Intermediate Neural Progenitors Research Articles

Developmental Genetic and Molecular Analysis of Drosophila Central Complex Lineages.

Complex behaviors are mediated by a diverse class of neurons and glia produced during development. Both neural stem cell-intrinsic and -extrinsic temporal cues regulate the appropriate number, molecular identity, and circuit assembly of neurons. The Drosophila central complex (CX) is a higher-order brain structure regulating various behaviors, including sensory-motor integration, celestial navigation, and sleep. Most neurons and glia in the adult CX are formed during larval development by 16 Type II neural stem cells (NSCs). Unlike Type I NSCs, which directly give rise to the ganglion mother cells (GMCs), Type II NSCs give rise to multiple intermediate neural progenitors (INPs), and each INP in turn generates multiple GMCs, hence fostering the generation of longer and more diverse lineages. This makes Type II NSCs a suitable model to unravel the molecular mechanisms regulating neural diversity in more complex lineages. In this review, we elaborate on the classification and identification of NSCs based on the types of division adopted and the molecular markers expressed in each type. In the end, we discuss genetic methods for lineage analysis and birthdating. We also explain the temporal expression of stem cell factors and genetic techniques to study how stem cell factors may regulate neural fate specification.

Lineage Analysis and Birthdating of Drosophila Central Complex Lineages.

From insects to humans, the nervous system generates complex behaviors mediated by distinct neural circuits that are composed of diverse cell types. During development, the spatiotemporal gene expression of the neural progenitors expands the diversity of neuronal and glial subtypes. Various neural stem cell-intrinsic and -extrinsic gene programs have been identified that are thought to play a major role in generating diverse neuronal and glial cell types. Drosophila has served as an excellent model system for discovering the fundamental principles of nervous system development and function. The sophisticated genetic tools allow us to link the origin and birth timing (the time when a particular neuron is born during development) of neuron types to unique neural stem cells (NSCs) and to a developmental time. In Drosophila, a special class of NSCs called Type II NSCs has adopted a more advanced division mode to generate lineages for the higher-order brain center, the central complex, which is an evolutionarily conserved brain region found in all insects. Type II NSCs, similar to the human outer radial glia, generate intermediate neural progenitors (INPs), which divide many times to produce about eight to 10 progeny. Both Type II NSCs and INPs express distinct transcription factors and RNA-binding proteins that have been proposed to regulate the specification of cell types populating the adult central complex. Here, we describe the recently invented lineage filtering system, called cell class-lineage intersection (CLIn), which enables the tracking and birthdating of the Type II NSC lineages. Using CLIn, one can easily generate clones of different Type II NSCs and identify not only the origins of neurons of interest but also their birth time.

Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex

The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. Here, we show that Imp/Syp are both present in INPs, but not always in opposing gradients. We find that newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. We find that neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. We conclude that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization.

Low-level repressive histone marks fine-tune gene transcription in neural stem cells.

Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor FruitlessC (FruC) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fruC function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. FruC negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fruC loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans.

Imp is required for timely exit from quiescence in Drosophila type II neuroblasts.

Stem cells must balance proliferation and quiescence, with excess proliferation favoring tumor formation, and premature quiescence preventing proper organogenesis. Drosophila brain neuroblasts are a model for investigating neural stem cell entry and exit from quiescence. Neuroblasts begin proliferating during embryogenesis, enter quiescence prior to larval hatching, and resume proliferation 12-30h after larval hatching. Here we focus on the mechanism used to exit quiescence, focusing on "type II" neuroblasts. There are 16 type II neuroblasts in the brain, and they undergo the same cycle of embryonic proliferation, quiescence, and proliferation as do most other brain neuroblasts. We focus on type II neuroblasts due to their similar lineage as outer radial glia in primates (both have extended lineages with intermediate neural progenitors), and because of the availability of specific markers for type II neuroblasts and their progeny. Here we characterize the role of Insulin-like growth factor II mRNA-binding protein (Imp) in type II neuroblast proliferation and quiescence. Imp has previously been shown to promote proliferation in type II neuroblasts, in part by acting antagonistically to another RNA-binding protein called Syncrip (Syp). Here we show that reducing Imp levels delays exit from quiescence in type II neuroblasts, acting independently of Syp, with Syp levels remaining low in both quiescent and newly proliferating type II neuroblasts. We conclude that Imp promotes exit from quiescence, a function closely related to its known role in promoting neuroblast proliferation.

