey) mouse which lacks functional Pax6, and a conditional Pax6 knock-out (Pax6cKO) mouse made using a Gsh2 Cre line that specifically reduces Pax6 expression in the ventral telencephalon and prethalamus. Using the Pax6Sey/Sey mouse I show that thalamocortical axons do not enter the ventral telencephalon in the absence of Pax6 and that a small number of axons incorrectly enter the hypothalamus. In addition axons found within the ventral telencephalon of the mutant do not originate from the thalamus but instead originate from cells within the ventral telencephalon itself. I have found that the expression of guidance molecule Robo2 is reduced in the Pax6Sey/Sey mouse, which may explain why thalamocortical axons enter the hypothalamus. When Pax6 expression is reduced at the prethalamus and ventral telencephalon using the Pax6cKO mouse I show that the majority of thalamocortical axons reach the cortex normally but some axons become disorganized within the thalamus. Pioneer axons which emanate from the prethalamus normally guide thalamocortical axons through the diencephalon but in the Pax6cKO I report that these axons are reduced which may explain the disorganization of thalamocortical axons within the thalamus. Taken together the data from these two models demonstrate that for the thalamocortical tract to form normally Pax6 expression is required in both the cells of the thalamus and in cells that lie along the route of the tract. In addition I provide evidence that Pax6 may influence axon guidance by controlling the expression of guidance molecules and the development of pioneer axon tracts.

The Pax6 encodes a highly conserved transcriptional regulator that contains two DNA binding domains, the paired domain (PD) and homeodomain (HD). In mammals, Pax6 is widely expressed in a complex spatiotemporal pattern during the development of the eye, olfactory bulbs and central nervous system and plays important roles in pattern formation, cell fate determination and cell cycle progression in these regions. Normal development requires Pax6 to be present in certain cells with correct levels, which implies that Pax6 expression is tightly controlled and that different levels need to be maintained across different regions as they develop. To gain better insight into the regulatory mechanisms of Pax6 expression, a series of tauGFP-Pax6 transgenic reporter mouse lines was previously generated in which the expression of tauGFP is under the control of putative Pax6 regulatory elements. Here, I have characterized the functional importance these regulatory elements by comparing the pattern of tauGFP expression with endogenous Pax6 expression in transgenic mice containing either complete or truncated versions of the reporter. I showed that the expression of tauGFP reporter exhibits the known Pax6 expression pattern in forebrain and eye, except for some minor discrepancy within the telecephalon. The loss of tauGFP expression within the eye and thalamus was observed in transgenic lines carrying truncated reporter sequences lacking the downstream regulatory region (DDR) of Pax6. Analysis of the pattern of GFP reporter expression in transgenic lines that vary in the extent of their putative Pax6 regulatory elements revealed the functional significance of these elements and also implied the existence of unknown distal regulatory elements, outside of the reporter sequences, which control Pax6 expression in the telecephalon. I went on to study a Pax6-dependent signaling pathway through which Pax6 controls progenitor cell proliferation in the developing telencephalon. Comparison of cell cycle parameters between Pax6+/+ and Pax6sey/sey progenitors suggested that correct levels of Pax6 are crucial in regulating progenitor cell proliferation. To address the possible molecular basis of the cell cycle defect observed in Pax6sey/sey embryos, the expression of a number of cell cycle genes was analyzed by qRT-PCR in the lateral cortex of Pax6+/+ and Pax6sey/sey embryos, which confirmed the significantly altered expression levels of these genes. Of them, Cdk6 was further identified as a direct target of Pax6 and the interaction of putative binding sites with Pax6 protein was confirmed by EMSA in vitro and by qChIP in vivo. In addition, the functional role of these Pax6 binding sites, through which Pax6 represses the transcription of Cdk6, was further evaluated by luciferase assays. Activation of Cdk6 is required for pRb phosphorylation as well as induction of the pRB/E2F pathway, and in turn promotes the G1-S cell-cycle transition. An increase in pRb phosphorylation accompanied by changes in pRb subcellular distribution and up-regulation of E2F downstream targets were observed in the cortex of Pax6sey/sey embryos. In contrast, a reduction of Cdk6 expression and pRb phosphorylation was found in HEK293 cells overexpressing Pax6. Collectively, these findings provided new insight into the molecular mechanism of Pax6-dependent regulation of progenitor cell proliferation in the developing telecephalon.

