Spatial and temporal regulation of cerebral cortex development by the transcription factor pax6

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 Required at the Telencephalic Pallial-Subpallial Boundary for the Generation of Neuronal Diversity in the Postnatal Limbic System

During embryogenesis, the pallial-subpallial boundary (PSB) divides the two main progenitor domains in the telencephalon: the pallium, the major source of excitatory neurons, and the subpallium, the major source of inhibitory neurons. The PSB is formed at the molecular interface between the pallial (high Pax6+) and subpallial (high Gsx2+) ventricular zone (VZ) compartments. Initially, the PSB contains cells that express both Pax6 and Gsx2, but during later stages of development this boundary is largely refined into two separate compartments. In this study we examined the developmental mechanisms underlying PSB boundary formation and the postnatal consequences of conditional loss of Pax6 function at the PSB on neuronal fate in the amygdala and olfactory bulb, two targets of PSB-derived migratory populations. Our cell fate and time-lapse imaging analyses reveal that the sorting of Pax6+ and Gsx2+ progenitors during embryogenesis is the result of a combination of changes in gene expression and cell movements. Interestingly, we find that in addition to giving rise to inhibitory neurons in the amygdala and olfactory bulb, Gsx2+ progenitors generate a subpopulation of amygdala excitatory neurons. Consistent with this finding, targeted conditional ablation of Pax6 in Gsx2+ progenitors results in discrete local embryonic patterning defects that are linked to changes in the generation of subsets of postnatal excitatory and inhibitory neurons in the amygdala and inhibitory neurons in the olfactory bulb. Thus, in PSB progenitors, Pax6 plays an important role in the generation of multiple subtypes of neurons that contribute to the amygdala and olfactory bulb

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not

Regulatory role of Pax6 on cell division cycle associated 7 and cortical progenitor cell proliferation

Forebrain development is controlled by a set of transcription factors which are expressed in dynamic spatiotemporal patterns in the embryonic forebrain and are known to regulate complex gene networks. Pax6 is a transcription factor that regulates corticogenesis and mutations affecting Pax6 protein levels cause neurodevelopmental defects in the eyes and forebrain in both humans and mice. In previous studies, it was shown that the graded expression pattern of Pax6 protein, which is high rostro-laterally to low caudo-medially in the cerebral cortex, is critical for its control of cell cycle progression and proliferation of cortical progenitors. However the underlying mechanisms are still unclear. Based on a microarray analysis carried out in our laboratory, a number of cell cycle-related candidate genes that may be affected by Pax6 have been identified. One such gene, Cell division cycle associated 7 (Cdca7) is expressed in a counter-gradient against that of Pax6. In my current study, I found that Cdca7 mRNA expression in the telencephalon is upregulated in Pax6 null (Small eye) mutants and downregulated in mice that overexpress PAX6 (PAX77) across developing time points from E12.5 to E15.5. There are several potential Pax6 binding motifs located in the genomic locus upstream of Cdca7. However, by chromatin immunoprecipitation, it is showed that none of the predicted binding sites are physically bound by Pax6. Promoter luciferase assays using fragments combining five suspected binding motifs show that Pax6 is functionally critical. Cdca7 is also identified as a Myc and E2F1 direct target and is upregulated in some tumours but its biological role is not fully understood. Current work using in utero electroporation to overexpress Cdca7 around the lateral telencephalon, where Cdca7 expression levels are normally low, tested the effects on the proliferation and differentiation of cortical progenitor cells in this region. In E12.5 mice embryos, overexpression of Cdca7 protein causes fewer intermediate progenitor cells and post-mitotic neurons to be produced but these effects were not found in E14.5 embryos. This result implies that Cdca7 may affect cell fate decision during cortical development

Spatial and temporal regulation of cerebral cortex development by the transcription factor pax6

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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The Generation of Superficial Cortical Layers Is Regulated by Levels of the Transcription Factor Pax6

The ventricular zone (VZ) of the embryonic dorsal telencephalon is a major site for generating cortical projection neurons. The transcription factor Pax6 is highly expressed in apical progenitors (APs) residing in the VZ from the earliest stages of corticogenesis. Previous studies mainly focused on Pax6−/− mice have implicated Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of superficial cortical layers. We analyzed the developing cortex of PAX77 transgenic mice that overexpress Pax6 in its normal domains of expression. We show that Pax6 overexpression increases cell cycle length of APs and drives the system toward 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, whereas radial migration and lamina specification of cortical neurons are not affected by Pax6 overexpression. Conditional deletion of Pax6 in cortical progenitors at midstages of corticogenesis, by using a tamoxifen-inducible Emx1-CreER line, affected both numbers and specification of late-born neurons in superficial layers of the mutant cortex. Our analyses suggest that correct levels of Pax6 are essential for normal production of superficial layers of the cortex