Neuroblast Division Research Articles

Histological changes in the olfactory bulb and rostral migratory stream due to interruption of olfactory input

ObjectivePeriglomerular and granule cells in the adult mammalian olfactory bulb modulate olfactory signal transmission. These cells originate from the subventricular zone, migrate to the olfactory bulb via the Rostral Migratory Stream (RMS), and differentiate into mature cells within the olfactory bulb throughout postnatal life. While the regulation of neuroblast development is known to be affected by external stimuli, there is a lack of information concerning changes that occur during the recovery process after injury caused by external stimuli. To address this gap in research, the present study conducted histological observations to investigate changes in the olfactory bulb and RMS occurring after the degeneration and regeneration of olfactory neurons. MethodsTo create a model of olfactory neurodegeneration, adult mice were administered methimazole intraperitoneally. Nasal tissue and whole brains were removed 3, 7, 14 and 28 days after methimazole administration, and EdU was administered 2 and 4 h before removal of these tissues to monitor dividing cells in the RMS. Methimazole-untreated mice were used as controls. Olfactory nerve fibers entering the olfactory glomerulus were observed immunohistochemically using anti-olfactory marker protein. In the brain tissue, the entire RMS was observed and the volume and total number of cells in the RMS were measured. In addition, the number of neuroblasts and dividing neuroblasts passing through the RMS were measured using anti-doublecortin and anti-EdU antibodies, respectively. Statistical analysis was performed using the Tukey test. ResultsOlfactory epithelium degenerated was observed after methimazole administration, and recovered after 28 days. In the olfactory glomeruli, degeneration of OMP fibers began after methimazole administration, and after day 14, OMP fibers were reduced or absent by day 28, and overall OMP positive fibers were less than 20%. Glomerular volume tended to decrease after methimazole administration and did not appear to recover, even 28 days after recovery of the olfactory epithelium. In the RMS, EdU-positive cells decreased on day 3 and began to increase on day 7. However, they did not recover to the same levels as the control methimazole-untreated mice even after 28 days. ConclusionThese results suggest that the division and maturation of neuroblasts migrating from the RMS was suppressed by olfactory nerve degeneration or the disruption of olfactory input.

Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila.

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Opposing effects of ecdysone signaling regulate neuroblast proliferation to ensure coordination of brain and organism development

Growth regulation must be robust to ensure correct final size, but also adaptative to adjust to less favorable environmental conditions. Developmental coordination between whole-organism and the brain is particularly important, as the brain is a critical organ with little adaptability. Brain growth mainly depends on neural stem cell (NSC) proliferation to generate differentiated neural cells, it is however unclear how organism developmental progression is coordinated with NSCs. Here we demonstrate that the steroid hormone ecdysone plays a multi-step, stage specific role in regulating Drosophila NSCs, the neuroblasts. We used animals that are unable to synthesize ecdysone, to show that the developmental milestone called “critical weight peak”, the peak that informs the body has reached minimum viable weight to survive metamorphosis, acts a checkpoint necessary to set neuroblast cell cycle pace during larval neurogenesis. The peaks of ecdysone that occur post-critical weight are no longer required to maintain neuroblast division rate. We additionally show that in a second stage, at the onset of pupariation, ecdysone is instead required to trigger neuroblast's proliferation exit and consequently the end of neurogenesis. We demonstrate that, without this signal from ecdysone, neuroblasts lose their ability to exit proliferation. Interestingly, although these neuroblasts proliferate for a longer period, the number of differentiated neurons is smaller compared to wild-type brains, suggesting a role for ecdysone in neuron maintenance. Our study provides insights into how neural stem cells coordinate their division rate with the pace of body growth, identifying a novel coordination mechanism between animal development and NSC proliferation.

Formation of the brain by stem cell divisions of large neuroblasts in Oikopleura dioica, a simple chordate.