The regulation of gene expression resulting in the formation of the mammalian cerebral cortex is tightly regulated by a group of transcription factors. The deletion of any one of these transcription factors results in numerous defects whose nature and severity depends on the role of the transcription factor in the regulation of complex gene regulatory networks involved in development. There is currently relatively little knowledge about the gene networks that these transcription factors control and how they exert their regulatory effects. The paired-box transcription factor Pax6 has been identified as a master regulator of gene networks involved in cortical development and its deletion results in numerous cortical defects such as an abnormally thin cortical plate and a vastly expanded proliferative zone. Previous work in our lab identified a list of candidate genes that are likely to be regulated by Pax6 in the developing cortex. Members of the Notch signalling pathway were potential Pax6 targets of particular interest since Notch signalling plays a crucial role in the maintenance of neural progenitor cells during development and consequently plays a critical role during corticogenesis. Our work aims to identify the regulatory relationship between Pax6 and Notch ligands Dll1 and Jag1 during cortical development. Analysis by flow cytometry and double labelling analysis of both gene and protein expression has provided insight into the relationship between Pax6 and Dll1 in progenitor cell subpopulations during cortical development. In situ hybridisation and qPCR results confirmed that loss of Pax6 causes loss of Dll1 expressing cells and downregulation of Jag1, indicating that both ligands are regulated by Pax6. Bioinformatic screening and analysis by luciferase assay suggests that Jag1 is a likely candidate to be a direct target of Pax6.

The development of the diencephalon can be summarised as a process in which cells that initially appear similar give rise to a complex structure that contains a variety of cell groups called nuclei. It involves two stages: the early patterning of the diencephalic prosomeres and the later formation of the individual nuclei. It has been shown that transcription factors and morphogens regulate the first stage but their further effects on the second stage remain unclear. The ventral lateral geniculate nucleus (vLGN) is involved in the visual system and is shown to have complex origins from the thalamus, the zona limitans intrathalamica (ZLI) and the prethalamus. The transcription factor Pax6 is involved in the development of brain structures including the cortex, the diencephalon and the major axonal tracts in the forebrain by playing a multifaceted role in patterning, proliferation, differentiation, migration and axon guidance. It is known that Pax6 is essential in setting up the prosomeric boundaries in the developing diencephalon but its role in the formation of individual nuclei has not yet been explored. By using a conditional Pax6 knock-out mouse driven by Zic4Cre with a green fluorescent protein (GFP) reporter showing the Cre activity, the formation of the thalamic nuclei vLGN, dorsal lateral geniculate nucleus (dLGN) and VP (ventral posterior nuclei) was examined in postnatal day 0 (P0) Pax6+/+, Pax6fl/+ and Pax6fl/fl pups. Using this mouse model, I found an increase in nuclear volume at the rostral level and a global decrease in cell density in the P0 Pax6fl/fl vLGN, whereas in the dLGN an increase of GFP+ve cell proportion was observed. In Pax6fl/+, I found an increase in GFP+ve cell proportion in the caudal part of the vLGN and across the dLGN. No significant change was observed in the VP in either the Pax6fl/+ or the Pax6fl/fl. The defects in the vLGN and dLGN could be caused by: 1. disruption of the expression of patterning factors such as Shh and Nkx2.2; 2. cell proliferation defcts and abnormal apoptosis; 3. ocular developmental defects; 4. failure in cell sorting/migration; 5. cell fate change. During my PhD, I tested the first three theories and explored the fourth but was not able to pursue the last due to the time limit of the project. To test the hypothesized mechanisms underlying those defects seen in the vLGN and dLGN, I performed BrdU labelling to study the time origin of cells that contribute to these two nuclei and discovered