IGF2 interacts with the imprinted gene Cdkn1c to promote terminal differentiation of neural stem cells.

Adult neurogenesis is supported by multipotent neural stem cells (NSCs) with unique properties and growth requirements. Adult NSCs constitute a reversibly quiescent cell population that can be activated by extracellular signals from the microenvironment in which they reside in vivo. Although genomic imprinting plays a role in adult neurogenesis through dose regulation of some relevant signals, the roles of many imprinted genes in the process remain elusive. Insulin-like growth factor 2 (IGF2) is encoded by an imprinted gene that contributes to NSC maintenance in the adult subventricular zone through a biallelic expression in only the vascular compartment. We show here that IGF2 additionally promotes terminal differentiation of NSCs into astrocytes, neurons and oligodendrocytes by inducing the expression of the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), encoding the cell cycle inhibitor p57. Using intraventricular infusion of recombinant IGF2 in a conditional mutant strain with Cdkn1c-deficient NSCs, we confirm that p57 partially mediates the differentiation effects of IGF2 in NSCs and that this occurs independently of its role in cell-cycle progression, balancing the relationship between astrogliogenesis, neurogenesis and oligodendrogenesis.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Dlk1 dosage regulates hippocampal neurogenesis and cognition.

Neurogenesis in the adult brain gives rise to functional neurons, which integrate into neuronal circuits and modulate neural plasticity. Sustained neurogenesis throughout life occurs in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and is hypothesized to be involved in behavioral/cognitive processes such as memory and in diseases. Genomic imprinting is of critical importance to brain development and normal behavior, and exemplifies how epigenetic states regulate genome function and gene dosage. While most genes are expressed from both alleles, imprinted genes are usually expressed from either the maternally or the paternally inherited chromosome. Here, we show that in contrast to its canonical imprinting in nonneurogenic regions, Delta-like homolog 1 (Dlk1) is expressed biallelically in the SGZ, and both parental alleles are required for stem cell behavior and normal adult neurogenesis in the hippocampus. To evaluate the effects of maternally, paternally, and biallelically inherited mutations within the Dlk1 gene in specific behavioral domains, we subjected Dlk1-mutant mice to a battery of tests that dissociate and evaluate the effects of Dlk1 dosage on spatial learning ability and on anxiety traits. Importantly, reduction in Dlk1 levels triggers specific cognitive abnormalities that affect aspects of discriminating differences in environmental stimuli, emphasizing the importance of selective absence of imprinting in this neurogenic niche.

Intergenerational transmission of the positive effects of physical exercise on brain and cognition.

Physical exercise has positive effects on cognition, but very little is known about the inheritance of these effects to sedentary offspring and the mechanisms involved. Here, we use a patrilineal design in mice to test the transmission of effects from the same father (before or after training) and from different fathers to compare sedentary- and runner-father progenies. Behavioral, stereological, and whole-genome sequence analyses reveal that paternal cognition improvement is inherited by the offspring, along with increased adult neurogenesis, greater mitochondrial citrate synthase activity, and modulation of the adult hippocampal gene expression profile. These results demonstrate the inheritance of exercise-induced cognition enhancement through the germline, pointing to paternal physical activity as a direct factor driving offspring's brain physiology and cognitive behavior.

Targeted deletion of a 170-kb cluster of LINE-1 repeats and implications for regional control.

Approximately half the mammalian genome is composed of repetitive sequences, and accumulating evidence suggests that some may have an impact on genome function. Here, we characterized a large array class of repeats of long-interspersed elements (LINE-1). Although widely distributed in mammals, locations of such arrays are species specific. Using targeted deletion, we asked whether a 170-kb LINE-1 array located at a mouse imprinted domain might function as a modulator of local transcriptional control. The LINE-1 array is lamina associated in differentiated ES cells consistent with its AT-richness, and although imprinting occurs both proximally and distally to the array, active LINE-1 transcripts within the tract are biallelically expressed. Upon deletion of the array, no perturbation of imprinting was observed, and abnormal phenotypes were not detected in maternal or paternal heterozygous or homozygous mutant mice. The array does not shield nonimprinted genes in the vicinity from local imprinting control. Reduced neural expression of protein-coding genes observed upon paternal transmission of the deletion is likely due to the removal of a brain-specific enhancer embedded within the LINE array. Our findings suggest that presence of a 170-kb LINE-1 array reflects the tolerance of the site for repeat insertion rather than an important genomic function in normal development.

Distinct fibroblast lineages determine dermal architecture in skin development and repair.

Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibres of the extracellular matrix (ECM). Even within a single tissue, fibroblasts exhibit considerable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing in mice, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle, which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesize the bulk of the fibrillar ECM, and the preadipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialization. Epidermal ß-catenin activation stimulates the expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

Cyclin-Dependent Kinase 4 Regulates Adult Neural Stem Cell Proliferation and Differentiation in Response to Insulin.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416.

Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis.

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts.

DLK1/PREF1 regulates nutrient metabolism and protects from steatosis.

