The transcriptional co-activator Yap1 promotes adult hippocampal neural stem cell activation.

Most adult hippocampal neural stem cells (NSCs) remain quiescent, with only a minor portion undergoing active proliferation and neurogenesis. The molecular mechanisms that trigger the transition from quiescence to activation are still poorly understood. Here, we found the activity of the transcriptional co-activator Yap1 to be enriched in active NSCs. Genetic deletion of Yap1 led to a significant reduction in the relative proportion of active NSCs, supporting a physiological role of Yap1 in regulating the transition from quiescence to activation. Overexpression of wild-type Yap1 in adult NSCs did not induce NSC activation, suggesting tight upstream control mechanisms, but overexpression of a gain-of-function mutant (Yap1-5SA) elicited cell cycle entry in NSCs and hilar astrocytes. Consistent with a role of Yap1 in NSC activation, single cell RNA sequencing revealed a partial induction of an activated NSC gene expression program. Furthermore, Yap1-5SA expression also induced expression of Taz and other key components of the Yap/Taz regulon that were previously identified in glioblastoma stem cell-like cells. Consequently, dysregulated Yap1 activity led to repression of hippocampal neurogenesis, aberrant cell differentiation, and partial acquisition of a glioblastoma stem cell-like signature.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Trnp1 organizes diverse nuclear membrane-less compartments in neural stem cells.

TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.

Repetitive injury and absence of monocytes promote astrocyte self-renewal and neurological recovery.

Unlike microglia and NG2 glia, astrocytes are incapable of migrating to sites of injury in the posttraumatic cerebral cortex, instead relying on proliferation to replenish their numbers and distribution in the affected region. However, neither the spectrum of their proliferative repertoire nor their postinjury distribution has been examined in vivo. Using a combination of different thymidine analogs and clonal analysis in a model of repetitive traumatic brain injury, we show for the first time that astrocytes that are quiescent following an initial injury can be coerced to proliferate after a repeated insult in the cerebral cortex grey matter. Interestingly, this process is promoted by invasion of monocytes to the injury site, as their genetic ablation (using CCR2-/- mice) increased the number of repetitively dividing astrocytes at the expense of newly proliferating astrocytes in repeatedly injured parenchyma. These differences profoundly affected both the distribution of astrocytes and recovery period for posttraumatic behavior deficits suggesting key roles of astrocyte self-renewal in brain repair after injury.

Smad4 and Trim33/Tif1γ redundantly regulate neural stem cells in the developing cortex.

During central nervous system (CNS) development, proliferation and differentiation of neural stem cells (NSCs) have to be regulated in a spatio-temporal fashion. Here, we report different branches of the transforming growth factor ß (TGFß) signaling pathway to be required for the brain area-specific control of NSCs. In the midbrain, canonical TGFß signaling via Smad4 regulates the balance between proliferation and differentiation of NSCs. Accordingly, Smad4 deletion resulted in horizontal expansion of NSCs due to increased proliferation, decreased differentiation, and decreased cell cycle exit. In the developing cortex, however, ablation of Smad4 alone did not have any effect on proliferation and differentiation of NSCs. In contrast, concomitant mutation of both Smad4 and Trim33 led to an increase in proliferative cells in the ventricular zone due to decreased cell cycle exit, revealing a functional redundancy of Smad4 and Trim33. Furthermore, in Smad4-Trim33 double mutant embryos, cortical NSCs generated an excess of deep layer neurons concurrent with a delayed and reduced production of upper layer neurons and, in addition, failed to undergo the neurogenic to gliogenic switch at the right developmental stage. Thus, our data disclose that in different regions of the developing CNS different aspects of the TGFß signaling pathway are required to ensure proper development.

Absence of the RING domain in MID1 results in patterning defects in the developing human brain.

The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked gene MID1 Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of the MID1 gene coding for the N-terminal RING domain.

Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain.

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.

Human Induced Pluripotent Stem Cell-Derived Pericytes as Scalable and Editable Source to Study Direct Lineage Reprogramming Into Induced Neurons.

Studying human somatic cell-to-neuron conversion using primary brain-derived cells as starting cell source is hampered by limitations and variations in human biopsy material. Thus, delineating the molecular variables that allow changing the identity of somatic cells, permit adoption of neuronal phenotypes, and foster maturation of induced neurons (iNs) is challenging. Based on our previous results that pericytes derived from the adult human cerebral cortex can be directly converted into iNs (Karow et al., 2018; Karow et al., 2012), we here introduce human induced pluripotent stem cell (hiPSC)-derived pericytes (hiPSC-pericytes) as a versatile and more uniform tool to study the pericyte-to-neuron conversion process. This strategy enables us to derive scalable cell numbers and allows for engineering of the starting cell population such as introducing reporter tools before differentiation into hiPSC-pericytes and subsequent iN conversion. Harvesting the potential of this approach, we established hiPSC-derived human-human neuronal cocultures that not only allow for independent manipulation of each coculture partner but also resulted in morphologically more mature iNs. In summary, we exploit hiPSC-based methods to facilitate the analysis of human somatic cell-to-neuron conversion.

Dentate gyrus astrocytes exhibit layer-specific molecular, morphological and physiological features.

Neuronal heterogeneity has been established as a pillar of higher central nervous system function, but glial heterogeneity and its implications for neural circuit function are poorly understood. Here we show that the adult mouse dentate gyrus (DG) of the hippocampus is populated by molecularly distinct astrocyte subtypes that are associated with distinct DG layers. Astrocytes localized to different DG compartments also exhibit subtype-specific morphologies. Physiologically, astrocytes in upper DG layers form large syncytia, while those in lower DG compartments form smaller networks. Astrocyte subtypes differentially express glutamate transporters, which is associated with different amplitudes of glutamate transporter-mediated currents. Key molecular and morphological features of astrocyte diversity in the mice DG are conserved in humans. This adds another layer of complexity to our understanding of brain network composition and function, which will be crucial for further studies on astrocytes in health and disease.

Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity.

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.

Cellular identity through the lens of direct lineage reprogramming.

Direct lineage reprogramming challenges our traditional view on basic aspects of cellular identity, and in particular on processes crucial for identity acquisition. This is partly because in direct lineage reprogramming but not during natural differentiation processes changing cellular identity can occur in the absence of mitosis. Only recently, technologies emerged to deconstruct the cellular and molecular processes governing the transitory states a cell passes through on the journey from its original identity to the new target cell fate. Here we discuss arising concepts on the nature of these transitory states and the challenges and decisions cells must conquer to reach their new cellular identity.

Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells.

In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem cells and is crucial to initiate the neurogenic lineage in the adult mouse subependymal zone. Conditional deletion of Smad4 in adult neural stem cells severely impairs neurogenesis, and this is phenocopied by infusion of Noggin, an extracellular antagonist of BMP. Smad4 deletion in stem, but not progenitor cells, as well as Noggin infusion lead to an increased number of Olig2-expressing progeny that migrate to the corpus callosum and differentiate into oligodendrocytes. Transplantation experiments further verified the cell-autonomous nature of this phenotype. Thus, BMP-mediated signaling via Smad4 is required to initiate neurogenesis from adult neural stem cells and suppress the alternative fate of oligodendrogliogenesis.

Direct pericyte-to-neuron reprogramming via unfolding of a neural stem cell-like program.

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.

Glial control of neurogenesis.

Glial cells are central components of all neurogenic niches in the embryonic as well as in the adult central nervous system. While neural stem cells (NSCs) themselves exhibit glial features the behavior of NSCs is also strongly influenced by niche glial cells. Recently, studies have begun to uncover a large variety of glial cell-extrinsic as well as intrinsic factors that play crucial roles in the control of NSCs and the regulation of the cellular output from the neurogenic niches. In this review, we focus on mechanisms underlying the formation of adult NSCs by embryonic radial glia cells, discuss the influence of niche glia cells on adult NSCs and examine how the neurogenic potential of glial cells is controlled.

Time-Specific Effects of Spindle Positioning on Embryonic Progenitor Pool Composition and Adult Neural Stem Cell Seeding.

The developmental mechanisms regulating the number of adult neural stem cells (aNSCs) are largely unknown. Here we show that the cleavage plane orientation in murine embryonic radial glia cells (RGCs) regulates the number of aNSCs in the lateral ganglionic eminence (LGE). Randomizing spindle orientation in RGCs by overexpression of Insc or a dominant-negative form of Lgn (dnLgn) reduces the frequency of self-renewing asymmetric divisions while favoring symmetric divisions generating two SNPs. Importantly, these changes during embryonic development result in reduced seeding of aNSCs. Interestingly, no effects on aNSC numbers were observed when Insc was overexpressed in postnatal RGCs or aNSCs. These data suggest a new mechanism for controlling aNSC numbers and show that the role of spindle orientation during brain development is highly time and region dependent.

Stage- and area-specific control of stem cells in the developing nervous system.

The spatiotemporal control of proliferation and differentiation in neural stem cells (NSCs) is essential to produce a functional nervous system (NS). Stem cells in different areas and at different time points during development have to produce different types of cells in a precise manner. Recent studies uncovered a plethora of cell extrinsic as well as intrinsic factors that play crucial roles in the area-specific and stage-specific control of NSCs. Moreover, recapitulation of the spatiotemporal specification of NSCs in vitro opens new avenues for future applications. In this review, we have selected some key molecules to discuss the mechanisms underlying the spatiotemporal regulation of NSC development.

Brain area-specific effect of TGF-beta signaling on Wnt-dependent neural stem cell expansion.

Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord.

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.