Stem cell division contributes to the generation of various cell types during animal development, especially a diverse pool of neural cells in the nervous system. One example is reiterated unequal stem cell divisions, in which a large stem cell undergoes a series of oriented unequal divisions to produce a chain of small daughter cells that differentiate. We show that reiterated unequal stem cell divisions are involved in the formation of the brain in simple chordate appendicularians (larvaceans). Two large neuroblasts in the anterior and middle of the brain-forming region of hatched larvae were observed. They produced at least 30 neural cells out of 96 total brain cells before completion of brain formation at 10 hours after fertilization by reiterated unequal stem cell divisions. The daughter cells of the anterior neuroblast were postmitotic, and the number was at least 19. The neuroblast produced small daughter neural cells posteriorly every 20 min. The neural cells first moved toward the dorsal side, turned in the anterior direction, aligned in a single line according to their birth order, and showed collective movement to accumulate in the anterior part of the brain. The anterior neuroblast originated from the right-anterior blastomeres of the eight-cell embryos and the right a222 blastomere of the 64-cell embryo. The posterior neuroblast also showed reiterated unequal stem cell divisions, and generated at least 11 neural cells. Sequential unequal stem cell divisions without stem cell growth have been observed in protostomes, such as insects and annelids. The results provide the first examples of this kind of stem cell division during brain formation in non-vertebrate deuterostomes.

A caspase-RhoGEF axis contributes to the cell size threshold for apoptotic death in developing Caenorhabditis elegans.

A cell's size affects the likelihood that it will die. But how is cell size controlled in this context and how does cell size impact commitment to the cell death fate? We present evidence that the caspase CED-3 interacts with the RhoGEF ECT-2 in Caenorhabditis elegans neuroblasts that generate "unwanted" cells. We propose that this interaction promotes polar actomyosin contractility, which leads to unequal neuroblast division and the generation of a daughter cell that is below the critical "lethal" size threshold. Furthermore, we find that hyperactivation of ECT-2 RhoGEF reduces the sizes of unwanted cells. Importantly, this suppresses the "cell death abnormal" phenotype caused by the partial loss of ced-3 caspase and therefore increases the likelihood that unwanted cells die. A putative null mutation of ced-3 caspase, however, is not suppressed, which indicates that cell size affects CED-3 caspase activation and/or activity. Therefore, we have uncovered novel sequential and reciprocal interactions between the apoptosis pathway and cell size that impact a cell's commitment to the cell death fate.

Peripheral astral microtubules ensure asymmetric furrow positioning in neural stem cells.

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.

A stochastic model of homeostasis: The roles of noise and nuclear positioning in deciding cell fate

SummaryWe study a population-based cellular model that starts from a single stem cell that divides stochastically to give rise to either daughter stem cells or differentiated daughter cells. There are three main components in the model: nucleus position, the underlying gene-regulatory network, and stochastic segregation of transcription factors in the daughter cells. The proportion of self-renewal and differentiated cell lines as a function of the nucleus position which in turn decides the plane of cleavage is studied. Both nuclear position and noise play an important role in determining the stem cell genealogies. We have observed both long and short genealogies in model simulation, and these compare well with experimental results from neuroblast and B-cell division. Symmetric divisions are observed in apical nuclei, while asymmetric division occurs when the nucleus is toward the base. In this model, the number of clones decreases over time, although the average clone size increases.

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.

Tens versus Four: Two Generations of Neural Progenitors in the Developing Mushroom Bodies of Muscina prolapsa Harris (Diptera, Muscidae)

Neurogenesis in the mushroom bodies was studied throughout the larval and pupal development of Muscina prolapsa. The existence of two generations of neural progenitors in the mushroom bodies of insects was established for the first time. Each larval mushroom body has four neuroblasts that divide as type I neuroblasts, producing a daughter neuroblast and a ganglion mother cell. The latter divides symmetrically into two cells that differentiate into Kenyon cells. In the young pupae, 8–11 cells surrounding each neuroblast of the mushroom body and morphologically indistinguishable from ganglion mother cells grow in size and transform into secondary neuroblasts, which repeatedly divide asymmetrically into a daughter secondary neuroblast and a typical ganglion mother cell. Divisions of secondary neuroblasts continue from the 3rd to the 7th day after puparium formation, and during the 7th day, secondary neuroblasts die massively through apoptosis. The fate of four neuroblasts of the larval mushroom body remains obscure.

The Case of the Scribble Polarity Module in Asymmetric Neuroblast Division in Development and Tumorigenesis.

The Scribble polarity module is composed by Scribble (Scrib), Discs large 1 (Dlg1) and Lethal (2) giant larvae (L(2)gl), a group of highly conserved neoplastic tumor suppressor genes (TSGs) from flies to humans. Even though the Scribble module has been profusely studied in epithelial cell polarity, the number of tissues and processes in which it is involved is increasingly growing. Here we discuss the role of the Scribble module in the asymmetric division of Drosophila neuroblasts (NBs), as well as the underlying mechanisms by which those TSGs act in this process. Finally, we also describe what we know about the consequences of mutating these genes in impairing the process of asymmetric NB division and promoting tumor-like overgrowth.

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability.

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates.

Mutations in ANKLE2, a ZIKA Virus Target, Disrupt an Asymmetric Cell Division Pathway in Drosophila Neuroblasts to Cause Microcephaly.

The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. We show that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. We suggest that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division.

Ankle2, a Target of Zika Virus, Controls Asymmetric Cell Division of Neuroblasts and Uncovers a Novel Microcephaly Pathway

Neuroblasts in flies divide asymmetrically by establishing polarity, distributing cell fate determinants asymmetrically, and positioning their spindle for cell division. The apical complex contains aPKC, Bazooka (Par3), and Par6, and its activity depends on L(2)gl. We show that Ankle2 interacts with L(2)gl and affects aPKC. Reducing Ankle2 levels disrupts ER and nuclear envelope morphology, releasing the kinase Ballchen/VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen/VRK1 or l(2)gl suppresses the loss of Ankle2 and restores viability and brain size. The Zika virus NS4A protein interacts with Drosophila Ankle2 and VRK1 in dividing neuroblasts. Human mutational studies implicate this neural cell division pathway in microcephaly and motor neuron disease. In summary, NS4A, ANKLE2, VRK1 and LLGL1 define a novel pathway that impinges on asymmetric determinants of neural stem cell division.

C. elegans PTEN and AMPK block neuroblast divisions by inhibiting a BMP-insulin-PP2A-MAPK pathway.

Caenorhabditis elegans that hatch in the absence of food stop their postembryonic development in a process called L1 arrest. Intriguingly, we find that the postembryonic Q neuroblasts divide and migrate during L1 arrest in mutants that have lost the energy sensor AMP-activated protein kinase (AMPK) or the insulin/IGF-1 signaling (IIS) negative regulator DAF-18/PTEN. We report that DBL-1/BMP works upstream of IIS to promote agonistic insulin-like peptides during L1 arrest. However, the abnormal Q cell divisions that occur during L1 arrest use a novel branch of the IIS pathway that is independent of the terminal transcription factor DAF-16/FOXO. Using genetic epistasis and drug interactions we show that AMPK functions downstream of, or in parallel with DAF-18/PTEN and IIS to inhibit PP2A function. Further, we show that PP2A regulates the abnormal Q cell divisions by activating the MPK-1/ERK signaling pathway via LIN-45/RAF, independently of LET-60/RAS. PP2A acts as a tumor suppressor in many oncogenic signaling cascades. Our work demonstrates a new role for PP2A that is needed to induce neuroblast divisions during starvation and is regulated by both insulin and AMPK.

