Mutant Cortex Research Articles

Aberrant generation of dentate gyrus granule cells is associated with epileptic susceptibility in p53 conditional knockout mice.

Neuronal apoptosis is a mechanism used to clear the cells of oxidative stress or DNA damage and refine the final number of neurons for a functional neuronal circuit. The tumor suppressor protein p53 is a key regulator of the cell cycle and serves as a checkpoint for eliminating neurons with high DNA damage, hyperproliferative signals or cellular stress. During development, p53 is largely expressed in progenitor cells. In the adult brain, p53 expression is restricted to the neurogenic niches where it regulates cell proliferation and self-renewal. To investigate the functional consequences of p53 deletion in the cortex and hippocampus, we generated a conditional mutant mouse (p53-cKO) in which p53 is deleted from pallial progenitors and their derivatives. Surprisingly, we did not find any significant change in the number of neurons in the mutant cortex or CA region of the hippocampus compared with control mice. However, p53-cKO mice exhibit more proliferative cells in the subgranular zone of the dentate gyrus and more granule cells in the granular cell layer. Glutamatergic synapses in the CA3 region are more numerous in p53-cKO mice compared with control littermates, which correlates with overexcitability and higher epileptic susceptibility in the mutant mice.

Pyramidal neurons proportionately alter the identity and survival of specific cortical interneuron subtypes.

The mammalian cerebral cortex comprises a complex neuronal network that maintains a delicate balance between excitatory neurons and inhibitory interneurons. Previous studies, including our own research, have shown that specific interneuron subtypes are closely associated with particular pyramidal neuron types, forming stereotyped local inhibitory microcircuits. However, the developmental processes that establish these precise networks are not well understood. Here we show that pyramidal neuron types are instrumental in driving the terminal differentiation and maintaining the survival of specific associated interneuron subtypes. In a wild-type cortex, the relative abundance of different interneuron subtypes aligns precisely with the pyramidal neuron types to which they synaptically target. In Fezf2 mutant cortex, characterized by the absence of layer 5 pyramidal tract neurons and an expansion of layer 6 intratelencephalic neurons, we observed a corresponding decrease in associated layer 5b interneurons and an increase in layer 6 subtypes. Interestingly, these shifts in composition are achieved through mechanisms specific to different interneuron types. While SST interneurons adjust their abundance to the change in pyramidal neuron prevalence through the regulation of programmed cell death, parvalbumin interneurons alter their identity. These findings illustrate two key strategies by which the dynamic interplay between pyramidal neurons and interneurons allows local microcircuits to be sculpted precisely. These insights underscore the precise roles of extrinsic signals from pyramidal cells in the establishment of interneuron diversity and their subsequent integration into local cortical microcircuits.

Pyramidal neurons proportionately alter the identity and survival of specific cortical interneuron subtypes.

The mammalian cerebral cortex comprises a complex neuronal network that maintains a delicate balance between excitatory neurons and inhibitory interneurons. Previous studies, including our own research, have shown that specific interneuron subtypes are closely associated with particular pyramidal neuron types, forming stereotyped local inhibitory microcircuits. However, the developmental processes that establish these precise networks are not well understood. Here we show that pyramidal neuron types are instrumental in driving the terminal differentiation and maintaining the survival of specific associated interneuron subtypes. In a wild-type cortex, the relative abundance of different interneuron subtypes aligns precisely with the pyramidal neuron types to which they synaptically target. In Fezf2 mutant cortex, characterized by the absence of layer 5 pyramidal tract neurons and an expansion of layer 6 intratelencephalic neurons, we observed a corresponding decrease in associated layer 5b interneurons and an increase in layer 6 subtypes. Interestingly, these shifts in composition are achieved through mechanisms specific to different interneuron types. While SST interneurons adjust their abundance to the change in pyramidal neuron prevalence through the regulation of programmed cell death, parvalbumin interneurons alter their identity. These findings illustrate two key strategies by which the dynamic interplay between pyramidal neurons and interneurons allows local microcircuits to be sculpted precisely. These insights underscore the precise roles of extrinsic signals from pyramidal cells in the establishment of interneuron diversity and their subsequent integration into local cortical microcircuits.

Analysis of genes differentially expressed in the cortex of mice with the Tbl1xr1Y446C/Y446C variant

Transducin β-like 1 X-linked receptor 1 (mouse Tbl1xr1) or TBL1X/Y related 1 (human TBL1XR1), part of the NCoR/SMRT corepressor complex, is involved in nuclear receptor signaling. Variants in TBL1XR1 cause a variety of neurodevelopmental disorders including Pierpont syndrome caused by the p.Tyr446Cys variant. We recently reported a mouse model carrying the Tbl1xr1Y446C/Y446C variant as a model for Pierpont syndrome.To obtain insight into mechanisms involved in altered brain development we studied gene expression patterns in the cortex of mutant and wild type (WT) mice, using RNA-sequencing, differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA), weighted gene correlation network analysis (WGCNA) and hub gene analysis. We validated results in mutated mouse cortex, as well as in BV2 and SK-N-AS cell lines, in both of which Tbl1xr1 was knocked down by siRNA.Two DEGs (adj.P. Val < 0.05) were found in the cortex, Mpeg1 (downregulated in mutant mice) and 2900052N01Rik (upregulated in mutant mice). GSEA, WGCNA and hub gene analysis demonstrated changes in genes involved in ion channel function and neuroinflammation in the cortex of the Tbl1xr1Y446C/Y446C mice. The lowered expression of ion channel genes Kcnh3 and Kcnj4 mRNA was validated in the mutant mouse cortex, and increased expression of TRIM9, associated with neuroinflammation, was confirmed in the SK-N-AS cell line.Conclusively, our results show altered expression of genes involved in ion channel function and neuroinflammation in the cortex of the Tbl1xr1Y446C/Y446C mice. These may partly explain the impaired neurodevelopment observed in individuals with Pierpont syndrome and related TBL1XR1-related disorders.

H3 Acetylation-Induced Basal Progenitor Generation and Neocortex Expansion Depends on the Transcription Factor Pax6.

Enrichment of basal progenitors (BPs) in the developing neocortex is a central driver of cortical enlargement. The transcription factor Pax6 is known as an essential regulator in generation of BPs. H3 lysine 9 acetylation (H3K9ac) has emerged as a crucial epigenetic mechanism that activates the gene expression program required for BP pool amplification. In this current work, we applied immunohistochemistry, RNA sequencing, chromatin immunoprecipitation and sequencing, and the yeast two-hybrid assay to reveal that the BP-genic effect of H3 acetylation is dependent on Pax6 functionality in the developing mouse cortex. In the presence of Pax6, increased H3 acetylation caused BP pool expansion, leading to enhanced neurogenesis, which evoked expansion and quasi-convolution of the mouse neocortex. Interestingly, H3 acetylation activation exacerbates the BP depletion and corticogenesis reduction effect of Pax6 ablation in cortex-specific Pax6 mutants. Furthermore, we found that H3K9 acetyltransferase KAT2A/GCN5 interacts with Pax6 and potentiates Pax6-dependent transcriptional activity. This explains a genome-wide lack of H3K9ac, especially in the promoter regions of BP-genic genes, in the Pax6 mutant cortex. Together, these findings reveal a mechanistic coupling of H3 acetylation and Pax6 in orchestrating BP production and cortical expansion through the promotion of a BP gene expression program during cortical development.

The L1 cell adhesion molecule constrains dendritic spine density in pyramidal neurons of the mouse cerebral cortex.

A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas (prefrontal cortex layer 2/3, motor cortex layer 5, visual cortex layer 4. The Ankyrin binding motif (FIGQY) in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome.

Layers 3 and 4 Neurons of the Bilateral Whisker-Barrel Cortex

In Robo3R3-5cKO mouse brain, rhombomere 3-derived trigeminal principal nucleus (PrV) neurons project bilaterally to the somatosensory thalamus. As a consequence, whisker-specific neural modules (barreloids and barrels) representing whiskers on both sides of the face develop in the sensory thalamus and the primary somatosensory cortex. We examined the morphological complexity of layer 4 barrel cells, their postsynaptic partners in layer 3, and functional specificity of layer 3 pyramidal cells.Layer 4 spiny stellate cells form much smaller barrels and their dendritic fields are more focalized and less complex compared to controls, while layer 3 pyramidal cells did not show notable differences. Using in vivo 2-photon imaging of a genetically encoded fluorescent [Ca2+] sensor, we visualized neural activity in the normal and Robo3R3-5cKO barrel cortex in response to ipsi- and contralateral single whisker stimulation. Layer 3 neurons in control animals responded only to their contralateral whiskers, while in the mutant cortex layer 3 pyramidal neurons showed both ipsi- and contralateral whisker responses. These results indicate that bilateral whisker map inputs stimulate different but neighboring groups of layer 3 neurons which normally relay contralateral whisker-specific information to other cortical areas.

Loss of BAF Complex in Developing Cortex Perturbs Radial Neuronal Migration in a WNT Signaling-Dependent Manner.

Radial neuronal migration is a key neurodevelopmental event indispensable for proper cortical laminar organization. Cortical neurons mainly use glial fiber guides, cell adhesion dynamics, and cytoskeletal remodeling, among other discrete processes, to radially trek from their birthplace to final layer positions. Dysregulated radial migration can engender cortical mis-lamination, leading to neurodevelopmental disorders. Epigenetic factors, including chromatin remodelers have emerged as formidable regulators of corticogenesis. Notably, the chromatin remodeler BAF complex has been shown to regulate several aspects of cortical histogenesis. Nonetheless, our understanding of how BAF complex regulates neuronal migration is limited. Here, we report that BAF complex is required for neuron migration during cortical development. Ablation of BAF complex in the developing mouse cortex caused alteration in the cortical gene expression program, leading to loss of radial migration-related factors critical for proper cortical layer formation. Of note, BAF complex inactivation in cortex caused defective neuronal polarization resulting in diminished multipolar-to-bipolar transition and eventual disruption of radial migration of cortical neurons. The abnormal radial migration and cortical mis-lamination can be partly rescued by downregulating WNT signaling hyperactivity in the BAF complex mutant cortex. By implication, the BAF complex modulates WNT signaling to establish the gene expression program required for glial fiber-dependent neuronal migration, and cortical lamination. Overall, BAF complex has been identified to be crucial for cortical morphogenesis through instructing multiple aspects of radial neuronal migration in a WNT signaling-dependent manner.

MACF1, Involved in the 1p34.2p34.3 Microdeletion Syndrome, is Essential in Cortical Progenitor Polarity and Brain Integrity.

1p34.2p34.3 deletion syndrome is characterized by an increased risk for autism. Microtubule Actin Crosslinking Factor 1 (MACF1) is one candidate gene for this syndrome. It is unclear, however, how MACF1 deletion is linked to brain development and neurodevelopmental deficits. Here we report on Macf1 deletion in the developing mouse cerebral cortex, focusing on radial glia polarity and morphological integrity, as these are critical factors in brain formation. We found that deleting Macf1 during cortical development resulted in double cortex/subcortical band heterotopia as well as disrupted cortical lamination. Macf1-deleted radial progenitors showed increased proliferation rates compared to control cells but failed to remain confined within their defined proliferation zone in the developing brain. The overproliferation of Macf1-deleted radial progenitors was associated with elevated cell cycle speed and re-entry. Microtubule stability and actin polymerization along the apical ventricular area were decreased in the Macf1 mutant cortex. Correspondingly, there was a disconnection between radial glial fibers and the apical and pial surfaces. Finally, we observed that Macf1-mutant mice exhibited social deficits and aberrant emotional behaviors. Together, these results suggest that MACF1 plays a critical role in cortical progenitor proliferation and localization by promoting glial fiber stabilization and polarization. Our findings may provide insights into the pathogenic mechanism underlying the 1p34.2p34.3 deletion syndrome.

Dyrk1a Mutations Cause Undergrowth of Cortical Pyramidal Neurons via Dysregulated Growth Factor Signaling

BackgroundMutations in DYRK1A are a cause of microcephaly, autism spectrum disorder, and intellectual disability; however, the underlying cellular and molecular mechanisms are not well understood. MethodsWe generated a conditional mouse model using Emx1-cre, including conditional heterozygous and homozygous knockouts, to investigate the necessity of Dyrk1a in the cortex during development. We used unbiased, high-throughput phosphoproteomics to identify dysregulated signaling mechanisms in the developing Dyrk1a mutant cortex as well as classic genetic modifier approaches and pharmacological therapeutic intervention to rescue microcephaly and neuronal undergrowth caused by Dyrk1a mutations. ResultsWe found that cortical deletion of Dyrk1a in mice causes decreased brain mass and neuronal size, structural hypoconnectivity, and autism-relevant behaviors. Using phosphoproteomic screening, we identified growth-associated signaling cascades dysregulated upon Dyrk1a deletion, including TrkB-BDNF (tyrosine receptor kinase B–brain-derived neurotrophic factor), an important regulator of ERK/MAPK (extracellular signal-regulated kinase/mitogen-activated protein kinase) and mTOR (mammalian target of rapamycin) signaling. Genetic suppression of Pten or pharmacological treatment with IGF-1 (insulin-like growth factor-1), both of which impinge on these signaling cascades, rescued microcephaly and neuronal undergrowth in neonatal mutants. ConclusionsAltogether, these findings identify a previously unknown mechanism through which Dyrk1a mutations disrupt growth factor signaling in the developing brain, thus influencing neuronal growth and connectivity. Our results place DYRK1A as a critical regulator of a biological pathway known to be dysregulated in humans with autism spectrum disorder and intellectual disability. In addition, these data position Dyrk1a within a larger group of autism spectrum disorder/intellectual disability risk genes that impinge on growth-associated signaling cascades to regulate brain size and connectivity, suggesting a point of convergence for multiple autism etiologies.

Ablation of Vti1a/1b Triggers Neural Progenitor Pool Depletion and Cortical Layer 5 Malformation in Late-embryonic Mouse Cortex

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders.

Early 17β-estradiol treatment reduces seizures but not abnormal behaviour in mice with expanded polyalanine tracts in the Aristaless related homeobox gene (ARX)

Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17β-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures. Treated PA2 mice had an earlier onset of seizures coupled with a subsequent reduction in seizures later in postnatal life, with a complete absence of any seizures during the analysis at 7 weeks of age. Despite the reduction in seizures, 17β-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (Log2FC > ± 0.5, P-value<0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17β-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. We conclude that 17β-estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome nor improve mortality or behavioural and cognitive deficits.

In vivo stress granule misprocessing evidenced in a FUS knock-in ALS mouse model.

Many RNA-binding proteins, including TDP-43, FUS, and TIA1, are stress granule components, dysfunction of which causes amyotrophic lateral sclerosis (ALS). However, whether a mutant RNA-binding protein disrupts stress granule processing in vivo in pathogenesis is unknown. Here we establish a FUS ALS mutation, p.R521C, knock-in mouse model that carries impaired motor ability and late-onset motor neuron loss. In disease-susceptible neurons, stress induces mislocalization of mutant FUS into stress granules and upregulation of ubiquitin, two hallmarks of disease pathology. Additionally, stress aggravates motor performance decline in the mutant mouse. By using two-photon imaging in TIA1-EGFP transduced animals, we document more intensely TIA1-EGFP-positive granules formed hours but cleared weeks after stress challenge in neurons in the mutant cortex. Moreover, neurons with severe granule misprocessing die days after stress challenge. Therefore, we argue that stress granule misprocessing is pathogenic in ALS, and the model we provide here is sound for further disease mechanistic study.

EphB2 receptor cell-autonomous forward signaling mediates auditory memory recall and learning-driven spinogenesis

While ephrin-B ligands and EphB receptors are expressed to high levels in the learning centers of the brain, it remains largely unknown how their trans-synaptic interactions contribute to memory. We find that EphB2 forward signaling is needed for contextual and sound-evoked memory recall and that constitutive over-activation of the receptor’s intracellular tyrosine kinase domain results in enhanced memory. Loss of EphB2 expression does not affect the number of neurons activated following encoding, although a reduction of neurons activated after the sound-cued retrieval test was detected in the auditory cortex and hippocampal CA1. Further, spine density and maturation was reduced in the auditory cortex of mutants especially in the neurons that were dual-activated during both encoding and retrieval. Our data demonstrates that trans-synaptic ephrin-B-EphB2 interactions and forward signaling facilitate neural activation and structural plasticity in learning-associated neurons involved in the generation of memories.

PlexinD1 and Sema3E determine laminar positioning of heterotopically projecting callosal neurons

The corpus callosum is the largest bundle of commissural fibres that transfer information between the two cerebral hemispheres. Callosal projection neurons (CPNs) are a diverse population of pyramidal neurons within the neocortex that mainly interconnect homotopic regions of the opposite cortices. Nevertheless, some CPNs are involved in heterotopic projections between distinct cortical areas or to subcortical regions such as the striatum. In this study, we showed that the axon guidance receptor PlexinD1 is expressed by a large proportion of heterotopically projecting CPNs in layer 5A of the primary somatosensory (S1) and motor (M1) areas. Retrograde tracing of M1 CPNs projecting to the contralateral striatum revealed the presence of ectopic neurons aberrantly located in layers 2/3 of Plxnd1 and Sema3e mutant cortices. These results showed that Sema3E/PlexinD1 signalling controls the laminar distribution of heterotopically projecting CPNs.

Extracellular Pax6 Regulates Tangential Cajal-Retzius Cell Migration in the Developing Mouse Neocortex.

The embryonic mouse cortex displays a striking low caudo-medial and high rostro-lateral graded expression of the homeoprotein transcription factor Pax6, which presents both cell autonomous and direct noncell autonomous activities. Through the genetic induction of anti-Pax6 single-chain antibody secretion, we have analyzed Pax6 noncell autonomous activity on the migration of cortical hem- and septum-derived Cajal-Retzius (CR) neurons by live imaging of flat mount developing cerebral cortices. Blocking extracellular Pax6 disrupts tangential CR cell migration patterns by decreasing the distance traveled and changing both directionality and depth at which CR cells migrate. Tracking of single CR cells in mutant cortices revealed that extracellular Pax6 neutralization enhances contact repulsion in medial regions yet reduces it in lateral regions. This study demonstrates that secreted Pax6 controls neuronal migration and distribution and suggests that it acts as a bona fide morphogen at an early stage of cerebral cortex development.