that E11.5 and E12.5 are the main ages when these cells and the GFP+ve subpopulation are born. Then I carried out experiments to examine the cell proliferation and cell apoptosis in the thalamus (pTH-R, rostral part of the progenitor zone of the thalamus, and pTH-C, caudal part of the progenitor zone of the thalamus) and the prethalamus (Pth) from E11.5 to E13.5 and found: 1. the proliferation rate decreased in the pTH-R in Pax6fl/+ at E11.5; 2. the growth fraction decreased in both pTH-C and pTH-R in E12.5 Pax6fl/fl; 3. there is no change in cell proliferation in the GFP+ve subpopulation; 4. no abnormal apoptosis is observed in either the whole cell population or the GFP+ve subpopulation. Judging by the amplitude of the change in proliferation in the pTH-R and pTH-C at E11.5 and E12.5, it is unlikely that these changes alone are responsible for the phenotypes seen in P0 vLGN and dLGN. Then I examined the expression patterns of Shh and Nkx2.2 and the expansion of both was observed in Pax6fl/fl at both E12.5 and E13.5, which could explain the volume change of the vLGN but not the change in the proportion of GFP+ve subpopulation in both the vLGN and dLGN. Then I continued to examine if the ocular input from the retinal ganglionic cells are severely affected by the deletion of Pax6 and found no gross change in the conditional mutants, which rejected the ocular developmental defects theory. At the end of my PhD, I performed a BrdU short-term survival experiment and a brain slice culture combined with live imaging experiment to explore the possibility of abnormal cell migration causing the vLGN and dLGN phenotypes and found that the cells moving along the border of the thalamus and prethalamus move faster in the Pax6fl/fl than in the Pax6fl/+, but rather than moving directly toward the lateral surface of the diencephalon, they take a detour. These findings indicate that the deletion of Pax6 causes minor changes in the proliferation of E11.5 to E13.5 diencephalon and expansion of regional marker expression such as Shh and Nkx2.2, which could potenially affect the volume and change the proportion of GFP+ve cells in P0 vLGN and dLGN. Migration defects caused by Pax6 could also contribute to the phenotype observed in those two nuclei. Potential cell fate change caused by Pax6 deletion could be another factor that contributes to the defects in the conditional mutants. More work needs to be done to test the migration defect and cell fate change hypotheses in future.

No description available.

Aniridia is a rare genetic disorder that affects the development of the eye and is caused in most cases by mutations in the PAX6 gene. Patients with a heterozygous mutation in their PAX6 gene are born without irises. Aniridia patients are also prone to other eye diseases over their lifetimes such as cataracts and glaucoma. Aniridia’s progressive nature suggests that therapeutic intervention aimed at restoring PAX6 expression may be effective at ameliorating the progression of this disease. PAX6 is necessary for the development and maintenance not only of the eye, but also the pancreas. Patients with aniridia have an increased likelihood of developing glucose intolerance and diabetes. Indeed, genetic studies in rodents have confirmed that haploinsufficient animals for Pax6 develop glucose intolerance due to an ongoing requirement for Pax6 expression in the pancreas and gut. This thesis is a proof-of-concept study designed to determine the effects of repressing miRNA regulation of murine Pax6. Pax6 is regulated by miRNA-7 and miRNA-375. I hypothesized that repression of miRNA-7 and miRNA-375 would restore Pax6 expression and that this strategy might be useful in treating some of the progressive symptoms that emerge in aniridia patients in adulthood. As a first step towards evaluating miRNA inhibition as a therapeutic strategy for the treatment of aniridia, my first objective was to confirm whether miRNA-7 and miRNA-375 regulate Pax6 expression in pancreatic cells and tissue. My second objective was to determine whether these miRNAs could be efficiently inhibited. My third objective was to determine whether repression of miRNA-7 or miRNA-375 alters endogenous PAX6 protein levels in pancreatic cell lines. My final objective was to determine whether target protectors, delivered to explants of pancreatic islets