Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis.

The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.

Jagged1/Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming.

While Notch signaling has been proposed to play a key role in fibrosis, the direct molecular pathways targeted by Notch signaling and the precise ligand and receptor pair that are responsible for kidney disease remain poorly defined. In this study, we found that JAG1 and NOTCH2 showed the strongest correlation with the degree of interstitial fibrosis in a genome-wide expression analysis of a large cohort of human kidney samples. Transcript analysis of mouse kidney disease models, including folic-acid (FA)-induced nephropathy, unilateral ureteral obstruction (UUO), or apolipoprotein L1 (APOL1)-associated kidney disease, indicated that Jag1 and Notch2 levels were higher in all analyzed kidney fibrosis models. Mice with tubule-specific deletion of Jag1 or Notch2 (Kspcre/Jag1flox/flox and Kspcre/Notch2flox/flox) had no kidney-specific alterations at baseline but showed protection from FA-induced kidney fibrosis. Tubule-specific genetic deletion of Notch1 and global knockout of Notch3 had no effect on fibrosis. In vitro chromatin immunoprecipitation experiments and genome-wide expression studies identified the mitochondrial transcription factor A (Tfam) as a direct Notch target. Re-expression of Tfam in tubule cells prevented Notch-induced metabolic and profibrotic reprogramming. Tubule-specific deletion of Tfam resulted in fibrosis. In summary, Jag1 and Notch2 play a key role in kidney fibrosis development by regulating Tfam expression and metabolic reprogramming.

Differential interactions between Notch and ID factors control neurogenesis by modulating Hes factor autoregulation.

During embryonic and adult neurogenesis, neural stem cells (NSCs) generate the correct number and types of neurons in a temporospatial fashion. Control of NSC activity and fate is crucial for brain formation and homeostasis. Neurogenesis in the embryonic and adult brain differ considerably, but Notch signaling and inhibitor of DNA-binding (ID) factors are pivotal in both. Notch and ID factors regulate NSC maintenance; however, it has been difficult to evaluate how these pathways potentially interact. Here, we combined mathematical modeling with analysis of single-cell transcriptomic data to elucidate unforeseen interactions between the Notch and ID factor pathways. During brain development, Notch signaling dominates and directly regulates Id4 expression, preventing other ID factors from inducing NSC quiescence. Conversely, during adult neurogenesis, Notch signaling and Id2/3 regulate neurogenesis in a complementary manner and ID factors can induce NSC maintenance and quiescence in the absence of Notch. Our analyses unveil key molecular interactions underlying NSC maintenance and mechanistic differences between embryonic and adult neurogenesis. Similar Notch and ID factor interactions may be crucial in other stem cell systems.

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).

miR379-410 cluster miRNAs regulate neurogenesis and neuronal migration by fine-tuning N-cadherin.

N-cadherin-mediated adhesion is essential for maintaining the tissue architecture and stem cell niche in the developing neocortex. N-cadherin expression level is precisely and dynamically controlled throughout development; however, the underlying regulatory mechanisms remain largely unknown. MicroRNAs (miRNAs) play an important role in the regulation of protein expression and subcellular localisation. In this study, we show that three miRNAs belonging to the miR379-410 cluster regulate N-cadherin expression levels in neural stem cells and migrating neurons. The overexpression of these three miRNAs in radial glial cells repressed N-cadherin expression and increased neural stem cell differentiation and neuronal migration. This phenotype was rescued when N-cadherin was expressed from a miRNA-insensitive construct. Transient abrogation of the miRNAs reduced stem cell differentiation and increased cell proliferation. The overexpression of these miRNAs specifically in newborn neurons delayed migration into the cortical plate, whereas the knockdown increased migration. Collectively, our results indicate a novel role for miRNAs of the miR379-410 cluster in the fine-tuning of N-cadherin expression level and in the regulation of neurogenesis and neuronal migration in the developing neocortex.

Striatal astrocytes produce neuroblasts in an excitotoxic model of Huntington's disease.