The Noonan Syndrome-linked Raf1L613V mutation drives increased glial number in the mouse cortex and enhanced learning.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.

Cajal-Retzius neurons are required for the development of the human hippocampal fissure.

Cajal-Retzius neurons (CRN) are the main source of Reelin in the marginal zone of the developing neocortex and hippocampus (HC). They also express the transcription factor p73 and are complemented by later-appearing GABAergic Reelin+ interneurons. The human dorsal HC forms at gestational week 10 (GW10), when it develops a rudimentary Ammonic plate and incipient dentate migration, although the dorsal hippocampal fissure (HF) remains shallow and contains few CRN. The dorsal HC transforms into the indusium griseum (IG), concurrently with the rostro-caudal appearance of the corpus callosum, by GW14-17. Dorsal and ventral HC merge at the site of the former caudal hem, which is located at the level of the future atrium of the lateral ventricle and closely connected with the choroid plexus. The ventral HC forms at GW11 in the temporal lobe. The ventral HF is wide open at GW14-16 and densely populated by large numbers of CRNs. These are in intimate contact with the meninges and meningeal blood vessels, suggesting signalling through diverse pathways. At GW17, the fissure deepens and begins to fuse, although it is still marked by p73/Reelin+ CRNs. The p73KO mouse illustrates the importance of p73 in CRN for HF formation. In the mutant, Tbr1/Reelin+ CRNs are born in the hem but do not leave it and subsequently disappear, so that the mutant cortex and HC lack CRN from the onset of corticogenesis. The HF is absent, which leads to profound architectonic alterations of the HC. To determine which p73 isoform is important for HF formation, isoform-specific TAp73- and DeltaNp73-deficient embryonic and early postnatal mice were examined. In both mutants, the number of CRNs was reduced, but each of their phenotypes was much milder than in the global p73KO mutant missing both isoforms. In the TAp73KO mice, the HF of the dorsal HC failed to form, but was present in the ventral HC. In the DeltaNp73KO mice, the HC had a mild patterning defect along with a shorter HF. Complex interactions between both isoforms in CRNs may contribute to their crucial activity in the developing brain.

Conditional Inactivation of Pen-2 in the Developing Neocortex Leads to Rapid Switch of Apical Progenitors to Basal Progenitors.

The transition of apical progenitors (APs) to basal progenitors (BPs) is an important neurogenic process during cortical expansion. Presenilin enhancer 2 (Pen-2, also named as Psenen) is a key subunit of γ-secretase and has been implicated in neurodevelopmental disease. However, it remains unknown how Pen-2 may regulate the maintenance of APs. To address this question, we generated a conditional KO (cKO) mouse in which Pen-2 is specifically inactivated in neural progenitor cells in the telencephalon. Both male and female embryos were used. We show that Pen-2 cKO cortices display remarkable depletion of Aps, but transient increase on BPs, compared with controls. We demonstrate that the proliferation rate of APs or BPs is not changed, but the switch of APs to BPs is dramatically accelerated in Pen-2 cKO cortices. Molecular analyses reveal decreased levels of Hes1 and Hes5 but increased levels of Ngn2 and NeuroD1 in Pen-2 KO cells. We report that expression of Notch1 intracellular domain in Pen-2 cKO cortices restores the population of APs and BPs. In summary, these findings highlight a central role of the Notch signaling in Pen-2-dependent maintenance of neural stem cells in the developing neocortex.SIGNIFICANCE STATEMENT Presenilin enhancer 2 (Pen-2) has been implicated in neurodevelopmental disease. However, mechanisms by which Pen-2 regulates cortical development are not understood. In this study, we generated neural progenitor cell-specific Pen-2 conditional KO mice. We observe depletion of apical progenitors and transiently increased the number of basal progenitors in the developing neocortex of Pen-2 mutant mice. Mechanistic analyses reveal decreased levels of Hes1 and Hes5, but increased levels of neurogenic transcription factors in Pen-2 mutant cortices, compared with controls. We demonstrate that reintroduction of Notch intracellular domain into mutant mice restores the population of apical progenitors to basal progenitors. The above findings strongly suggest that the Pen-2-Notch pathway plays an essential role in the maintenance of neural stem cells during cortical development.

The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1.

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.