Cortical Progenitors Research Articles

Centrosome regulation and function in mammalian cortical neurogenesis.

As the primary microtubule-organizing center in animal cells, centrosomes regulate microtubule cytoskeleton to support various cellular behaviors. They also serve as the base for nucleating primary cilia, the hub of diverse signaling pathways. Cells typically possess one centrosome that contains two inequal centrioles and undergoes semi-conservative duplication during cell division, resulting in two centrosomes with an inherent asymmetry in age and properties. While the centrosome is ubiquitously present, mutations of centrosome proteins are strongly associated with human microcephaly characterized by a small cerebral cortex, underscoring the importance of an intact centrosome in supporting cortical neurogenesis. Here we review recent advances on centrosome regulation and function in mammalian cortical neural progenitors and discuss the implications for a better understanding of cortical neurogenesis and related disease mechanisms.

Rack1 is essential for corticogenesis by preventing p21-dependent senescence in neural stem cells.

Normal neurodevelopment relies on intricate signaling pathways that balance neural stem cell (NSC) self-renewal, maturation, and survival. Disruptions lead to neurodevelopmental disorders, including microcephaly. Here, we implicate the inhibition of NSC senescence as a mechanism underlying neurogenesis and corticogenesis. We report that the receptor for activated C kinase (Rack1), a family member of WD40-repeat (WDR) proteins, is highly enriched in NSCs. Deletion of Rack1 in developing cortical progenitors leads to a microcephaly phenotype. Strikingly, the absence of Rack1 decreases neurogenesis and promotes a cellular senescence phenotype in NSCs. Mechanistically, the senescence-related p21 signaling pathway is dramatically activated in Rack1 null NSCs, and removal of p21 significantly rescues the Rack1-knockout phenotype invivo. Finally, Rack1 directly interacts with Smad3 to suppress the activation of transforming growth factor (TGF)-β/Smad signaling pathway, which plays a critical role in p21-mediated senescence. Our data implicate Rack1-driven inhibition of p21-induced NSC senescence as a critical mechanism behind normal cortical development.

An Early Cortical Progenitor-Specific Mechanism Regulates Thalamocortical Innervation.

The cortical subplate is critical in regulating the entry of thalamocortical sensory afferents into the cortex. These afferents reach the subplate at embryonic day (E)15.5 in the mouse, but "wait" for several days, entering the cortical plate postnatally. We report that when transcription factor LHX2 is lost in E11.5 cortical progenitors, which give rise to subplate neurons, thalamocortical afferents display premature, exuberant ingrowth into the E15.5 cortex. Embryonic mutant subplate neurons are correctly positioned below the cortical plate, but they display an altered transcriptome and immature electrophysiological properties during the waiting period. The sensory thalamus in these cortex-specific Lhx2 mutants displays atrophy and by postnatal day (P) 7, sensory innervation to the cortex is nearly eliminated leading to a loss of the somatosensory barrels. Strikingly, these phenotypes do not manifest if LHX2 is lost in postmitotic subplate neurons, and the transcriptomic dysregulation in the subplate resulting from postmitotic loss of LHX2 is vastly distinct from that seen when LHX2 is lost in progenitors. These results demonstrate a mechanism operating in subplate progenitors that has profound consequences on the growth of thalamocortical axons into the cortex.SIGNIFICANCE STATEMENT Thalamocortical nerves carry sensory information from the periphery to the cortex. When they first grow into the embryonic cortex, they "wait" at the subplate, a structure critical for the guidance and eventual connectivity of thalamic axons with their cortical targets. How the properties of subplate neurons are regulated is unclear. We report that transcription factor LHX2 is required in the progenitor "mother" cells of the cortical primordium when they are producing their "daughter" subplate neurons, in order for the thalamocortical pathway to wait at the subplate. Without LHX2 function in subplate progenitors, thalamocortical axons grow past the subplate, entering the cortical plate prematurely. This is followed by their eventual attrition and, consequently, a profound loss of sensory innervation of the mature cortex.

Loss of CC2D1A in Glutamatergic Neurons Results in Autistic-Like Features in Mice.