In the adult brain, subsets of astrocytic cells residing in well-defined neurogenic niches constitutively generate neurons throughout life. Brain lesions can stimulate neurogenesis in otherwise non-neurogenic regions, but whether local astrocytic cells generate neurons in these conditions is unresolved. Here, through genetic and viral lineage tracing in mice, we demonstrate that striatal astrocytes become neurogenic following an acute excitotoxic lesion. Similar to astrocytes of adult germinal niches, these activated parenchymal progenitors express nestin and generate neurons through the formation of transit amplifying progenitors. These results shed new light on the neurogenic potential of the adult brain parenchyma.

Notch: an interactive player in neurogenesis and disease.

Notch signaling is evolutionarily conserved from Drosophila to human. It plays critical roles in neural stem cell maintenance and neurogenesis in the embryonic brain as well as in the adult brain. Notch functions greatly depend on careful regulation and cross-talk with other regulatory mechanisms. Deregulation of Notch signaling is involved in many neurodegenerative diseases and brain disorders. Here, we summarize the fundamental role of Notch in neuronal development and specification and discuss how epigenetic regulation and pathway cross-talk contribute to Notch function. In addition, we cover aberrant alterations of Notch signaling in the diseased brain. The aim of this review is to provide an insight into how Notch signaling works in different contexts to control neurogenesis and its potential effects in diagnoses and therapies of neurodegeneration, brain tumors and disorders.

Notch and Neurogenesis.

Neurogenesis is the process of forming neurons and is essential during vertebrate development to produce most of the neurons of the adult brain. However, neurogenesis continues throughout life at distinct locations in the vertebrate brain. Neural stem cells (NSCs) are the origin of both embryonic and adult neurogenesis, but their activity and fate are tightly regulated by their local milieu or niche. In this chapter, we will discuss the role of Notch signaling in the control of neurogenesis and regeneration in the embryo and adult. Notch-dependence is a common feature among NSC populations, we will discuss how differences in Notch signaling might contribute to heterogeneity among adult NSCs. Understanding the fate of multiple NSC populations with distinct functions could be important for effective brain regeneration.

Notch-Tnf signalling is required for development and homeostasis of arterial valves.

AIMS: Congenital anomalies of arterial valves are common birth defects, leading to valvar stenosis. With no pharmaceutical treatment that can prevent the disease progression, prosthetic replacement is the only choice of treatment, incurring considerable morbidity and mortality. Animal models presenting localized anomalies and stenosis of congenital arterial valves similar to that of humans are critically needed research tools to uncover developmental molecular mechanisms underlying this devastating human condition. METHODS AND RESULTS: We generated and characterized mouse models with conditionally altered Notch signalling in endothelial or interstitial cells of developing valves. Mice with inactivation of Notch1 signalling in valvar endothelial cells (VEC) developed congenital anomalies of arterial valves including bicuspid aortic valves and valvar stenosis. Notch1 signalling in VEC was required for repressing proliferation and activating apoptosis of valvar interstitial cells (VIC) after endocardial-to-mesenchymal transformation (EMT). We showed that Notch signalling regulated Tnfα expression in vivo, and Tnf signalling was necessary for apoptosis of VIC and post-EMT development of arterial valves. Furthermore, activation or inhibition of Notch signalling in cultured pig aortic VEC-promoted or suppressed apoptosis of VIC, respectively. CONCLUSION: We have now met the need of critical animal models and shown that Notch-Tnf signalling balances proliferation and apoptosis for post-EMT development of arterial valves. Our results suggest that mutations in its components may lead to congenital anomaly of aortic valves and valvar stenosis in humans.

GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors.

Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. We show that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. Our data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis.

Baculovirus-based genome editing in primary cells.

Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).

Transcriptional Hallmarks of Heterogeneous Neural Stem Cell Niches of the Subventricular Zone.

Throughout postnatal life in mammals, neural stem cells (NSCs) are located in the subventricular zone (SVZ) of the lateral ventricles. The greatest diversity of neuronal and glial lineages they generate occurs during early postnatal life in a region-specific manner. In order to probe heterogeneity of the postnatal SVZ, we microdissected its dorsal and lateral walls at different postnatal ages and isolated NSCs and their immediate progeny based on their expression of Hes5-EGFP/Prominin1 and Ascl1-EGFP, respectively. Whole genome comparative transcriptome analysis revealed transcriptional regulators as major hallmarks that sustain postnatal SVZ regionalization. Manipulation of single genes encoding for locally enriched transcription factors (loss-of-function or ectopic gain-of-function in vivo) influenced NSC specification indicating that the fate of regionalized postnatal SVZ-NSCs can be readily modified. These findings reveal the pronounced transcriptional heterogeneity of the postnatal SVZ and provide targets to recruit region-specific lineages in regenerative contexts. Stem Cells 2015;33:2232-2242.

Early phenotypic asymmetry of sister oligodendrocyte progenitor cells after mitosis and its modulation by aging and extrinsic factors.

Oligodendrocyte progenitor cells (OPCs) persist in the adult central nervous system and guarantee oligodendrocyte turnover throughout life. It remains obscure how OPCs avoid exhaustion during adulthood. Similar to stem cells, OPCs could self-maintain by undergoing asymmetric divisions generating a mixed progeny either keeping a progenitor phenotype or proceeding to differentiation. To address this issue, we examined the distribution of stage-specific markers in sister OPCs during mitosis and later after cell birth, and assessed its correlation with distinct short-term fates. In both the adult and juvenile cerebral cortex a fraction of dividing OPCs gives rise to sister cells with diverse immunophenotypic profiles and short-term behaviors. Such heterogeneity appears as cells exit cytokinesis, but does not derive from the asymmetric segregation of molecules such as NG2 or PDGFRa expressed in the mother cell. Rather, rapid downregulation of OPC markers and upregulation of molecules associated with lineage progression contributes to generate early sister OPC asymmetry. Analyses during aging and upon exposure to physiological (i.e., increased motor activity) and pathological (i.e., trauma or demyelination) stimuli showed that both intrinsic and environmental factors contribute to determine the fraction of symmetric and asymmetric OPC pairs and the phenotype of the OPC progeny as soon as cells exit mitosis.

Molecular diversity subdivides the adult forebrain neural stem cell population.

Neural stem cells (NSCs) in the ventricular domain of the subventricular zone (V-SVZ) of rodents produce neurons throughout life while those in humans become largely inactive or may be lost during infancy. Most adult NSCs are quiescent, express glial markers, and depend on Notch signaling for their self-renewal and the generation of neurons. Using genetic markers and lineage tracing, we identified subpopulations of adult V-SVZ NSCs (type 1, 2, and 3) indicating a striking heterogeneity including activated, brain lipid binding protein (BLBP, FABP7) expressing stem cells. BLBP(+) NSCs are mitotically active components of pinwheel structures in the lateral ventricle walls and persistently generate neurons in adulthood. BLBP(+) NSCs express epidermal growth factor (EGF) receptor, proliferate in response to EGF, and are a major clonogenic population in the SVZ. We also find BLBP expressed by proliferative ventricular and subventricular progenitors in the fetal and postnatal human brain. Loss of BLBP(+) stem/progenitor cells correlates with reduced neurogenesis in aging rodents and postnatal humans. These findings of molecular heterogeneity and proliferative differences subdivide the NSC population and have implications for neurogenesis in the forebrain of mammals during aging.

SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA.

Neural stem cells (NSCs) are multipotent and correct fate determination is crucial to guarantee brain formation and homeostasis. How NSCs are instructed to generate neuronal or glial progeny is not well understood. Here, we addressed how murine adult hippocampal NSC fate is regulated and described how scaffold attachment factor B (SAFB) blocks oligodendrocyte production to enable neuron generation. We found that SAFB prevents NSC expression of the transcription factor nuclear factor I/B (NFIB) by binding to sequences in the Nfib mRNA and enhancing Drosha-dependent cleavage of the transcripts. We show that increasing SAFB expression prevents oligodendrocyte production by multipotent adult NSCs, and conditional deletion of Safb increases NFIB expression and oligodendrocyte formation in the adult hippocampus. Our results provide novel insights into a mechanism that controls Drosha functions for selective regulation of NSC fate by modulating the post-transcriptional destabilization of Nfib mRNA in a lineage-specific manner.

DeePhys: A machine learning-assisted platform for electrophysiological phenotyping of human neuronal networks.

Reproducible functional assays to study in vitro neuronal networks represent an important cornerstone in the quest to develop physiologically relevant cellular models of human diseases. Here, we introduce DeePhys, a MATLAB-based analysis tool for data-driven functional phenotyping of in vitro neuronal cultures recorded by high-density microelectrode arrays. DeePhys is a modular workflow that offers a range of techniques to extract features from spike-sorted data, allowing for the examination of functional phenotypes both at the individual cell and network levels, as well as across development. In addition, DeePhys incorporates the capability to integrate novel features and to use machine-learning-assisted approaches, which facilitates a comprehensive evaluation of pharmacological interventions. To illustrate its practical application, we apply DeePhys to human induced pluripotent stem cell-derived dopaminergic neurons obtained from both patients and healthy individuals and showcase how DeePhys enables phenotypic screenings.

Neurogenic subventricular zone stem/progenitor cells are Notch1-dependent in their active but not quiescent state.

The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.

Notch signaling as a master regulator of adult neurogenesis.

Neurogenesis ceases in most regions of the mammalian brain before or shortly after birth, however, in a few restricted brain regions, the production of new neurons proceeds into adulthood. Neural stem cells (NSCs) in these neurogenic zones are integrated into niches that control their activity and fate. Most stem cells in the adult brain are mitotically inactive and these cells can remain quiescent for months or even years. One of the key questions is what are the molecular mechanisms that regulate NSC maintenance and differentiation. Notch signaling has been shown to be a critical regulator of stem cell activity and maintenance in many tissues including in the nervous system. In this mini-review we discuss the roles of Notch signaling and the functions of the different Notch receptors and ligands in regulating neurogenesis in the adult murine brain. We review the functions of Notch signaling components in controlling NSC quiescence and entry into cell cycle and neurogenesis.

Targeting neuronal lysosomal dysfunction caused by ß-glucocerebrosidase deficiency with an enzyme-based brain shuttle construct.

Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to ß-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.

The small GTPase RhoA is required to maintain spinal cord neuroepithelium organization and the neural stem cell pool.

The regulation of adherens junctions (AJs) is critical for multiple events during CNS development, including the formation and maintenance of the neuroepithelium. We have addressed the role of the small GTPase RhoA in the developing mouse nervous system using tissue-specific conditional gene ablation. We show that, in the spinal cord neuroepithelium, RhoA is essential to localize N-cadherin and ß-catenin to AJs and maintain apical-basal polarity of neural progenitor cells. Ablation of RhoA caused the loss of AJs and severe abnormalities in the organization of cells within the neuroepithelium, including decreased neuroepithelial cell proliferation and premature cell-cycle exit, reduction of the neural stem cell pool size, and the infiltration of neuroepithelial cells into the lumen of the ventricle. We also show that, in the absence of RhoA, its effector, mammalian diaphanous-related formin1 (mDia1), does not localize to apical AJs in which it likely stabilizes intracellular adhesion by promoting local actin polymerization and microtubule organization. Furthermore, expressing a dominant-negative form of mDia1 in neural stem/progenitor cells results in a similar phenotype compared with that of the RhoA conditional knock-out, namely the loss of AJs and apical polarity. Together, our data show that RhoA signaling is necessary for AJ regulation and for the maintenance of mammalian neuroepithelium organization preventing precocious cell-cycle exit and differentiation.