HES1, two programs: promoting the quiescence and proliferation of adult neural stem cells.

Adult neural stem cells are mostly quiescent and only rarely enter the cell cycle to self-renew and generate neuronal or glial progenies. The Notch signaling pathway is essential for both the quiescent and proliferative states of neural stem cells. However, these are mutually exclusive cellular states; thus, how Notch promotes both of these programs within adult neural stem cells has remained unclear. In this issue of Genes & Development, Sueda and colleagues (pp. 511-523) use an extensive repertoire of mouse genetic tools and techniques to demonstrate that it is the levels and dynamic expression of the Notch transcriptional effector Hairy and Enhancer of Split 1 that enables this dual role.

Polycomb repressive complex 2 is critical for mouse cortical glutamatergic neuron development.

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.

Wnt/ß-catenin signalling is dispensable for adult neural stem cell homeostasis and activation.

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/ß-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here, we used Wnt/ß-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/ß-catenin signalling. Wnt/ß-catenin stimulation instructed the neuronal differentiation of proliferating NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, deletion of ß-catenin in NSCs did not affect either their activation or maintenance of their stem cell characteristics. Together, these results indicate that, although NSCs do respond to Wnt/ß-catenin stimulation in a dose-dependent and state-specific manner, Wnt/ß-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/ß-catenin signalling in adult hippocampal NSCs.

Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression.

Our understanding of the transcriptional programme underpinning adult hippocampal neurogenesis is incomplete. In mice, under basal conditions, adult hippocampal neural stem cells (AH-NSCs) generate neurons and astrocytes, but not oligodendrocytes. The factors limiting oligodendrocyte production, however, remain unclear. Here, we reveal that the transcription factor NFIX plays a key role in this process. NFIX is expressed by AH-NSCs, and its expression is sharply upregulated in adult hippocampal neuroblasts. Conditional ablation of Nfix from AH-NSCs, coupled with lineage tracing, transcriptomic sequencing and behavioural studies collectively reveal that NFIX is cell-autonomously required for neuroblast maturation and survival. Moreover, a small number of AH-NSCs also develop into oligodendrocytes following Nfix deletion. Remarkably, when Nfix is deleted specifically from intermediate progenitor cells and neuroblasts using a Dcx-creERT2 driver, these cells also display elevated signatures of oligodendrocyte gene expression. Together, these results demonstrate the central role played by NFIX in neuroblasts within the adult hippocampal stem cell neurogenic niche in promoting the maturation and survival of these cells, while concomitantly repressing oligodendrocyte gene expression signatures.

NFIX-Mediated Inhibition of Neuroblast Branching Regulates Migration Within the Adult Mouse Ventricular-Subventricular Zone.

Understanding the migration of newborn neurons within the brain presents a major challenge in contemporary biology. Neuronal migration is widespread within the developing brain but is also important within the adult brain. For instance, stem cells within the ventricular-subventricular zone (V-SVZ) and the subgranular zone of dentate gyrus of the adult rodent brain produce neuroblasts that migrate to the olfactory bulb and granule cell layer of the dentate gyrus, respectively, where they regulate key brain functions including innate olfactory responses, learning, and memory. Critically, our understanding of the factors mediating neuroblast migration remains limited. The transcription factor nuclear factor I X (NFIX) has previously been implicated in embryonic cortical development. Here, we employed conditional ablation of Nfix from the adult mouse brain and demonstrated that the removal of this gene from either neural stem and progenitor cells, or neuroblasts, within the V-SVZ culminated in neuroblast migration defects. Mechanistically, we identified aberrant neuroblast branching, due in part to increased expression of the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), as a factor contributing to abnormal migration in Nfix-deficient adult mice. Collectively, these data provide new insights into how neuroblast migration is regulated at a transcriptional level within the adult brain.

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development.

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development.

A morphology independent approach for identifying dividing adult neural stem cells in the mouse hippocampus.

BACKGROUND: Type 1 adult hippocampal neural stem cells (AH-NSCs) continue to generate neurons throughout life, albeit at a very low rate. The relative quiescence of this population of cells has led to many studies investigating factors that may increase their division. Current methods of identifying dividing AH-NSCs in vivo require the identification and tracing of radial processes back to nuclei within the subgranular zone. However, caveats to this approach include the time-intensive nature of identifying AH-NSCs with such a process, as well as the fact that this approach ignores the relatively more active population of horizontally oriented AH-NSCs that also reside in the subgranular zone. RESULTS: Here we describe, and then verify using Hes5::GFP mice, that labeling for the cell cycle marker Ki67 and selection against the intermediate progenitor cell marker TBR2 (Ki67+ve ; TBR2-ve nuclei) is sufficient to identify dividing horizontally and radially oriented AH-NSCs in the adult mouse hippocampus. CONCLUSIONS: These findings provide a simple and accurate way to quantify dividing AH-NSCs in vivo using a morphology-independent approach that will facilitate studies into neurogenesis within the hippocampal stem cell niche of the adult brain. Developmental Dynamics 247:194-200, 2018. © 2017 Wiley Periodicals, Inc.

Transcriptional regulation of Nfix by NFIB drives astrocytic maturation within the developing spinal cord.

During mouse spinal cord development, ventricular zone progenitor cells transition from producing neurons to producing glia at approximately embryonic day 11.5, a process known as the gliogenic switch. The transcription factors Nuclear Factor I (NFI) A and B initiate this developmental transition, but the contribution of a third NFI member, NFIX, remains unknown. Here, we reveal that ventricular zone progenitor cells within the spinal cord express NFIX after the onset of NFIA and NFIB expression, and after the gliogenic switch has occurred. Mice lacking NFIX exhibit normal neurogenesis within the spinal cord, and, while early astrocytic differentiation proceeds normally, aspects of terminal astrocytic differentiation are impaired. Finally, we report that, in the absence of Nfia or Nfib, there is a marked reduction in the spinal cord expression of NFIX, and that NFIB can transcriptionally activate Nfix expression in vitro. These data demonstrate that NFIX is part of the downstream transcriptional program through which NFIA and NFIB coordinate gliogenesis within the spinal cord. This hierarchical organisation of NFI protein expression and function during spinal cord gliogenesis reveals a previously unrecognised auto-regulatory mechanism within this gene family.

Nuclear factor one transcription factors as epigenetic regulators in cancer.

Tumour heterogeneity poses a distinct obstacle to therapeutic intervention. While the initiation of tumours across various physiological systems is frequently associated with signature mutations in genes that drive proliferation and bypass senescence, increasing evidence suggests that tumour progression and clonal diversity is driven at an epigenetic level. The tumour microenvironment plays a key role in driving diversity as cells adapt to demands imposed during tumour growth, and is thought to drive certain subpopulations back to a stem cell-like state. This stem cell-like phenotype primes tumour cells to react to external cues via the use of developmental pathways that facilitate changes in proliferation, migration and invasion. Because the dynamism of this stem cell-like state requires constant chromatin remodelling and rapid alterations at regulatory elements, it is of great therapeutic interest to identify the cell-intrinsic factors that confer these epigenetic changes that drive tumour progression. The nuclear factor one (NFI) family are transcription factors that play an important role in the development of many mammalian organ systems. While all four family members have been shown to act as either oncogenes or tumour suppressors across various cancer models, evidence has emerged implicating them as key epigenetic regulators during development and within tumours. Notably, NFIs have also been shown to regulate chromatin accessibility at distal regulatory elements that drive tumour cell dissemination and metastasis. Here we summarize the role of the NFIs in cancer, focusing largely on the potential mechanisms associated with chromatin remodelling and epigenetic modulation of gene expression.

Short-pulse actively Q-switched Er:YAG lasers.

We report the shortest duration pulses obtained to date from an actively Q-switched Er:YAG laser pumped by a low spectral and spatial brightness laser diode. The 14.5 ns, 6 mJ pulses were obtained using a 1470 nm laser diode end-pumped co-planar folded zigzag slab architecture. We also present an analytical model that accurately predicts the pulse energy-duration product achievable from virtually all Q-switched Er:YAG lasers and high repetition rate quasi-three-level Q-switched lasers in general.

Compact cavity-dumped Q-switched Er:YAG laser.

We report a compact cavity-dumped Q-switched Er:YAG laser that produces pulses with 4.5 ns full width at half-maximum duration and 10 mJ energy. To the best of our knowledge, the resulting 2 MW peak power is the highest reported to date from a 1645 nm Er:YAG laser.

NFIX Regulates Proliferation and Migration Within the Murine SVZ Neurogenic Niche.

Transcription factors of the nuclear factor one (NFI) family play a pivotal role in the development of the nervous system. One member, NFIX, regulates the development of the neocortex, hippocampus, and cerebellum. Postnatal Nfix(-/-) mice also display abnormalities within the subventricular zone (SVZ) lining the lateral ventricles, a region of the brain comprising a neurogenic niche that provides ongoing neurogenesis throughout life. Specifically, Nfix(-/-) mice exhibit more PAX6-expressing progenitor cells within the SVZ. However, the mechanism underlying the development of this phenotype remains undefined. Here, we reveal that NFIX contributes to multiple facets of SVZ development. Postnatal Nfix(-/-) mice exhibit increased levels of proliferation within the SVZ, both in vivo and in vitro as assessed by a neurosphere assay. Furthermore, we show that the migration of SVZ-derived neuroblasts to the olfactory bulb is impaired, and that the olfactory bulbs of postnatal Nfix(-/-) mice are smaller. We also demonstrate that gliogenesis within the rostral migratory stream is delayed in the absence of Nfix, and reveal that Gdnf (glial-derived neurotrophic factor), a known attractant for SVZ-derived neuroblasts, is a target for transcriptional activation by NFIX. Collectively, these findings suggest that NFIX regulates both proliferation and migration during the development of the SVZ neurogenic niche.

Nuclear factor one transcription factors: Divergent functions in developmental versus adult stem cell populations.

Nuclear factor one (NFI) transcription factors are a group of site-specific DNA-binding proteins that are emerging as critical regulators of stem cell biology. During development NFIs promote the production of differentiated progeny at the expense of stem cell fate, with Nfi null mice exhibiting defects such as severely delayed brain and lung maturation, skeletomuscular defects and renal abnormalities, phenotypes that are often consistent with patients with congenital Nfi mutations. Intriguingly, recent research suggests that in adult tissues NFI factors play a qualitatively different role than during development, with NFIs serving to promote the survival and maintenance of slow-cycling adult stem cell populations rather than their differentiation. Here we review the role of NFI factors in development, largely focusing on their role as promoters of stem cell differentiation, and attempt to reconcile this with the emerging role of NFIs in adult stem cell niches.

NFIB-mediated repression of the epigenetic factor Ezh2 regulates cortical development.

Epigenetic mechanisms are essential in regulating neural progenitor cell self-renewal, with the chromatin-modifying protein Enhancer of zeste homolog 2 (EZH2) emerging as a central player in promoting progenitor cell self-renewal during cortical development. Despite this, how Ezh2 is itself regulated remains unclear. Here, we demonstrate that the transcription factor nuclear factor IB (NFIB) plays a key role in this process. Nfib(-/-) mice exhibit an increased number of proliferative ventricular zone cells that express progenitor cell markers and upregulation of EZH2 expression within the neocortex and hippocampus. NFIB binds to the Ezh2 promoter and overexpression of NFIB represses Ezh2 transcription. Finally, key downstream targets of EZH2-mediated epigenetic repression are misregulated in Nfib(-/-) mice. Collectively, these results suggest that the downregulation of Ezh2 transcription by NFIB is an important component of the process of neural progenitor cell differentiation during cortical development.

Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179.

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.

A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Familial Alzheimer's Disease Mutations in PSEN1 Lead to Premature Human Stem Cell Neurogenesis.

Mutations in presenilin 1 (PSEN1) or presenilin 2 (PSEN2), the catalytic subunit of γ-secretase, cause familial Alzheimer's disease (fAD). We hypothesized that mutations in PSEN1 reduce Notch signaling and alter neurogenesis. Expression data from developmental and adult neurogenesis show relative enrichment of Notch and γ-secretase expression in stem cells, whereas expression of APP and ß-secretase is enriched in neurons. We observe premature neurogenesis in fAD iPSCs harboring PSEN1 mutations using two orthogonal systems: cortical differentiation in 2D and cerebral organoid generation in 3D. This is partly driven by reduced Notch signaling. We extend these studies to adult hippocampal neurogenesis in mutation-confirmed postmortem tissue. fAD cases show mutation-specific effects and a trend toward reduced abundance of newborn neurons, supporting a premature aging phenotype. Altogether, these results support altered neurogenesis as a result of fAD mutations and suggest that neural stem cell biology is affected in aging and disease.

Long-term self-renewing stem cells in the adult mouse hippocampus identified by intravital imaging.

Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.

Coordinated changes in cellular behavior ensure the lifelong maintenance of the hippocampal stem cell population.

Neural stem cell numbers fall rapidly in the hippocampus of juvenile mice but stabilize during adulthood, ensuring lifelong hippocampal neurogenesis. We show that this stabilization of stem cell numbers in young adults is the result of coordinated changes in stem cell behavior. Although proliferating neural stem cells in juveniles differentiate rapidly, they increasingly return to a resting state of shallow quiescence and progress through additional self-renewing divisions in adulthood. Single-cell transcriptomics, modeling, and label retention analyses indicate that resting cells have a higher activation rate and greater contribution to neurogenesis than dormant cells, which have not left quiescence. These changes in stem cell behavior result from a progressive reduction in expression of the pro-activation protein ASCL1 because of increased post-translational degradation. These cellular mechanisms help reconcile current contradictory models of hippocampal neural stem cell (NSC) dynamics and may contribute to the different rates of decline of hippocampal neurogenesis in mammalian species, including humans.