Biallelic loss-of-function mutations in Coiled-coil and C2 domain containing 1A (CC2D1A) cause autosomal recessive intellectual disability, sometimes comorbid with other neurodevelopmental disabilities, such as autism spectrum disorder (ASD) and seizures. We recently reported that conditional deletion of Cc2d1a in glutamatergic neurons of the postnatal mouse forebrain leads to impaired hippocampal synaptic plasticity and cognitive function. However, the pathogenic origin of the autistic features of CC2D1A deficiency remains elusive. Here, we confirmed that CC2D1A is highly expressed in the cortical zones during embryonic development. Taking advantage of Cre-LoxP-mediated gene deletion strategy, we generated a novel line of Cc2d1a conditional knockout (cKO) mice by crossing floxed Cc2d1a mice with Emx1-Cre mice, in which CC2D1A is ablated specifically in glutamatergic neurons throughout all embryonic and adult stages. We found that CC2D1A deletion leads to a trend toward decreased number of cortical progenitor cells at embryonic day 12.5 and alters the cortical thickness on postnatal day 10. In addition, male Cc2d1a cKO mice display autistic-like phenotypes including self-injurious repetitive grooming and aberrant social interactions. Loss of CC2D1A also results in decreased complexity of apical dendritic arbors of medial prefrontal cortex (mPFC) layer V pyramidal neurons and increased synaptic excitation/inhibition (E/I) ratio in the mPFC. Notably, chronic treatment with minocycline rescues behavioral and morphological abnormalities, as well as E/I changes, in male Cc2d1a cKO mice. Together, these findings indicate that male Cc2d1a cKO mice recapitulate autistic-like phenotypes of human disorder and suggest that minocycline has both structural and functional benefits in treating ASD.

Human-Specific Genes, Cortical Progenitor Cells, and Microcephaly.

Over the past few years, human-specific genes have received increasing attention as potential major contributors responsible for the 3-fold difference in brain size between human and chimpanzee. Accordingly, mutations affecting these genes may lead to a reduction in human brain size and therefore, may cause or contribute to microcephaly. In this review, we will concentrate, within the brain, on the cerebral cortex, the seat of our higher cognitive abilities, and focus on the human-specific gene ARHGAP11B and on the gene family comprising the three human-specific genes NOTCH2NLA, -B, and -C. These genes are thought to have significantly contributed to the expansion of the cerebral cortex during human evolution. We will summarize the evolution of these genes, as well as their expression and functional role during human cortical development, and discuss their potential relevance for microcephaly. Furthermore, we will give an overview of other human-specific genes that are expressed during fetal human cortical development. We will discuss the potential involvement of these genes in microcephaly and how these genes could be studied functionally to identify a possible role in microcephaly.

DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction.

Diaphanous (DIAPH) three (DIAPH3) is a member of the formin proteins that have the capacity to nucleate and elongate actin filaments and, therefore, to remodel the cytoskeleton. DIAPH3 is essential for cytokinesis as its dysfunction impairs the contractile ring and produces multinucleated cells. Here, we report that DIAPH3 localizes at the centrosome during mitosis and regulates the assembly and bipolarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton and multipolar spindles. DIAPH3 deficiency disrupts the expression and/or stability of several proteins including the kinetochore-associated protein SPAG5. DIAPH3 and SPAG5 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies DIAPH3 deficiency, whereas its overexpression rescues the DIAHP3 knockdown phenotype. Conditional inactivation of Diaph3 in mouse cerebral cortex profoundly disrupts neurogenesis, depleting cortical progenitors and neurons, leading to cortical malformation and autistic-like behavior. Our data uncover the uncharacterized functions of DIAPH3 and provide evidence that this protein belongs to a molecular toolbox that links microtubule dynamics during mitosis to aneuploidy, cell death, fate determination defects, and cortical malformation.

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.

FGF-MAPK signaling regulates human deep-layer corticogenesis.

SummaryDespite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

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.

PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors.

ABSTRACTRadial glia (RG) in the neocortex sequentially generate distinct subtypes of projection neurons, accounting for the diversity and complex assembly of cortical neural circuits. Mechanisms that drive the rapid and precise temporal progression of RG are beginning to be elucidated. Here, we reveal that the RG-specific transcriptional regulator PRDM16 promotes the transition of early to late phase of neurogenesis in the mouse neocortex. Loss of Prdm16 delays the timely progression of RG, leading to defective cortical laminar organization. Our genomic analyses demonstrate that PRDM16 regulates a subset of genes that are dynamically expressed between early and late neurogenesis. We show that PRDM16 suppresses target gene expression through limiting chromatin accessibility of permissive enhancers. We further confirm that crucial target genes regulated by PRDM16 are neuronal specification genes, cell cycle regulators and molecules required for neuronal migration. These findings provide evidence to support the finding that neural progenitors temporally shift the gene expression program to achieve neural cell diversity.

Differentiation of IMR32 Neuroblastoma Is Accompanied by a Global Change in the Transcriptome.

Neuroblastoma is one of the most common cancers in infants and is often multidrug-resistant. One of the methods of treating neuroblastomas is to create conditions for their differentiation. In this work, we performed a full-transcriptome analysis of gene expression in an undifferentiated and differentiated in vitro human neuroblastoma cell line IMR-32 and identified the signaling pathways and biological processes that undergo the greatest changes during differentiation. The results obtained show that a complex heterogeneous population of nerve cells is formed at different stages of differentiation. In the cell population of differentiating neuroblastoma, the expression of genes in which cortical neuronal progenitor cells are enriched increases; at the same time, there are cells expressing markers of early postmitotic neurons. Cells differentiate in several different directions according to the type of synaptic mediator. At the same time, the differentiation of IMR-32 cells is accompanied by an increase in the transcription of genes that suppress the differentiation of nerve cells, Sox2 and PROM1, the expression of which is normally suppressed during in vivo differentiation.

Dissecting the impact of regional identity and the oncogenic role of human-specific NOTCH2NL in an hESC model of H3.3G34R-mutant glioma

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

Differential roles of ARID1B in excitatory and inhibitory neural progenitors in the developing cortex

Genetic evidence indicates that haploinsufficiency of ARID1B causes intellectual disability (ID) and autism spectrum disorder (ASD), but the neural function of ARID1B is largely unknown. Using both conditional and global Arid1b knockout mouse strains, we examined the role of ARID1B in neural progenitors. We detected an overall decrease in the proliferation of cortical and ventral neural progenitors following homozygous deletion of Arid1b, as well as altered cell cycle regulation and increased cell death. Each of these phenotypes was more pronounced in ventral neural progenitors. Furthermore, we observed decreased nuclear localization of β-catenin in Arid1b-deficient neurons. Conditional homozygous deletion of Arid1b in ventral neural progenitors led to pronounced ID- and ASD-like behaviors in mice, whereas the deletion in cortical neural progenitors resulted in minor cognitive deficits. This study suggests an essential role for ARID1B in forebrain neurogenesis and clarifies its more pronounced role in inhibitory neural progenitors. Our findings also provide insights into the pathogenesis of ID and ASD.

Neonatal cortical astrocytes possess intrinsic potential in neuronal conversion in defined media

Astrocytes are multifunctional brain cells responsible for maintaining the health and function of the central nervous system. Accumulating evidence suggests that astrocytes might be complementary source across different brain regions to supply new neurons during adult neurogenesis. In this study, we found that neonatal mouse cortical astrocytes can be directly converted into neurons when exposed to neurogenic differentiation culture conditions, with insulin being the most critical component. Detailed comparison studies between mouse cortical astrocytes and neuronal progenitor cells (NPCs) demonstrated the converted neuronal cells originate indeed from the astrocytes rather than NPCs. The neurons derived from mouse cortical astrocytes display typical neuronal morphologies, express neuronal markers and possess typical neuronal electrophysiological properties. More importantly, these neurons can survive and mature in the mouse brain in vivo. Finally, by comparing astrocytes from different brain regions, we found that only cortical astrocytes but not astrocytes from other brain regions such as hippocampus and cerebellum can be converted into neurons under the current condition. Altogether, our findings suggest that neonatal astrocytes from certain brain regions possess intrinsic potential to differentiate/transdifferentiate into neurons which may have clinical relevance in the future.

The essential role of primary cilia in cerebral cortical development and disorders.

Primary cilium, first described in the 19th century in different cell types and organisms by Alexander Ecker, Albert Kolliker, Aleksandr Kowalevsky, Paul Langerhans, and Karl Zimmermann (Ecker, 1844; Kolliker, 1854; Kowalevsky, 1867; Langerhans, 1876; Zimmermann, 1898), play an essential modulatory role in diverse aspects of nervous system development and function. The primary cilium, sometimes referred to as the cell's 'antennae', can receive wide ranging inputs from cellular milieu, including morphogens, growth factors, neuromodulators, and neurotransmitters. Its unique structural and functional organization bequeaths it the capacity to hyper-concentrate signaling machinery in a restricted cellular domain approximately one-thousandth the volume of cell soma. Thus enabling it to act as a signaling hub that integrates diverse developmental and homestatic information from cellular milieu to regulate the development and function of neural cells. Dysfunction of primary cilia contributes to the pathophysiology of several brain malformations, intellectual disabilities, epilepsy, and psychiatric disorders. This review focuses on the most essential contributions of primary cilia to cerebral cortical development and function, in the context of neurodevelopmental disorders and malformations. It highlights the recent progress made in identifying the mechanisms underlying primary cilia's role in cortical progenitors, neurons and glia, in health and disease. A future challenge will be to translate these insights and advances into effective clinical treatments for ciliopathies.

The Extracellular Matrix in the Evolution of Cortical Development and Folding.

The evolution of the mammalian cerebral cortex leading to humans involved a remarkable sophistication of developmental mechanisms. Specific adaptations of progenitor cell proliferation and neuronal migration mechanisms have been proposed to play major roles in this evolution of neocortical development. One of the central elements influencing neocortex development is the extracellular matrix (ECM). The ECM provides both a structural framework during tissue formation and to present signaling molecules to cells, which directly influences cell behavior and movement. Here we review recent advances in the understanding of the role of ECM molecules on progenitor cell proliferation and neuronal migration, and how these contribute to cerebral cortex expansion and folding. We discuss how transcriptomic studies in human, ferret and mouse identify components of ECM as being candidate key players in cortex expansion during development and evolution. Then we focus on recent functional studies showing that ECM components regulate cortical progenitor cell proliferation, neuron migration and the mechanical properties of the developing cortex. Finally, we discuss how these features differ between lissencephalic and gyrencephalic species, and how the molecular evolution of ECM components and their expression profiles may have been fundamental in the emergence and evolution of cortex folding across mammalian phylogeny.

Altered cleavage plane orientation with increased genomic aneuploidy produced by receptor-mediated lysophosphatidic acid (LPA) signaling in mouse cerebral cortical neural progenitor cells

The brain is composed of cells having distinct genomic DNA sequences that arise post-zygotically, known as somatic genomic mosaicism (SGM). One form of SGM is aneuploidy—the gain and/or loss of chromosomes—which is associated with mitotic spindle defects. The mitotic spindle orientation determines cleavage plane positioning and, therefore, neural progenitor cell (NPC) fate during cerebral cortical development. Here we report receptor-mediated signaling by lysophosphatidic acid (LPA) as a novel extracellular signal that influences cleavage plane orientation and produces alterations in SGM by inducing aneuploidy during murine cortical neurogenesis. LPA is a bioactive lipid whose actions are mediated by six G protein-coupled receptors, LPA1–LPA6. RNAscope and qPCR assessment of all six LPA receptor genes, and exogenous LPA exposure in LPA receptor (Lpar)-null mice, revealed involvement of Lpar1 and Lpar2 in the orientation of the mitotic spindle. Lpar1 signaling increased non-vertical cleavage in vivo by disrupting cell–cell adhesion, leading to breakdown of the ependymal cell layer. In addition, genomic alterations were significantly increased after LPA exposure, through production of chromosomal aneuploidy in NPCs. These results identify LPA as a receptor-mediated signal that alters both NPC fate and genomes during cortical neurogenesis, thus representing an extracellular signaling mechanism that can produce stable genomic changes in NPCs and their progeny. Normal LPA signaling in early life could therefore influence both the developing and adult brain, whereas its pathological disruption could contribute to a range of neurological and psychiatric diseases, via long-lasting somatic genomic alterations.

Behavior and lineage progression of neural progenitors in the mammalian cortex.

The cerebral cortex is a central structure in the mammalian brain that enables higher cognitive functions and intellectual skills. It is the hallmark of the mammalian nervous system with enormous complexity, consisting of a large number of neurons and glia that are diverse in morphology, molecular expression, biophysical properties, circuit connectivity and physiological function. Cortical neurons and glia are generated by neural progenitor cells during development. Ensuring the correct cell cycle kinetics, fate behavior and lineage progression of neural progenitor cells is essential to determine the number and types of neurons and glia in the cerebral cortex, which together constitute neural circuits for brain function. In this review, we discuss recent findings on mammalian cortical progenitor cell types and their lineage behaviors in generating neurons and glia, cortical evolution and expansion, and advances in brain organoid technology that allow the modeling of human cortical development under normal and disease conditions.

Transcriptome Analysis Identifies SenZfp536, a Sense LncRNA that Suppresses Self-renewal of Cortical Neural Progenitors.

Long non-coding RNAs (lncRNAs) regulate transcription to control development and homeostasis in a variety of tissues and organs. However, their roles in the development of the cerebral cortex have not been well elucidated. Here, a bioinformatics pipeline was applied to delineate the dynamic expression and potential cis-regulating effects of mouse lncRNAs using transcriptome data from 8 embryonic time points and sub-regions of the developing cerebral cortex. We further characterized a sense lncRNA, SenZfp536, which is transcribed downstream of and partially overlaps with the protein-coding gene Zfp536. Both SenZfp536 and Zfp536 were predominantly expressed in the proliferative zone of the developing cortex. Zfp536 was cis-regulated by SenZfp536, which facilitates looping between the promoter of Zfp536 and the genomic region that transcribes SenZfp536. Surprisingly, knocking down or activating the expression of SenZfp536 increased or compromised the proliferation of cortical neural progenitor cells (NPCs), respectively. Finally, overexpressing Zfp536 in cortical NPCs reversed the enhanced proliferation of cortical NPCs caused by SenZfp536 knockdown. The study deepens our understanding of how lncRNAs regulate the propagation of cortical NPCs through cis-regulatory mechanisms.

16p11.2 microdeletion imparts transcriptional alterations in human iPSC-derived models of early neural development.

Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of 'footprint'-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information-available with the cell lines by request from the Simons Foundation Autism Research Initiative-with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.