Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling.

Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N6-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis.

mSWI/SNF (BAF) Complexes Are Indispensable for the Neurogenesis and Development of Embryonic Olfactory Epithelium.

Neurogenesis is a key developmental event through which neurons are generated from neural stem/progenitor cells. Chromatin remodeling BAF (mSWI/SNF) complexes have been reported to play essential roles in the neurogenesis of the central nervous system. However, whether BAF complexes are required for neuron generation in the olfactory system is unknown. Here, we identified onscBAF and ornBAF complexes, which are specifically present in olfactory neural stem cells (oNSCs) and olfactory receptor neurons (ORNs), respectively. We demonstrated that BAF155 subunit is highly expressed in both oNSCs and ORNs, whereas high expression of BAF170 subunit is observed only in ORNs. We report that conditional deletion of BAF155, a core subunit in both onscBAF and ornBAF complexes, causes impaired proliferation of oNSCs as well as defective maturation and axonogenesis of ORNs in the developing olfactory epithelium (OE), while the high expression of BAF170 is important for maturation of ORNs. Interestingly, in the absence of BAF complexes in BAF155/BAF170 double-conditional knockout mice (dcKO), OE is not specified. Mechanistically, BAF complex is required for normal activation of Pax6-dependent transcriptional activity in stem cells/progenitors of the OE. Our findings unveil a novel mechanism mediated by the mSWI/SNF complex in OE neurogenesis and development.

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.

Regulation of Cell Delamination During Cortical Neurodevelopment and Implication for Brain Disorders.

Cortical development is dependent on key processes that can influence apical progenitor cell division and progeny. Pivotal among such critical cellular processes is the intricate mechanism of cell delamination. This indispensable cell detachment process mainly entails the loss of apical anchorage, and subsequent migration of the mitotic derivatives of the highly polarized apical cortical progenitors. Such apical progenitor derivatives are responsible for the majority of cortical neurogenesis. Many factors, including transcriptional and epigenetic/chromatin regulators, are known to tightly control cell attachment and delamination tendency in the cortical neurepithelium. Activity of these molecular regulators principally coordinate morphogenetic cues to engender remodeling or disassembly of tethering cellular components and external cell adhesion molecules leading to exit of differentiating cells in the ventricular zone. Improper cell delamination is known to frequently impair progenitor cell fate commitment and neuronal migration, which can cause aberrant cortical cell number and organization known to be detrimental to the structure and function of the cerebral cortex. Indeed, some neurodevelopmental abnormalities, including Heterotopia, Schizophrenia, Hydrocephalus, Microcephaly, and Chudley-McCullough syndrome have been associated with cell attachment dysregulation in the developing mammalian cortex. This review sheds light on the concept of cell delamination, mechanistic (transcriptional and epigenetic regulation) nuances involved, and its importance for corticogenesis. Various neurodevelopmental disorders with defective (too much or too little) cell delamination as a notable etiological underpinning are also discussed.

BAF (mSWI/SNF) complex regulates mediolateral cortical patterning in the developing forebrain.

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny.

Mapping of domain-mediated protein-protein interaction by SPOT peptide assay.

Identification of peptides mediating protein-protein interaction (PPI) is crucial for understanding the function of interlinked proteins in cellular processes and amino acid-associated diseases. Traditional PPI assays are laborious, involving the generation of many truncated proteins. SPOT peptide assay allows high-throughput detection of domains essential for PPI by synthesizing several hundred peptides on a cellulose membrane. Here, we present a rapid SPOT peptide protocol for identifying the binding motifs, which mediate interaction between the chromatin remodeling factors BAF155/BAF170 and the epigenetic factor Kdm6b. For complete details on the use and execution of this protocol, please refer to Narayanan et al. (2015).

Emerging Role of m6 A Methylome in Brain Development: Implications for Neurological Disorders and Potential Treatment.

Dynamic modification of RNA affords proximal regulation of gene expression triggered by non-genomic or environmental changes. One such epitranscriptomic alteration in RNA metabolism is the installation of a methyl group on adenosine [N6-methyladenosine (m6A)] known to be the most prevalent modified state of messenger RNA (mRNA) in the mammalian cell. The methylation machinery responsible for the dynamic deposition and recognition of m6A on mRNA is composed of subunits that play specific roles, including reading, writing, and erasing of m6A marks on mRNA to influence gene expression. As a result, peculiar cellular perturbations have been linked to dysregulation of components of the mRNA methylation machinery or its cofactors. It is increasingly clear that neural tissues/cells, especially in the brain, make the most of m6A modification in maintaining normal morphology and function. Neurons in particular display dynamic distribution of m6A marks during development and in adulthood. Interestingly, such dynamic m6A patterns are responsive to external cues and experience. Specific disturbances in the neural m6A landscape lead to anomalous phenotypes, including aberrant stem/progenitor cell proliferation and differentiation, defective cell fate choices, and abnormal synaptogenesis. Such m6A-linked neural perturbations may singularly or together have implications for syndromic or non-syndromic neurological diseases, given that most RNAs in the brain are enriched with m6A tags. Here, we review the current perspectives on the m6A machinery and function, its role in brain development and possible association with brain disorders, and the prospects of applying the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas13b system to obviate m6A-related neurological anomalies.

Intranuclear immunostaining-based FACS protocol from embryonic cortical tissue.

Cell sorting can be used to purify cell populations for cell type-specific molecular probing. Fluorescence-activated cell sorting (FACS) coupled with high-throughput sequencing affords molecular signature identification for specific cell types. FACS has many challenges that limit comprehensive cell purification from the brain, leading to incomplete molecular characterization. Here, we present the intranuclear immunostaining-based FACS protocol with several modified steps, which allows optimized nuclei/cell sorting from mouse or human embryonic cortical tissue for distinct downstream molecular investigation of basal intermediate progenitors.

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.

Molecular Profiling Reveals Involvement of ESCO2 in Intermediate Progenitor Cell Maintenance in the Developing Mouse Cortex.

Intermediate progenitor cells (IPCs) are neocortical neuronal precursors. Although IPCs play crucial roles in corticogenesis, their molecular features remain largely unknown. In this study, we aimed to characterize the molecular profile of IPCs. We isolated TBR2-positive (+) IPCs and TBR2-negative (-) cell populations in the developing mouse cortex. Comparative genome-wide gene expression analysis of TBR2+ IPCs versus TBR2- cells revealed differences in key factors involved in chromatid segregation, cell-cycle regulation, transcriptional regulation, and cell signaling. Notably, mutation of many IPC genes in human has led to intellectual disability and caused a wide range of cortical malformations, including microcephaly and agenesis of corpus callosum. Loss-of-function experiments in cortex-specific mutants of Esco2, one of the novel IPC genes, demonstrate its critical role in IPC maintenance, and substantiate the identification of a central genetic determinant of IPC biogenesis. Our data provide novel molecular characteristics of IPCs in the developing mouse cortex.

Conditional Loss of BAF (mSWI/SNF) Scaffolding Subunits Affects Specification and Proliferation of Oligodendrocyte Precursors in Developing Mouse Forebrain.

Oligodendrocytes are responsible for axon myelination in the brain and spinal cord. Generation of oligodendrocytes entails highly regulated multistage neurodevelopmental events, including proliferation, differentiation and maturation. The chromatin remodeling BAF (mSWI/SNF) complex is a notable regulator of neural development. In our previous studies, we determined the indispensability of the BAF complex scaffolding subunits BAF155 and BAF170 for neurogenesis, whereas their role in gliogenesis is unknown. Here, we show that the expression of BAF155 and BAF170 is essential for the genesis of oligodendrocytes during brain development. We report that the ablation of BAF155 and BAF170 in the dorsal telencephalic (dTel) neural progenitors or in oligodendrocyte-producing progenitors in the ventral telencephalon (vTel) in double-conditional knockout (dcKO) mouse mutants, perturbed the process of oligodendrogenesis. Molecular marker and cell cycle analyses revealed impairment of oligodendrocyte precursor specification and proliferation, as well as overt depletion of oligodendrocytes pool in dcKO mutants. Our findings unveil a central role of BAF155 and BAF170 in oligodendrogenesis, and thus substantiate the involvement of the BAF complex in the production of oligodendrocytes in the forebrain.

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.

H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion.

Increase in the size of human neocortex­acquired in evolution­accounts for the unique cognitive capacity of humans. This expansion reflects the evolutionarily enhanced proliferative ability of basal progenitors (BPs), including the basal radial glia and basal intermediate progenitors (bIPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in bIP amplification and cortical expansion. Through epigenetic profiling of sorted bIPs, we show that histone H3 lysine 9 acetylation (H3K9ac) is low in murine bIPs and high in human bIPs. Elevated H3K9ac preferentially increases bIP proliferation, increasing the size and folding of the normally smooth mouse neocortex. H3K9ac drives bIP amplification by increasing expression of the evolutionarily regulated gene, Trnp1, in developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture.

RBM15 Modulates the Function of Chromatin Remodeling Factor BAF155 Through RNA Methylation in Developing Cortex.

Chromatin remodeling factor BAF155 is an important regulator of many biological processes. As a core and scaffold subunit of the BAF (SWI/SNF-like) complex, BAF155 is capable of regulating the stability and function of the BAF complex. The spatiotemporal expression of BAF155 during embryogenesis is essential for various aspects of organogenesis, particularly in the brain development. However, our understanding of the mechanisms that regulate the expression and function of BAF155 is limited. Here, we report that RBM15, a subunit of the m6A methyltransferase complex, interacts with BAF155 mRNA and mediates BAF155 mRNA degradation through the mRNA methylation machinery. Ablation of endogenous RBM15 expression in cultured neuronal cells and in the developing cortex augmented the expression of BAF155. Conversely, RBM15 overexpression decreased BAF155 mRNA and protein levels, and perturbed BAF155 functions in vivo, including repression of BAF155-dependent transcriptional activity and delamination of apical radial glial progenitors as a hallmark of basal radial glial progenitor genesis. Furthermore, we demonstrated that the regulation of BAF155 by RBM15 depends on the activity of the mRNA methylation complex core catalytic subunit METTL3. Altogether, our findings reveal a new regulatory avenue that elucidates how BAF complex subunit stoichiometry and functional modulation are achieved in mammalian cells.

Structural brain anomalies in patients with FOXG1 syndrome and in Foxg1+/- mice.

OBJECTIVE: FOXG1 syndrome is a rare neurodevelopmental disorder associated with heterozygous FOXG1 variants or chromosomal microaberrations in 14q12. The study aimed at assessing the scope of structural cerebral anomalies revealed by neuroimaging to delineate the genotype and neuroimaging phenotype associations. METHODS: We compiled 34 patients with a heterozygous (likely) pathogenic FOXG1 variant. Qualitative assessment of cerebral anomalies was performed by standardized re-analysis of all 34 MRI data sets. Statistical analysis of genetic, clinical and neuroimaging data were performed. We quantified clinical and neuroimaging phenotypes using severity scores. Telencephalic phenotypes of adult Foxg1+/- mice were examined using immunohistological stainings followed by quantitative evaluation of structural anomalies. RESULTS: Characteristic neuroimaging features included corpus callosum anomalies (82%), thickening of the fornix (74%), simplified gyral pattern (56%), enlargement of inner CSF spaces (44%), hypoplasia of basal ganglia (38%), and hypoplasia of frontal lobes (29%). We observed a marked, filiform thinning of the rostrum as recurrent highly typical pattern of corpus callosum anomaly in combination with distinct thickening of the fornix as a characteristic feature. Thickening of the fornices was not reported previously in FOXG1 syndrome. Simplified gyral pattern occurred significantly more frequently in patients with early truncating variants. Higher clinical severity scores were significantly associated with higher neuroimaging severity scores. Modeling of Foxg1 heterozygosity in mouse brain recapitulated the associated abnormal cerebral morphology phenotypes, including the striking enlargement of the fornix. INTERPRETATION: Combination of specific corpus callosum anomalies with simplified gyral pattern and hyperplasia of the fornices is highly characteristic for FOXG1 syndrome.

Transcriptional and Epigenetic Control of Mammalian Olfactory Epithelium Development.

The postnatal mammalian olfactory epithelium (OE) represents a major aspect of the peripheral olfactory system. It is a pseudostratified tissue that originates from the olfactory placode and is composed of diverse cells, some of which are specialized receptor neurons capable of transducing odorant stimuli to afford the perception of smell (olfaction). The OE is known to offer a tractable miniature model for studying the systematic generation of neurons and glia that typify neural tissue development. During OE development, stem/progenitor cells that will become olfactory sensory neurons and/or non-neuronal cell types display fine spatiotemporal expression of neuronal and non-neuronal genes that ensures their proper proliferation, differentiation, survival, and regeneration. Many factors, including transcription and epigenetic factors, have been identified as key regulators of the expression of such requisite genes to permit normal OE morphogenesis. Typically, specific interactive regulatory networks established between transcription and epigenetic factors/cofactors orchestrate histogenesis in the embryonic and adult OE. Hence, investigation of these regulatory networks critical for OE development promises to disclose strategies that may be employed in manipulating the stepwise transition of olfactory precursor cells to become fully differentiated and functional neuronal and non-neuronal cell types. Such strategies potentially offer formidable means of replacing injured or degenerated neural cells as therapeutics for nervous system perturbations. This review recapitulates the developmental cellular diversity of the olfactory neuroepithelium and discusses findings on how the precise and cooperative molecular control by transcriptional and epigenetic machinery is indispensable for OE ontogeny.

ATP-Dependent Chromatin Remodeling During Cortical Neurogenesis.

The generation of individual neurons (neurogenesis) during cortical development occurs in discrete steps that are subtly regulated and orchestrated to ensure normal histogenesis and function of the cortex. Notably, various gene expression programs are known to critically drive many facets of neurogenesis with a high level of specificity during brain development. Typically, precise regulation of gene expression patterns ensures that key events like proliferation and differentiation of neural progenitors, specification of neuronal subtypes, as well as migration and maturation of neurons in the developing cortex occur properly. ATP-dependent chromatin remodeling complexes regulate gene expression through utilization of energy from ATP hydrolysis to reorganize chromatin structure. These chromatin remodeling complexes are characteristically multimeric, with some capable of adopting functionally distinct conformations via subunit reconstitution to perform specific roles in major aspects of cortical neurogenesis. In this review, we highlight the functions of such chromatin remodelers during cortical development. We also bring together various proposed mechanisms by which ATP-dependent chromatin remodelers function individually or in concert, to specifically modulate vital steps in cortical neurogenesis.

Epigenetic Regulation by BAF Complexes Limits Neural Stem Cell Proliferation by Suppressing Wnt Signaling in Late Embryonic Development.

During early cortical development, neural stem cells (NSCs) divide symmetrically to expand the progenitor pool, whereas, in later stages, NSCs divide asymmetrically to self-renew and produce other cell types. The timely switch from such proliferative to differentiative division critically determines progenitor and neuron numbers. However, the mechanisms that limit proliferative division in late cortical development are not fully understood. Here, we show that the BAF (mSWI/SNF) complexes restrict proliferative competence and promote neuronal differentiation in late corticogenesis. Inactivation of BAF complexes leads to H3K27me3-linked silencing of neuronal differentiation-related genes, with concurrent H3K4me2-mediated activation of proliferation-associated genes via de-repression of Wnt signaling. Notably, the deletion of BAF complexes increased proliferation of neuroepithelial cell-like NSCs, impaired neuronal differentiation, and exerted a Wnt-dependent effect on neocortical and hippocampal development. Thus, these results demonstrate that BAF complexes act as both activators and repressors to control global epigenetic and gene expression programs in late corticogenesis.

Chromatin Remodeling BAF155 Subunit Regulates the Genesis of Basal Progenitors in Developing Cortex.

The abundance of basal progenitors (BPs), basal radial glia progenitors (bRGs) and basal intermediate progenitors (bIPs), in primate brain has been correlated to the high degree of cortical folding. Here we examined the role of BAF155, a subunit of the chromatin remodeling BAF complex, in generation of cortical progenitor heterogeneity. The conditional deletion of BAF155 led to diminished bIP pool and increased number of bRGs, due to delamination of apical RGs. We found that BAF155 is required for normal activity of neurogenic transcription factor PAX6, thus controlling the expression of genes that are involved in bIP specification, cell-cell interaction, and establishment of adherens junction. In a PAX6-dependent manner, BAF155 regulates the expression of the CDC42 effector protein CEP4, thereby controlling progenitor delamination. Furthermore, BAF155-dependent chromatin remodeling seems to exert a specific role in the genesis of BPs through the regulation of human RG-specific genes (such as Foxn4) that possibly acquired evolutionary significance.