OTHR-41. Amplification of the PLAG family genes – PLAGL1 and PLAGL2 – is a key feature of a novel embryonal CNS tumor type

Pediatric central nervous system (CNS) tumors differ substantially from their adult counterparts, are marked by considerable molecular and clinical heterogeneity, and diagnosis through histopathology alone can be challenging. Using DNA methylation-based CNS tumor classification in combination with copy number and RNAseq analysis, we identify a rare, novel pediatric CNS tumor type (n=32) which is characterized by focal high-level amplification and consecutive overexpression of one of the PLAG family genes – PLAGL1 or PLAGL2. It is epigenetically divergent from other known tumor types such as high-grade gliomas, medulloblastomas, embryonal tumors, or CNS sarcomas. The wide range of original histopathologic diagnosis rendered attests to their polyphenotypic nature in terms of morphology. We suggest that these tumors may arise from early to intermediate neural progenitor cells with some neuronal commitment. Using ChIPseq data, we show that both PLAGL1 and PLAGL2 act as transcription factors for: i) the oncogenic kinase RET, a potential drug target, that was overexpressed in our cohort; ii) components of the Wnt/β-Catenin pathway; iii) a set of imprinted genes, reported to regulate the imprinted gene network in mouse models, that was deregulated in the PLAGL-amplified tumors. Consequently, a 250-gene expression PLAGL-signature indicated dysregulation of imprinting control and differentiation/development as a prominent feature. We report differences regarding age and sex distribution between PLAGL1- and PLAGL2-amplified tumors and shed light on differences in clinical behavior and outcomes between these subtypes in male and female patients. PLAGL1-amplified tumors were more prevalent in school-age children and teenagers, while PLAGL2-amplified cases occurred in very young patients. Kaplan-Meier analysis showed a trend towards a more favorable outcome in patients with PLAGL1-amplified tumors and in female patients. Survival rates remained constant after 5 years with a five-/ten-year overall survival of 75% for PLAGL1, 24% for PLAGL2, 18% for male patients, and 88% for female patients.

NanoDam identifies Homeobrain (ARX) and Scarecrow (NKX2.1) as conserved temporal factors in the Drosophila central brain and visual system.

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for missing factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously tagged proteins invivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam revealed that Homeobrain and Scarecrow (ARX and NKX2.1 orthologs) are also temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell-type-specific genome-wide profiling with temporal resolution and is easily adapted for use in higher organisms.

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless.

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.

Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine.

A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicineresearch for both neurological and psychiatric disorders.

Regulation of Neural Stem Cell Competency and Commitment during Indirect Neurogenesis.

Indirect neurogenesis, during which neural stem cells generate neurons through intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. The mechanisms that specify intermediate progenitor identity and that regulate stem cell competency to generate intermediate progenitors remain poorly understood despite their roles in indirect neurogenesis. Well-characterized lineage hierarchy and available powerful genetic tools for manipulating gene functions make fruit fly neural stem cell (neuroblast) lineages an excellent in vivo paradigm for investigating the mechanisms that regulate neurogenesis. Type II neuroblasts in fly larval brains repeatedly undergo asymmetric divisions to generate intermediate neural progenitors (INPs) that undergo limited proliferation to increase the number of neurons generated per stem cell division. Here, we review key regulatory genes and the mechanisms by which they promote the specification and generation of INPs, safeguarding the indirect generation of neurons during fly larval brain neurogenesis. Homologs of these regulators of INPs have been shown to play important roles in regulating brain development in vertebrates. Insight into the precise regulation of intermediate progenitors will likely improve our understanding of the control of indirect neurogenesis during brain development and brain evolution.

Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting

BackgroundMany transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers.ResultsIn this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions.ConclusionThis analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future.

Ulk4, a Newly Discovered Susceptibility Gene for Schizophrenia, Regulates Corticogenesis in Mice.

Schizophrenia (SCZ) is a chronic and severe mental disease that affects around 1% of the population. The precise etiology of SCZ still remains largely unknown, and no conclusive mechanisms are firmly established. Recent advances in epidemiological and clinical investigation support an overwhelmingly strong neurodevelopmental origin for SCZ. Here, we demonstrated that Unc-51-like kinase 4 (Ulk4), a novel risk factor for major mental disorders including schizophrenia, is involved in the corticogenesis. Deletion of Ulk4 in mice led to significantly thinner layers of II–III, and V in the cerebral cortex, which was confirmed in conditional Ulk4 deletion mice achieved by Cre-loxp strategy. This abnormality might be caused by decreased intermediate neural progenitors and increased apoptosis. Thus, our data suggest that Ulk4 manipulates the behaviors of neural progenitors during brain development and, when functionally defective, leads to the reduction of specific cortical layers. This anomaly may increase predisposition to a range of neurodevelopmental disorders, including SCZ.

Homeodomain protein Six4 prevents the generation of supernumerary Drosophila type II neuroblasts and premature differentiation of intermediate neural progenitors.

In order to boost the number and diversity of neurons generated from neural stem cells, intermediate neural progenitors (INPs) need to maintain their homeostasis by avoiding both dedifferentiation and premature differentiation. Elucidating how INPs maintain homeostasis is critical for understanding the generation of brain complexity and various neurological diseases resulting from defects in INP development. Here we report that Six4 expressed in Drosophila type II neuroblast (NB) lineages prevents the generation of supernumerary type II NBs and premature differentiation of INPs. We show that loss of Six4 leads to supernumerary type II NBs likely due to dedifferentiation of immature INPs (imINPs). We provide data to further demonstrate that Six4 inhibits the expression and activity of PntP1 in imINPs in part by forming a trimeric complex with Earmuff and PntP1. Furthermore, knockdown of Six4 exacerbates the loss of INPs resulting from the loss of PntP1 by enhancing ectopic Prospero expression in imINPs, suggesting that Six4 is also required for preventing premature differentiation of INPs. Taken together, our work identified a novel transcription factor that likely plays important roles in maintaining INP homeostasis.

Mushroom Bodies of Stick and Leaf Insects (Phasmatodea: Insecta): Structure and Sources of Formation

It has been established that mushroom bodies of stick insects are formed by Kenyon cells collected in numerous groups of cells the processes of which originally form individual bundles going into the calyx. The majority of such bundles lose their identity already in the cellular layer, and therefore, complex bundles usually enter the calyx. The merging of bundles progresses in the calyx, and even the peduncle keeps a fascicular structure. Only lobes have a classical concentric structure. In the embryonic period, mushroom bodies are formed by two proliferative centers with one neuroblast per center. It is supposed that the neuroblasts of mushroom bodies of stick insects are neuroblasts of type II and that groups of Kenyon cells are generated by intermediate neural progenitors.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3−/−) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3−/− embryos. Slc52a3−/− mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Changes in Cellular Composition of Dentate Gyrus in Response to Intermittent Hypoxia

Adult neurogenesis allows for the continual generation of neurons throughout adulthood. It is implicated in the maintenance of normal hippocampal function and is tightly regulated by oxygen homeostasis. Sleep apnea causes periodic cessations in breathing and leads to intermittent hypoxia (IH), which can impair hippocampal based learning and memory. This ongoing study examines how IH influences neural precursors and remodels the neurogenic niche in the hippocampus. We hypothesize that IH destabilizes the pool of intermediate neural progenitors (INPs) and recruits microglia into the area. Our findings indicate that the number of INPs is reduced by IH. Moreover, while IH does not change the number of microglia in the molecular and granular layers of the dentate gyrus, microglia appear to be uniquely recruited into the neurogenic niche (i.e., the subgranular zone) following IH. These findings suggest that IH‐dependent changes in hippocampal adult neurogenesis may stimulate microglial activity which in turn further impact normal hippocampal function. Such effects may play a significant role in influencing hippocampal physiology during untreated sleep apnea.Support or Funding InformationThis work was supported by NIH PO 1 HL 144454, NIH R01 NS10742101 awarded to AJG, and a grant from The BSD Office of Diversity & Inclusion at The University of Chicago awarded to AJG.

Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1.

Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. Here we show that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, we could block tumour formation completely by re-expressing Asense (homologue of human ASCL1), which we show is a direct target of Tailless. Our results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells.

Fzr/Cdh1 Promotes the Differentiation of Neural Stem Cell Lineages in Drosophila.

How stem cells and progenitors balance between self-renewal and differentiation is a central issue of stem cell biology. Here, we describe a novel and essential function of Drosophila Fzr/Cdh1, an evolutionary conserved protein, during the differentiation of neural stem cell (NSC) lineages in the central nervous system. We show that Fzr, a known co-activator of Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, promotes the production of neurons from neural progenitors called ganglion mother cells (GMCs). However, knockdown of APC/C subunit Ida or another APC/C co-activator CDC20 does not similarly impair GMC-neuron transition. We also observe a concomitant loss of differentiation factor Prospero expression and ectopic accumulation of mitotic kinase Polo in fzr mutant clones, strongly supporting the impairment of GMC to neuron differentiation. Besides functioning in GMCs, Fzr is also present in NSCs to facilitate the production of intermediate neural progenitors from NSCs. Taken together, Fzr plays a novel function in promoting differentiation programs during Drosophila NSC lineage development. Given that human Fzr is inactivated in multiple types of human cancers including brain tumors and that Fzr regulates neurotoxicity in various models of neurodegenerative diseases, our study on the role of Fzr in turning off proliferation in neuronal cells may provide insights into how Fzr deficits may contribute to human neurodegenerative diseases and tumors.

Adult neurogenesis in crayfish: Origin, expansion, and migration of neural progenitor lineages in a pseudostratified neuroepithelium.

Two decades after the discovery of adult-born neurons in the brains of decapod crustaceans, the deutocerebral proliferative system (DPS) producing these neural lineages has become a model of adult neurogenesis in invertebrates. Studies on crayfish have provided substantial insights into the anatomy, cellular dynamics, and regulation of the DPS. Contrary to traditional thinking, recent evidence suggests that the neurogenic niche in the crayfish DPS lacks self-renewing stem cells, its cell pool being instead sustained via integration of hemocytes generated by the innate immune system. Here, we investigated the origin, division and migration patterns of the adult-born neural progenitor (NP) lineages in detail. We show that the niche cell pool is not only replenished by hemocyte integration but also by limited numbers of symmetric cell divisions with some characteristics reminiscent of interkinetic nuclear migration. Once specified in the niche, first generation NPs act as transit-amplifying intermediate NPs that eventually exit and produce multicellular clones as they move along migratory streams toward target brain areas. Different clones may migrate simultaneously in the streams but occupy separate tracks and show spatio-temporally flexible division patterns. Based on this, we propose an extended DPS model that emphasizes structural similarities to pseudostratified neuroepithelia in other arthropods and vertebrates. This model includes hemocyte integration and intrinsic cell proliferation to synergistically counteract niche cell pool depletion during the animal's lifespan. Further, we discuss parallels to recent findings on mammalian adult neurogenesis, as both systems seem to exhibit a similar decoupling of proliferative replenishment divisions and consuming neurogenic divisions.

Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors.

Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.