through an adeno-associated virus (AAV) vector, could be used to restore Pax6 expression in murine haploinsufficient islets. From this study, I have confirmed that miRNA-7 and miRNA-375 regulate Pax6 in pancreatic cells that these miRNAs can be specifically inhibited, and that inhibition leads to an increase in Pax6 on both the reporter and protein levels. I have shown that target protectors against the miRNA-7 and miRNA-375 binding sites within the Pax6 3’UTR are effective at increasing the levels of PAX6 protein in pancreatic cell lines. Finally, I have also shown that a target protector against the miRNA-7 binding site can increase PAX6 protein levels in islets from murine haploinsufficient islets to near wild-type levels. My thesis lays the groundwork for the development of anti-miRNA-based therapies aimed at restoring PAX6 expression in the eye and pancreas. / Graduate

Pax6 encodes a highly conserved transcriptional regulator that is widely expressed during development of the eye, olfactory bulbs and central nervous system. Pax6-/- mice exhibit severe brain defects, lack eyes and nasal structures, and die at birth. Included among the functions of Pax6 are cell adhesion, cell cycle progression, axon guidance and boundary formation. The pallial-subpallial boundary (PSPB) is both a physical and gene expression boundary separating dorsal and ventral telencephalon. Pax6 is required for this boundary to develop. In Pax6-/- embryos, genes which normally have a sharp border of expression at the PSPB become ectopically expressed and the radial glial fasicles that make up the physical component of the boundary fail to form. There is also an increase in the number of interneurons migrating dorsally across the boundary to enter the cortex while corticofugal axons struggle to cross the PSPB and enter the ventral telencephalon. Here a novel tauGFP-Pax6 reporter mouse, DTy54, is described in which cells capable of expressing Pax6 are tauGFP positive. In general the expression pattern of tauGFP corresponds well with the known Pax6 expression pattern in the eye and forebrain and the gradient of cortical Pax6 expression from high rostro-laterally to low caudo-medially is also recapitulated by tauGFP. The cytoskeletal localisation of the tauGFP also labels cellular processes and the axons projecting from Pax6 positive cells such as those forming the optic nerve can be clearly seen. At E10.5 the forebrain expression patterns of tauGFP and Pax6 correspond exactly, but at later stages tauGFP expression can be seen in areas negative for Pax6. This can be seen at E12.5 in the ventral telencephalon and in both the dorsal and ventral telencephalon at E15.5. Pax6 and tauGFP expression colocalise more closely in the diencephalon. In situ hybridization analysis of Pax6 and tauGFP transcripts suggests that many of the discrepancies in expression seen at the protein level are due to a longer protein half-life for tauGFP than for Pax6. The expression of tauGFP allows the PSPB to be accurately dissected. The cells from this region can then be sorted by FACS to isolate cells expressing high levels of tauGFP and enrich for the Pax6 positive population. Microarray analysis of gene expression is this population of cells in Pax6+/+.DTy54+ and Pax6sey/sey. DTy54+ embryos is described here. This analysis identified many genes that show a significant change in expression at the PSPB in the absence of Pax6 expression including Ngn2, Lhx6, Neurod6 and CyclinD1 and 2. The biological processes and molecular functions in which these genes are involved were examined to provide insight into the role of Pax6 in this population of cells. Several processes previously reported to be regulated by Pax6 were identified together with a number of novel processes with which Pax6 has not formerly been associated. Some of these include cell cycle, neurogenesis, transcription and metabolic and signalling pathways. This study has also identified many novel downstream targets of Pax6, such as Sema3G and PlexinA4, which will help to elucidate the genetic basis for the Pax6sey/sey phenotype at the PSPB. The changes in expression levels of Ngn2, Lhx6 and Gsh2, identified by microarray, were validated by in situ hybridization, which showed a good correspondence with the microarray results.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.