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance and obesity, as well as progressive liver dysfunction. Recent animal studies have underscored the importance of hepatic growth hormone (GH) signaling in the development of NAFLD. The imprinted Delta-like homolog 1 (Dlk1)/preadipocyte factor 1 (Pref1) gene encodes a complex protein producing both circulating and membrane-tethered isoforms whose expression dosage is functionally important because even modest elevation during embryogenesis causes lethality. DLK1 is up-regulated during embryogenesis, during suckling, and in the mother during pregnancy. We investigated the normal role for elevated DLK1 dosage by overexpressing Dlk1 from endogenous control elements. This increased DLK1 dosage caused improved glucose tolerance with no primary defect in adipose tissue expansion even under extreme metabolic stress. Rather, Dlk1 overexpression caused reduced fat stores, pituitary insulin-like growth factor 1 (IGF1) resistance, and a defect in feedback regulation of GH. Increased circulatory GH culminated in a switch in whole body fuel metabolism and a reduction in hepatic steatosis. We propose that the function of DLK1 is to shift the metabolic mode of the organism toward peripheral lipid oxidation and away from lipid storage, thus mediating important physiological adaptations associated with early life and with implications for metabolic disease resistance.

Isolation, culture and analysis of adult subependymal neural stem cells.

Individual cells dissected from the subependymal neurogenic niche of the adult mouse brain proliferate in medium containing basic fibroblast growth factor (bFGF) and/or epidermal growth factor (EGF) as mitogens, to produce multipotent clonal aggregates called neurospheres. These cultures constitute a powerful tool for the study of neural stem cells (NSCs) provided that they allow the analysis of their features and potential capacity in a controlled environment that can be modulated and monitored more accurately than in vivo. Clonogenic and population analyses under mitogen addition or withdrawal allow the quantification of the self-renewing and multilineage potency of these cells and the identification of the mechanisms involved in these properties. Here, we describe a set of procedures developed and/or modified by our group including several experimental options that can be used either independently or in combination for the ex vivo assessment of cell properties of NSCs obtained from the adult subependymal niche.

The cyclin-dependent kinase inhibitor p27 kip1 regulates radial stem cell quiescence and neurogenesis in the adult hippocampus.

Members of the cyclin-dependent kinase (CDK)-inhibitory protein (CIP)/kinase-inhibitory protein (KIP) family of cyclin-dependent kinase inhibitors regulate proliferation and cell cycle exit of mammalian cells. In the adult brain, the CIP/KIP protein p27(kip1) has been related to the regulation of intermediate progenitor cells located in neurogenic niches. Here, we uncover a novel function of p27(kip1) in the adult hippocampus as a dual regulator of stem cell quiescence and of cell-cycle exit of immature neurons. In vivo, p27(kip1) is detected in radial stem cells expressing SOX2 and in newborn neurons of the dentate gyrus. In vitro, the Cdkn1b gene encoding p27(kip1) is transcriptionally upregulated by quiescence signals such as BMP4. The nuclear accumulation of p27(kip1) protein in adult hippocampal stem cells encompasses the BMP4-induced quiescent state and its overexpression is able to block proliferation. p27(kip1) is also expressed in immature neurons upon differentiation of adult hippocampal stem cell cultures. Loss of p27(kip1) leads to an increase in proliferation and neurogenesis in the adult dentate gyrus, which results from both a decrease in the percentage of radial stem cells that are quiescent and a delay in cell cycle exit of immature neurons. Analysis of animals carrying a disruption in the cyclin-CDK interaction domain of p27(kip1) indicates that the CDK inhibitory function of the protein is necessary to control the activity of radial stem cells. Thus, we report that p27(kip1) acts as a central player of the molecular program that keeps adult hippocampal stem cells out of the cell cycle.

Paracrine regulation of neural stem cells in the subependymal zone.

Stem cells maintain their self-renewal and multipotency capacities through a self-organizing network of transcription factors and intracellular pathways activated by extracellular signaling from the microenvironment or "niche" in which they reside in vivo. In the adult mammalian brain new neurons continue to be generated throughout life of the organisms and this lifelong process of neurogenesis is supported by a reservoir of neural stem cells in the germinal regions. The discovery of adult neurogenesis in the mammalian brain has sparked great interest in defining the conditions that guide neural stem cell (NSC) maintenance and differentiation into the great variety of neuronal and glial subtypes. Here we review current knowledge regarding the paracrine regulation provided by the components of the niche and its function, focusing on the main germinal region of the adult central nervous system (CNS), the subependymal zone (SEZ).

Aberrations of Genomic Imprinting in Glioblastoma Formation.

In human glioblastoma (GBM), the presence of a small population of cells with stem cell characteristics, the glioma stem cells (GSCs), has been described. These cells have GBM potential and are responsible for the origin of the tumors. However, whether GSCs originate from normal neural stem cells (NSCs) as a consequence of genetic and epigenetic changes and/or dedifferentiation from somatic cells remains to be investigated. Genomic imprinting is an epigenetic marking process that causes genes to be expressed depending on their parental origin. The dysregulation of the imprinting pattern or the loss of genomic imprinting (LOI) have been described in different tumors including GBM, being one of the earliest and most common events that occurs in human cancers. Here we have gathered the current knowledge of the role of imprinted genes in normal NSCs function and how the imprinting process is altered in human GBM. We also review the changes at particular imprinted loci that might be involved in the development of the tumor. Understanding the mechanistic similarities in the regulation of genomic imprinting between normal NSCs and GBM cells will be helpful to identify molecular players that might be involved in the development of human GBM.

Adult Neural Stem Cells Are Alerted by Systemic Inflammation through TNF-α Receptor Signaling.

Adult stem cells (SCs) transit between the cell cycle and a poorly defined quiescent state. Single neural SCs (NSCs) with quiescent, primed-for-activation, and activated cell transcriptomes have been obtained from the subependymal zone (SEZ), but the functional regulation of these states under homeostasis is not understood. Here, we develop a multilevel strategy to analyze these NSC states with the aim to uncover signals that regulate their level of quiescence/activation. We show that transitions between states occur in vivo and that activated and primed, but not quiescent, states can be captured and studied in culture. We also show that peripherally induced inflammation promotes a transient activation of primed NSCs (pNSCs) mediated by tumor necrosis factor α (TNF-α) acting through its receptor, TNF receptor 2 (TNFR2), and a return to quiescence in a TNF receptor 1 (TNFR1)-dependent manner. Our data identify a signaling pathway promoting NSC alertness and add to the emerging concept that SCs can respond to the systemic milieu.

Telomere shortening in neural stem cells disrupts neuronal differentiation and neuritogenesis.

Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and self-renewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.

Pigment epithelium-derived factor is a niche signal for neural stem cell renewal.

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.

Beyond protein-coding genes.

A long non-coding RNA called lnc-NR2F1 regulates several neuronal genes, including some involved in autism and intellectual disabilities.

TET3 prevents terminal differentiation of adult NSCs by a non-catalytic action at Snrpn.

Ten-eleven-translocation (TET) proteins catalyze DNA hydroxylation, playing an important role in demethylation of DNA in mammals. Remarkably, although hydroxymethylation levels are high in the mouse brain, the potential role of TET proteins in adult neurogenesis is unknown. We show here that a non-catalytic action of TET3 is essentially required for the maintenance of the neural stem cell (NSC) pool in the adult subventricular zone (SVZ) niche by preventing premature differentiation of NSCs into non-neurogenic astrocytes. This occurs through direct binding of TET3 to the paternal transcribed allele of the imprinted gene Small nuclear ribonucleoprotein-associated polypeptide N (Snrpn), contributing to transcriptional repression of the gene. The study also identifies BMP2 as an effector of the astrocytic terminal differentiation mediated by SNRPN. Our work describes a novel mechanism of control of an imprinted gene in the regulation of adult neurogenesis through an unconventional role of TET3.

Genomic Imprinting and the Regulation of Postnatal Neurogenesis.

Most genes required for mammalian development are expressed from both maternally and paternally inherited chromosomal homologues. However, there are a small number of genes known as "imprinted genes" that only express a single allele from one parent, which is repressed on the gene from the other parent. Imprinted genes are dependent on epigenetic mechanisms such as DNA methylation and post-translational modifications of the DNA-associated histone proteins to establish and maintain their parental identity. In the brain, multiple transcripts have been identified which show parental origin-specific expression biases. However, the mechanistic relationship with canonical imprinting is unknown. Recent studies on the postnatal neurogenic niches raise many intriguing questions concerning the role of genomic imprinting and gene dosage during postnatal neurogenesis, including how imprinted genes operate in concert with signalling cues to contribute to newborn neurons' formation during adulthood. Here we have gathered the current knowledge on the imprinting process in the neurogenic niches. We also review the phenotypes associated with genetic mutations at particular imprinted loci in order to consider the impact of imprinted genes in the maintenance and/or differentiation of the neural stem cell pool in vivo and during brain tumour formation.

Isolation, long-term expansion, and differentiation of murine neural stem cells.

Stem cells are capable of extensive self-renewal while preserving the ability to generate cell progeny that can differentiate into different cell types. Here, we describe some methods for the isolation of neural stem cells (NSCs) from the adult murine subependymal zone (SEZ), their extensive culturing and the assessment of their full developmental potential, particularly with respect to their differentiation capacity. The procedure includes chemically defined conditions such as absence of serum and addition of specific growth factors, in which differentiated cells die and are rapidly eliminated from the culture. In contrast, undifferentiated precursors become hypertrophic and proliferate, forming clonal spherical clusters called "neurospheres." Experimental manipulation of NSCs identifies populations of cells with differential restriction in their self-renewal potential and introduces a great interest in defining the conditions that guide their differentiation into a variety of neuronal and glial subtypes, aspects that have important implications for their use in future clinical purposes.