SummaryThe mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct.

Pax6 encodes a highly conserved transcriptional regulator with two DNA-binding motifs, a paired domain and a paired-like homeodomain. Humans carrying PAX6 loss-of-function mutations suffer from abnormal development of the eyes (congenital aniridia) and brain. Small eye mice carrying Pax6 loss-of-function mutations provide a good model for these human conditions. Their analysis has demonstrated the critical importance of this transcription factor in multiple cell types and at several key stages of forebrain development. In the forebrain, Pax6 is critical for the establishment of the pallial-subpallial boundary, which separates dorsal (future cerebral cortex) and ventral (future striatum) telencephalic regions. Levels of Pax6 expression are critically important for cortical progenitor proliferation and its presence in a rostro-lateral(high) to caudo-medial(low) gradient in the cortex is necessary to establish rostro-lateral identities. Furthermore, axon guidance is disrupted in Pax6⁻/⁻ mutants: the majority of thalamocortical axons fail to enter the ventral telencephalon and those that do are unable to innervate their cortical targets. The extent to which the effects of Pax6 later in development are secondary to its effects in early patterning and proliferation remains largely unknown. This is likely to be clarified by future studies on the molecular mechanisms of action of Pax6 and, in particular, the identification of its downstream target genes. Such studies should also help generate an increasingly coherent understanding of how this pleiotropic transcription factor becomes involved in so many facets of neural development.

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.

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.

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 post-natal 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 post-natal 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.

Postnatal ΔO, a modifiable factor in neonatal care, impairs cortical development in a rodent model with preferential disadvantage to superficial neurons.

new expression domain for Wt1 during gastrulation. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell based therapies.Gene regulatory networks are key elements for understanding how the different central nervous system regions differentiate. In recent years our group has done an extensive molecular characterization of the chicken pretectal region, obtaining a precise map of AP and DV molecular codes with respect to differential histogenetic pattern (Ferran et al., 2007; 2009, in press). As in other CNS regions, such as the spinal cord, Bmps signals from the roof plate and Shh signals from the floor and basal plates are clearcut candidates for governing the dorso-ventral patterning of the pretectum. The Msx1 transcription factor plays a role in diencephalic development, acting as intermediary between Bmp and Wnt (Bach et al., 2006), but it is still unclear how its

Background and aims: Chronic lung disease of prematurity is strongly associated with adverse neurodevelopmental outcome. Oxygen therapy has been implicated as a factor leading to preterm brain injury. Preterm infants with intrauterine growth restriction (IUGR) are at increased risk of both chronic respiratory morbidity and neurodevelopmental impairment. We determined the effect of postnatal variable hyperoxia on the motor cortex in a rodent model of IUGR. Methods: Sprague-Dawley dams were fed 18% or 9% protein diet from E15 to P7. Pups were reared in air or variable hyperoxia from birth until P7. Brains were weighed and sections through the motor cortex were stained with cresyl violet. The thickness of the superficial (II-IV) and deep (V-VI) layers of the motor cortex was measured using ImageJ analysis. Staining for neuronal subtypes was performed using immunofluorescence and cell numbers were quantified. Results: Protein restricted pups had smaller brains than normally grown pups at P7 (p< 0.0001) but cortical thickness was preserved. Postnatal variable hyperoxia did not further affect brain weight at P7 in either group but did result in a decrease in cortical thickness in growth restricted pups (p=0.003). Thinning was observed in both superficial (p=0.01) and deep layers (p=0.03). However total numbers of superficial, callosal, corticothalamic and corticospinal neurons were preserved. Conclusions: Postnatal variable hyperoxia, a modifiable factor in neonatal care, impairs cortical growth without reducing total brain weight or neuron numbers in a preterm model of IUGR. This implies that the reduction in cortical thickness may be secondary to impaired cortical circuitry.