A functional study of all 40 Caenorhabditis elegans insulin-like peptides

The human genome encodes 10 insulin-like genes, whereas the Caenorhabditis elegans genome remarkably encodes 40 insulin-like genes. Knockout strategies to determine the roles of all the insulin/insulin-like peptide ligands (INS) in C. elegans has been challenging due to functional redundancy. Here, we individually overexpressed each of the 40 ins genes pan-neuronally, and monitored multiple phenotypes including: L1 arrest life span, neuroblast divisions under L1 arrest, dauer formation, and fat accumulation, as readouts to characterize the functions of each INS in vivo Of the 40 INS peptides, we found functions for 35 INS peptides and functionally categorized each as agonists, antagonists, or of pleiotropic function. In particular, we found that 9 of 16 agonistic INS peptides shortened L1 arrest life span and promoted neuroblast divisions during L1 arrest. Our study revealed that a subset of β-class INS peptides that contain a distinct F peptide sequence are agonists. Our work is the first to categorize the structures of INS peptides and relate these structures to the functions of all 40 INS peptides in vivo Our findings will promote the study of insulin function on development, metabolism, and aging-related diseases.

Caenorhabditis elegans ced-3 Caspase Is Required for Asymmetric Divisions That Generate Cells Programmed To Die.

Caspases have functions other than in apoptosis. Here, we report that Caenorhabditis elegans CED-3 caspase regulates asymmetric cell division. Many of the 131 cells that are “programmed” to die during C. elegans development are the smaller daughter of a neuroblast that divides asymmetrically by size and fate. We have previously shown that CED-3 caspase is activated in such neuroblasts, and that before neuroblast division, a gradient of CED-3 caspase activity is formed in a ced-1 MEGF10 (multiple EGF-like domains 10)-dependent manner. This results in the nonrandom segregation of active CED-3 caspase or “apoptotic potential” into the smaller daughter. We now show that CED-3 caspase is necessary for the ability of neuroblasts to divide asymmetrically by size. In addition, we provide evidence that a pig-1 MELK (maternal embryonic leucine zipper kinase)-dependent reciprocal gradient of “mitotic potential” is formed in the QL.p neuroblast, and that CED-3 caspase antagonizes this mitotic potential. Based on these findings, we propose that CED-3 caspase plays a critical role in the asymmetric division by size and fate of neuroblasts, and that this contributes to the reproducibility and robustness with which the smaller daughter cell is produced and adopts the apoptotic fate. Finally, the function of CED-3 caspase in this context is dependent on its activation through the conserved egl-1 BH3-only, ced-9 Bcl-2, and ced-4 Apaf-1 pathway. In mammals, caspases affect various aspects of stem cell lineages. We speculate that the new nonapoptotic function of C. elegans CED-3 caspase in asymmetric neuroblast division is relevant to the function(s) of mammalian caspases in stem cells.

Myc and the Tip60 chromatin remodeling complex control neuroblast maintenance and polarity in Drosophila.

Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino in regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

Cell scientist to watch – Guangshuo Ou

ABSTRACT Guangshuo moved from China to the USA in 2001 for his PhD at the University of California, Davis. There, he worked on motor proteins in ciliogenesis in C. elegans in the laboratory of Jonathan Scholey. He then joined the laboratory of Ron Vale at the University of California, San Francisco, and started to develop tools to investigate the mechanisms of neuroblast division, migration and differentiation in C. elegans larvae. Guangshuo returned to China in 2011 to establish his own research group at the Institute of Biophysics at the Chinese Academy of Sciences with a Junior One Thousand Talent Plan Award. Two years later, he moved to the School of Life Sciences at Tsinghua University in Beijing, and he became a principal investigator of the Joint Center for Life Sciences at Tsinghua and Peking Universities. Guangshuo became a Journal of Cell Science Editor in 2017. His laboratory continues to investigate the mechanisms regulating neural progenitors in C. elegans, using live-imaging, biochemical and genetic tools.

Centrosome Inheritance Does Not Regulate Cell Fate in Granule Neuron Progenitors of the Developing Cerebellum.

An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited.