Administration of 5-bromo-2'-deoxyuridine interferes with neuroblast proliferation and promotes apoptotic cell death in the rat cerebellar neuroepithelium.

The current study was conducted to assess whether a single administration of 5-bromo-2'-deoxyuridine (BrdU) interferes with cell proliferation and leads to the activation of apoptotic cellular events in the prenatal cerebellum. BrdU effects across a wide range of doses (25-300 µg/g b.w.) were analyzed using immunohistochemical and ultrastructural procedures. The pregnant rats were injected with BrdU at embryonic day 13, and their fetuses were sacrificed from 5 to 35 hr after exposure. The quantification of several parameters such as the density of mitotic figures, and BrdU and proliferating cell nuclear antigen (PCNA)-reactive cells showed that, in comparison with the saline injected rats, the administration of BrdU impairs the proliferative behavior of neuroepithelial cells. The above-mentioned parameters were significantly reduced in rats injected with 100 µg/g b.w. of BrdU. The reduction was more evident using 200 µg/g b.w. The most severe effects were found with 300 µg/g b.w. of BrdU. The present findings also revealed that high doses of BrdU lead to the activation of apoptotic cellular events as evidenced by both terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for active caspase-3. In comparison with saline rats, many apoptotic cells were found in rats injected with 100 µg/g b.w. of BrdU. The number of dying cells increased with 200 µg/g b.w. The most important number of apoptotic cells were observed in animals injected with 300 µg/g b.w. of BrdU. Ultrastructural studies confirmed the presence of neuroblasts at different stages of apoptosis.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

Compartment and cell-type specific hypoxia responses in the developing Drosophila brain.

Environmental factors such as the availability of oxygen are instructive cues that regulate stem cell maintenance and differentiation. We used a genetically encoded biosensor to monitor the hypoxic state of neural cells in the larval brain of Drosophila The biosensor reveals brain compartment and cell-type specific levels of hypoxia. The values correlate with differential tracheolation that is observed throughout development between the central brain and the optic lobe. Neural stem cells in both compartments show the strongest hypoxia response while intermediate progenitors, neurons and glial cells reveal weaker responses. We demonstrate that the distance between a cell and the next closest tracheole is a good predictor of the hypoxic state of that cell. Our study indicates that oxygen availability appears to be the major factor controlling the hypoxia response in the developing Drosophila brain and that cell intrinsic and cell-type specific factors contribute to modulate the response in an unexpected manner.This article has an associated First Person interview with the first author of the paper.

CCL2/CCR2 system in neuroepithelial radial glia progenitor cells: involvement in stimulatory, sexually dimorphic effects of maternal ethanol on embryonic development of hypothalamic peptide neurons.

BACKGROUND: Clinical and animal studies show that alcohol consumption during pregnancy produces lasting behavioral disturbances in offspring, including increased alcohol drinking, which are linked to inflammation in the brain and disturbances in neurochemical systems that promote these behaviors. These include the neuropeptide, melanin-concentrating hormone (MCH), which is mostly expressed in the lateral hypothalamus (LH). Maternal ethanol administration at low-to-moderate doses, while stimulating MCH neurons without affecting apoptosis or gliogenesis, increases in LH the density of neurons expressing the inflammatory chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 and their colocalization with MCH. These neural effects associated with behavioral changes are reproduced by maternal CCL2 administration, reversed by a CCR2 antagonist, and consistently stronger in females than males. The present study investigates in the embryo the developmental origins of this CCL2/CCR2-mediated stimulatory effect of maternal ethanol exposure on MCH neurons. METHODS: Pregnant rats from embryonic day 10 (E10) to E15 during peak neurogenesis were orally administered ethanol at a moderate dose (2 g/kg/day) or peripherally injected with CCL2 or CCR2 antagonist to test this neuroimmune system's role in ethanol's actions. Using real-time quantitative PCR, immunofluorescence histochemistry, in situ hybridization, and confocal microscopy, we examined in embryos at E19 the CCL2/CCR2 system and MCH neurons in relation to radial glia progenitor cells in the hypothalamic neuroepithelium where neurons are born and radial glia processes projecting laterally through the medial hypothalamus that provide scaffolds for neuronal migration into LH. RESULTS: We demonstrate that maternal ethanol increases radial glia cell density and their processes while stimulating the CCL2/CCR2 system and these effects are mimicked by maternal administration of CCL2 and blocked by a CCR2 antagonist. While stimulating CCL2 colocalization with radial glia and neurons but not microglia, ethanol increases MCH neuronal number near radial glia cells and making contact along their processes projecting into LH. Further tests identify the CCL2/CCR2 system in NEP as a primary source of ethanol's sexually dimorphic actions. CONCLUSIONS: These findings provide new evidence for how an inflammatory chemokine pathway functions within neuroprogenitor cells to mediate ethanol's long-lasting, stimulatory effects on peptide neurons linked to adolescent drinking behavior.

Deficiency of the oxidative stress-responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy.

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Altered Signaling in CB1R-5-HT2AR Heteromers in Olfactory Neuroepithelium Cells of Schizophrenia Patients is Modulated by Cannabis Use.

Schizophrenia (SCZ) has been associated with serotonergic and endocannabinoid systems dysregulation, but difficulty in obtaining in vivo neurological tissue has limited its exploration. We investigated CB1R-5-HT2AR heteromer expression and functionality via intracellular pERK and cAMP quantification in olfactory neuroepithelium (ON) cells of SCZ patients non-cannabis users (SCZ/nc), and evaluated whether cannabis modulated these parameters in patients using cannabis (SCZ/c). Results were compared vs healthy controls non-cannabis users (HC/nc) and healthy controls cannabis users (HC/c). Further, antipsychotic effects on heteromer signaling were tested in vitro in HC/nc and HC/c. Results indicated that heteromer expression was enhanced in both SCZ groups vs HC/nc. Additionally, pooling all 4 groups together, heteromer expression correlated with worse attentional performance and more neurological soft signs (NSS), indicating that these changes may be useful markers for neurocognitive impairment. Remarkably, the previously reported signaling properties of CB1R-5-HT2AR heteromers in ON cells were absent, specifically in SCZ/nc treated with clozapine. These findings were mimicked in cells from HC/nc exposed to clozapine, suggesting a major role of this antipsychotic in altering the quaternary structure of the CB1R-5-HT2AR heteromer in SCZ/nc patients. In contrast, cells from SCZ/c showed enhanced heteromer functionality similar to HC/c. Our data highlight a molecular marker of the interaction between antipsychotic medication and cannabis use in SCZ with relevance for future studies evaluating its association with specific neuropsychiatric alterations.

Why is folate effective in preventing neural tube closure defects?

Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure.

Cellular generation of mechanical forces required to close the presumptive spinal neural tube, the 'posterior neuropore' (PNP), involves interkinetic nuclear migration (INM) and apical constriction. Both processes change the apical surface area of neuroepithelial cells, but how they are biomechanically integrated is unknown. Rho kinase (Rock; herein referring to both ROCK1 and ROCK2) inhibition in mouse whole embryo culture progressively widens the PNP. PNP widening is not caused by increased mechanical tension opposing closure, as evidenced by diminished recoil following laser ablation. Rather, Rock inhibition diminishes neuroepithelial apical constriction, producing increased apical areas in neuroepithelial cells despite diminished tension. Neuroepithelial apices are also dynamically related to INM progression, with the smallest dimensions achieved in cells positive for the pan-M phase marker Rb phosphorylated at S780 (pRB-S780). A brief (2 h) Rock inhibition selectively increases the apical area of pRB-S780-positive cells, but not pre-anaphase cells positive for phosphorylated histone 3 (pHH3+). Longer inhibition (8 h, more than one cell cycle) increases apical areas in pHH3+ cells, suggesting cell cycle-dependent accumulation of cells with larger apical surfaces during PNP widening. Consequently, arresting cell cycle progression with hydroxyurea prevents PNP widening following Rock inhibition. Thus, Rock-dependent apical constriction compensates for the PNP-widening effects of INM to enable progression of closure.This article has an associated First Person interview with the first authors of the paper.

Screening of pure synthetic coating substrates for induced pluripotent stem cells and iPSC-derived neuroepithelial progenitors with short peptide based integrin array.

Simple and pure synthetic coating substrates are needed to overcome the disadvantages of traditional coating products like animal derived Matrigel in stem cell research. Since integrins are of great importance in cell adhesion and cell-ECM communication, in this study, a commercially available integrin array established by synthetic integrin binding peptides is used to screen coating substrates for iPSCs and NEPs. The results showed that binding peptides of integrin α5ß1, αVß1, αMß2 and αIIbß3 supported cell adhesion of iPSCs, while α5ß1, αVß1 and αIIbß3 binding peptides supported NEPs adhesion. Additionally, integrin α5ß1 binding peptide was revealed to support rapid expansion of iPSCs and iPSC-derived NEPs, as well as the process of NEPs generation, with equal efficiency as Matrigel. In this work, we demonstrated that by supporting stem cell growth in an integrin dependent manner, the integrin array and coating system has the potential to develop more precise and efficient systems in neurological disease modeling.

CBP/p300 inhibitor C646 prevents high glucose exposure induced neuroepithelial cell proliferation.

BACKGROUND: Maternal diabetes related neural tube defects (NTDs) are a result of oxidative stress and apoptosis. However, the molecular mechanism behind the pathogenesis is not fully understood. Here, we report that high glucose exposure-induced epigenetic changes influence histone H4 acetylation and neuroepithelial cell proliferation. We also show that the acetyltransferase inhibitor C646 can prevent high glucose induced changes in histone H4 acetylation and neuroepithelial cell proliferation. METHODS: By using LC-MS/MS as an unbiased approach, we screened the histone acetylation profile in an E9 neuroepithelial cell line (NE-4C) under high glucose exposure. We further explored the mechanism in cells in vitro and in maternal diabetes-induced mouse embryos in vivo. RESULTS: We identified 35 core histone acetylation marks in normal E9 neuroepithelial cells, whereas high glucose exposure resulted in novel acetylation sites on H4K31 and H4K44. Acetylation levels of embryonic development associated H4K5/K8/K12/K16 increased in neuroepithelial cells exposed to high glucose in vitro and in brain tissue from maternal diabetes induced exencephalic embryos in vivo. Further, mRNA level of histone acetyltransferase CBP encoded gene Crebbp was significantly increased both in vitro and in vivo. The addition of C646, a selective inhibitor for CBP/p300, significantly rescued increase of H4K5/K8/K12/K16 acetylation levels and H3S10pi-labeled neuroepithelial cell proliferation induced by high glucose exposure. CONCLUSION: Our data provide complementary insights for potential mechanisms of maternal diabetes induced NTDs.

Signaling-Dependent Control of Apical Membrane Size and Self-Renewal in Rosette-Stage Human Neuroepithelial Stem Cells.

In the developing nervous system, neural stem cells are polarized and maintain an apical domain facing a central lumen. The presence of apical membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling apical membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls apical membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and apical membrane components.

Elasticity-based boosting of neuroepithelial nucleokinesis via indirect energy transfer from mother to daughter.

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle-dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 µm from the surface, which is compressed and elastic because of the apical surface's contractility, laterally pushes the densely neighboring processes of non-M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor's daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development.

Poly (ethylene-co-vinyl alcohol) is a suitable substrate for human olfactory neuroepithelial cell differentiation in vitro through a defined regulatory pathway.

Olfactory dysfunction significantly influences patients' life quality, but currently has no adequate treatment. Poly (ethylene-co-vinyl alcohol) (EVAL) mediates cell adhesion, growth and modulates differentiation of neural stem cells. However, whether EVAL is a suitable substrate to establish an in vitro culture system that can promote development and differentiation of human olfactory neuroepithelial cells (HONCs) remains unexplored. This study isolates and cultures HONCs on controls and EVAL films for 21 days. The effects of treatment are assessed using immunocytochemistry, microarray analysis, quantitative PCR, ELISA and western blots following culturing. Most of the cell morphology on controls is epithelial and expresses markers of sustentacular cells (SCs), cadherin-1 and cytokeratin18, whereas the main population on EVAL presents as morphology with extended thin processes and possesses markers of mature olfactory sensory neurons (OSNs), olfactory marker protein (OMP). Microarray analyses reveal neuropeptide Y (NPY) and amphiregulin (AREG) are the two important regulating factors on EVAL films. HONCs cultured on EVAL films enhance the development of mature OSNs through NPY signaling, and significantly decrease the growth of SCs by blocking epidermal growth factor receptor (EGFR) activation. EVAL is a potential biomaterial to serve as an ideal substrate for treating olfactory dysfunction in the future. STATEMENT OF SIGNIFICANCE: Olfaction not only contributes to enjoyments of food, but provides a clue to escape from dangerous environmental hazards. However, loss of smell is commonly progressive and there is no good prognostic approach for olfactory dysfunction. Here, we use poly (ethylene-co-vinyl alcohol) (EVAL) to establish an in vitro culture system that promotes development and differentiation of human olfactory neuroepithelial cells. We show that EVAL not only enhances the development of mature olfactory sensory neurons through neuronpeptide Y signaling, but significantly protects the olfactory neuroepithelium from metaplasia by inhibiting EGFR activation. Therefore, EVAL is a potential biomaterial to serve as an ideal substrate for treating olfactory dysfunction in the future.

Inhibition of thymidylate synthase affects neural tube development in mice.

Thymidylate synthase (TYMS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP). Our aim was to investigate the role of dTMP dysmetabolism via inhibition of TYMS by an inhibitor, 5-fluorouracil (5-FU) in the occurrence of neural tube defects (NTDs). We found that a high incidence of NTDs occurred after treatment with 5-FU at 12.5 mg/kg body weight. TYMS activity was significantly inhibited with decreased dTMP and accumulation of dUMP after 5-FU injection. The proliferation of neuroepithelial cells were markedly inhibited in NTDs compared with control. Expressions of proliferating cell nuclear antigen and phosphohistone H3 were significantly decreased in NTDs, while phosphorylated replication protein A2, P53 and Caspase3 were significantly increased in NTDs compared with control. These results indicated that inhibition of TYMS affected the balance between proliferation and apoptosis in neuroepithelial cells, which might shed some lights on the mechanisms involved in NTDs.

Promotion of olfactory receptor neuron differentiation of olfactory neuroepithelial cells by using chitosan solution.

BACKGROUND: Olfactory dysfunction significantly influences patients' quality of life. Chitosan has been reported to support neuron and Schwann cell growth and even leads to orient axonal growth. However, researchers have yet to explore whether chitosan solution can promote differentiation of olfactory receptor neurons of the olfactory neuroepithelium and be used for treating olfactory dysfunction. OBJECTIVE: To evaluate the effect of chitosan solution on the differentiation of olfactory neuroepithelial cells. METHOD: Olfactory neuroepithelial cells were isolated from embryonic day 17 of Wistar rats and then cultured with and without soluble chitosan for 9 days. The concentration of chitosan solution was set at 0.1 mg/mL. The effects of treatment were assessed by immunocytochemistry and Western blot after culturing. RESULTS: The morphologic analysis indicated that olfactory neuroepithelial cells treated with chitosan exhibited bipolar shape with asymmetric processes. In addition, from days 3 to 9, the expression level of ßIII tubulin gradually reduced, but the expression level of olfactory marker protein significantly rose at day 9 in the chitosan groups (p < 0.05). Importantly, chitosan-treated olfactory neuroepithelial cells expressed more signal transduction apparatuses, olfactory neuron specific-G protein and adenylate cyclase 3, than those without chitosan treatment at day 9. Western blot analysis also further confirmed the results (p < 0.05). CONCLUSION: Experimental results revealed that soluble chitosan promoted differentiation of olfactory neuroepithelial cells based on its role in olfactory receptor neuron differentiation, neurite outgrowth, and signal transduction apparatus expressions.

Embryonic origin and lineage hierarchies of the neural progenitor subtypes building the zebrafish adult midbrain.

Neurogenesis in the post-embryonic vertebrate brain varies in extent and efficiency between species and brain territories. Distinct neurogenesis modes may account for this diversity, and several neural progenitor subtypes, radial glial cells (RG) and neuroepithelial progenitors (NE), have been identified in the adult zebrafish brain. The neurogenic sequences issued from these progenitors, and their contribution to brain construction, remain incompletely understood. Here we use genetic tracing techniques based on conditional Cre recombination and Tet-On neuronal birthdating to unravel the neurogenic sequence operating from NE progenitors in the zebrafish post-embryonic optic tectum. We reveal that a subpopulation of her5-positive NE cells of the posterior midbrain layer stands at the top of a neurogenic hierarchy involving, in order, the amplification pool of the tectal proliferation zone (TPZ), followed by her4-positive RG cells with transient neurogenic activity. We further demonstrate that the adult her5-positive NE pool is issued in lineage from an identically located NE pool expressing the same gene in the embryonic neural tube. Finally, we show that these features are reminiscent of the neurogenic sequence and embryonic origin of the her9-positive progenitor NE pool involved in the construction of the lateral pallium at post-embryonic stages. Together, our results highlight the shared recruitment of an identical neurogenic strategy by two remote brain territories, where long-lasting NE pools serve both as a growth zone and as the life-long source of young neurogenic RG cells.

Face off against ROS: Tcof1/Treacle safeguards neuroepithelial cells and progenitor neural crest cells from oxidative stress during craniofacial development.

One-third of all congenital birth defects affect the head and face, and most craniofacial anomalies are considered to arise through defects in the development of cranial neural crest cells. Cranial neural crest cells give rise to the majority of craniofacial bones, cartilages and connective tissues. Therefore, understanding the events that control normal cranial neural crest and subsequent craniofacial development is important for elucidating the pathogenetic mechanisms of craniofacial anomalies and for the exploring potential therapeutic avenues for their prevention. Treacher Collins syndrome (TCS) is a congenital disorder characterized by severe craniofacial anomalies. An animal model of TCS, generated through mutation of Tcof1, the mouse (Mus musculus) homologue of the gene primarily mutated in association with TCS in humans, has recently revealed significant insights into the pathogenesis of TCS. Apoptotic elimination of neuroepithelial cells including neural crest cells is the primary cause of craniofacial defects in Tcof1 mutant embryos. However, our understanding of the mechanisms that induce tissue-specific apoptosis remains incomplete. In this review, we describe recent advances in our understanding of the pathogenesis TCS. Furthermore, we discuss the role of Tcof1 in normal embryonic development, the correlation between genetic and environmental factors on the severity of craniofacial abnormalities, and the prospect for prenatal prevention of craniofacial anomalies.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Accelerated Three-Dimensional Neuroepithelium Formation from Human Embryonic Stem Cells and Its Use for Quantitative Differentiation to Human Retinal Pigment Epithelium.

Successful applications of pluripotent stem cells to cell-based therapies will rely on rapid and efficient methods to differentiate cells toward the target cell type. While methods have been developed for the generation of some medically relevant cell types including retinal pigment epithelium (RPE) cells, such protocols are lengthy and result in a heterogeneous cell mixture of RPE and non-RPE cells, requiring manual subselection and expansion. Such considerations have significant limiting impact of therapeutic applicability. Here we describe the accelerated three-dimensional neuroepithelial cyst culture of human embryonic stem cells (hESCs) and its utility to achieve quantitative production of RPE cell sheet with no manual selection in 30 days.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using L-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with L-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae.