FGF signaling gradient maintains symmetrical proliferative divisions of midbrain neuronal progenitors

For the correct development of the central nervous system, the balance between self-renewing and differentiating divisions of the neuronal progenitors must be tightly regulated. To maintain their self-renewing identity, the progenitors need to retain both apical and basal interfaces. However, the identities of fate-determining signals which cells receive via these connections, and the exact mechanism of their action, are poorly understood. The conditional inactivation of Fibroblast growth factor (FGF) receptors 1 and 2 in the embryonic mouse midbrain–hindbrain area results in premature neuronal differentiation. Here, we aim to elucidate the connection between FGF signaling and neuronal progenitor maintenance. Our results reveal that the loss of FGF signaling leads to downregulation of Hes1 and upregulation of Ngn2, Dll1, and p57 in the ventricular zone (VZ) cells, and that this increased neurogenesis occurs cell-autonomously. Yet the cell cycle progression, apico-basal-polarity, cell–cell connections, and the positioning of mitotic spindle in the mutant VZ appear unaltered. Interestingly, FGF8-protein is highly concentrated in the basal lamina. Thus, FGFs may act through basal processes of neuronal progenitors to maintain their progenitor status. Indeed, midbrain neuronal progenitors deprived in vitro of FGFs switched from symmetrical proliferative towards symmetrical neurogenic divisions. We suggest that FGF signaling in the midbrain VZ is cell-autonomously required for the maintenance of symmetrical proliferative divisions via Hes1-mediated repression of neurogenic genes.     

Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate

Neurogenesis is widespread in the zebrafish adult brain through the maintenance of active germinal niches. To characterize which progenitor properties correlate with this extensive neurogenic potential, we set up a method that allows progenitor cell transduction and tracing in the adult zebrafish brain using GFP-encoding retro- and lentiviruses. The telencephalic germinal zone of the zebrafish comprises quiescent radial glial progenitors and actively dividing neuroblasts. Making use of the power of clonal viral vector-based analysis, we demonstrate that these progenitors follow different division modes and fates: neuroblasts primarily undergo a limited amplification phase followed by symmetric neurogenic divisions; by contrast, radial glia are capable at the single cell level of both self-renewing and generating different cell types, and hence exhibit bona fide neural stem cell (NSC) properties in vivo. We also show that radial glial cells predominantly undergo symmetric gliogenic divisions, which amplify this NSC pool and may account for its long-lasting maintenance. We further demonstrate that blocking Notch signaling results in a significant increase in proliferating cells and in the numbers of clones, but does not affect clone composition, demonstrating that Notch primarily controls proliferation rather than cell fate. Finally, through long-term tracing, we illustrate the functional integration of newborn neurons in forebrain adult circuitries. These results characterize fundamental aspects of adult progenitor cells and neurogenesis, and open the way to using virus-based technologies for stable genetic manipulations and clonal analyses in the zebrafish adult brain.     

Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.     

G protein betagamma subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors

Neurons in the developing mammalian brain are generated from progenitor cells in the proliferative ventricular zone, and control of progenitor division is essential to produce the correct number of neurons during neurogenesis. Here we establish that Gbetagamma subunits of heterotrimeric G proteins are required for proper mitotic-spindle orientation of neural progenitors in the developing neocortex. Interfering with Gbetagamma function in progenitors causes a shift in spindle orientation from apical-basal divisions to planar divisions. This results in hyperdifferentiation of progenitors into neurons as a consequence of both daughter cells adopting a neural fate instead of the normal asymmetric cell fates. Silencing AGS3, a nonreceptor activator of Gbetagamma, results in defects similar to the impairment of Gbetagamma, providing evidence that AGS3-Gbetagamma signaling in progenitors regulates apical-basal division and asymmetric cell-fate decisions. Furthermore, our observations indicate that the cell-fate decision of daughter cells is coupled to mitotic-spindle orientation in progenitors.     

Wnt3a Promotes Hippocampal Neurogenesis by Shortening Cell Cycle Duration of Neural Progenitor Cells

The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67⁺ proliferating cells and Tuj1⁺ differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1⁺ cells to the total cells, and (2) the ratio of BrdU⁺ Tuj1⁺ cells to the total BrdU⁺ cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.     

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.     

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a beta-tubulin III(-) progenitor and a beta-tubulin III(+) neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb. When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin(+) progenitor and a Nestin(-) neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation. At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without. Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.     

Neurogenesis and asymmetric cell division

The astonishing cellular diversity in the central nervous system (CNS) arises from neural progenitors which can undergo different modes of symmetric and asymmetric divisions to self-renew as well as produce differentiated neuronal and glial progeny. Drosophila CNS neural progenitor cells, neuroblasts, have been utilised as a model to stimulate the understanding of the processes of asymmetric division, generation of neuronal lineages and, more recently, stem cell biology in vertebrates. Here we review some recent developments involving Drosophila and mammalian neural progenitor cells, highlighting some similarities and differences in the mechanisms that regulate their divisions during neurogenesis.     

Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord

Background

Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/Principal Findings

In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/Significance

Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.     

The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development

The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation.     

Mapping neurogenesis onset in the optic tectum of Xenopus laevis

Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior‐posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increasing restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1328–1341, 2016     

Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex

The development of the mammalian neocortex requires radial and tangential migration of cells. Radial migration of differentiated neurons from the ventricular zone (VZ) is well established. It is hypothesised that an earlier phase of tangential migration of mitotically active cells lays down a widespread periodically spaced set of progenitors that generate radial arrays of postmitotic neurons. We use a transgenic cell lineage marker to label and observe the behaviour of progenitors before and during the early stages of neurogenesis. Using optical projection tomography (OPT), we show that individual progenitor cells generate many radially arrayed columns of periodically spaced cells. Column positions indicate the paths taken by these progenitor cells as they migrate, often over long distances, through the proliferative zone. Clonally related cells can be distributed in both hemispheres, suggesting progenitor cells cross the midline in the anterior neural plate. We observe a dramatic and rapid decline in the number of labelled clones after E13.5, indicating that there is extensive cell death at this time.