RESUMO
The peripheral nervous system consists of ganglia, nerve trunks, plexuses, and nerve endings, that transmit afferent and efferent information. Regeneration after a peripheral nerve damage is sluggish and imperfect. Peripheral nerve injury frequently causes partial or complete loss of motor and sensory function, physical impairment, and neuropathic pain, all of which have a negative impact on patients' quality of life. Because the mechanism of peripheral nerve injury and healing is still unclear, the therapeutic efficacy is limited. As peripheral nerve injury research has processed, an increasing number of studies have revealed that biological scaffolds work in tandem with progenitor cells to repair peripheral nerve injury. Here, we fabricated collagen chitosan nerve conduit bioscaffolds together with collagen and then filled neuroepithelial stem cells (NESCs). Scanning electron microscopy showed that the NESCs grew well on the scaffold surface. Compared to the control group, the NESCs group contained more cells with bigger diameters and myelinated structures around the axons. Our findings indicated that a combination of chitosan-collagen bioscaffold and neural stem cell transplantation can facilitate the functional restoration of peripheral nerve tissue, with promising future applications and research implications.
Assuntos
Quitosana , Colágeno , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Alicerces Teciduais , Quitosana/química , Regeneração Nervosa/fisiologia , Colágeno/química , Animais , Alicerces Teciduais/química , Traumatismos dos Nervos Periféricos/terapia , Ratos , Células Neuroepiteliais/citologia , Células-Tronco Neurais/citologia , Nervos Periféricos/fisiologia , Nervo Isquiático/fisiologiaRESUMO
The number of neural stem cells reflects the total number of neurons in the mature brain. As neural stem cells arise from neuroepithelial cells, the neuroepithelial cell population must be expanded to secure a sufficient number of neural stem cells. However, molecular mechanisms that regulate timely differentiation from neuroepithelial to neural stem cells are largely unclear. Here, we show that TCF4/Daughterless is a key factor that determines the timing of the differentiation in Drosophila. The neuroepithelial cells initiated but never completed the differentiation in the absence of TCF4/Daughterless. We also found that TCF4/Daughterless binds to the Notch locus, suggesting that Notch is one of its downstream candidate genes. Consistently, Notch expression was ectopically induced in the absence of TCF4/Daughterless. Furthermore, ectopic activation of Notch signaling phenocopied loss of TCF4/Daughterless. Our findings demonstrate that TCF4/Daughterless directly inactivates Notch signaling pathway, resulting in completion of the differentiation from neuroepithelial cells into neural stem cells with optimal timing. Thus, the present results suggest that TCF4/Daughterless is essential for determining whether to move to the next state or stay in the current state in differentiating neuroepithelial cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proteínas de Drosophila , Células-Tronco Neurais , Células Neuroepiteliais , Receptores Notch , Transdução de Sinais , Animais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Receptores Notch/metabolismo , Receptores Notch/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Células Neuroepiteliais/metabolismo , Células Neuroepiteliais/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/citologia , Fatores de Tempo , Drosophila/metabolismoRESUMO
In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor ß (TGF-ß) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.
Assuntos
Encéfalo , Proliferação de Células , Proteínas de Drosophila , Larva , Células-Tronco Neurais , Células Neuroepiteliais , Isoformas de Proteínas , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Encéfalo/metabolismo , Células Neuroepiteliais/metabolismo , Células Neuroepiteliais/citologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Transdução de Sinais , Receptores de Ativinas/metabolismo , Receptores de Ativinas/genéticaRESUMO
The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.
Every cell in the body can be traced back to a stem cell. For instance, most cells in the adult brains of fruit flies come from a type of stem cell known as a neuroblast. This includes neurons and glial cells (which support and protect neurons) in the optic lobe, the part of the brain that processes visual information. The numbers of neurons and glia in the optic lobe are tightly regulated such that when the right numbers are reached, the neuroblasts stop making more and are terminated. But how and when this occurs is poorly understood. To investigate, Nguyen and Cheng studied when neuroblasts disappear in the optic lobe over the course of development. This revealed that the number of neuroblasts dropped drastically 12 to 18 hours after the fruit fly larvae developed in to pupae, and were completely gone by 30 hours in to pupae life. Further experiments revealed that the timing of this decrease is influenced by neuroepithelium cells, the pool of stem cells that generate neuroblasts during the early stages of development. Nguyen and Cheng found that speeding up this transition so that neuroblasts arise from the neuroepithelium earlier, led neuroblasts to disappear faster from the optic lobe; whereas delaying the transition caused neuroblasts to persist for much longer. Thus, the time at which neuroblasts are born determines when they are terminated. Furthermore, Nguyen and Cheng showed that the neuroblasts were lost through a combination of means. This includes dying via a process called apoptosis, dividing to form two mature neurons, or switching to a glial cell fate. These findings provide a deeper understanding of the mechanisms regulating stem cell pools and their conversion to different cell types, a process that is crucial to the proper development of the brain. How cells divide to form the optic lobe of fruit flies is similar to how new neurons arise in the mammalian brain. Understanding how and when stem cells in the fruit fly brain stop proliferating could therefore provide new insights in to the development of the human brain.
Assuntos
Apoptose , Diferenciação Celular , Proteínas de Drosophila , Células-Tronco Neurais , Células Neuroepiteliais , Neurogênese , Animais , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Neurogênese/fisiologia , Células Neuroepiteliais/fisiologia , Células Neuroepiteliais/citologia , Neuroglia/fisiologia , Neuroglia/citologia , Drosophila/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Drosophila melanogaster/citologia , Lobo Óptico de Animais não Mamíferos/citologia , Lobo Óptico de Animais não Mamíferos/crescimento & desenvolvimento , Pupa/crescimento & desenvolvimento , Proteínas de Ligação a DNA , Fatores de TranscriçãoRESUMO
In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.
Assuntos
Tubo Neural , Medula Espinal , Animais , Medula Espinal/embriologia , Tubo Neural/embriologia , Crista Neural/embriologia , Crista Neural/citologia , Crista Neural/fisiologia , Diferenciação Celular/fisiologia , Neuroglia/fisiologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/fisiologia , HumanosRESUMO
Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection. For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012).
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células Neuroepiteliais/citologia , Células Cultivadas , Humanos , Neuroglia/citologia , Neurônios/citologiaRESUMO
Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Defeitos do Tubo Neural/embriologia , Tubo Neural/embriologia , Animais , Polaridade Celular , Forma Celular , Proteínas do Citoesqueleto , Expressão Gênica , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Morfogênese , Mutação , Proteínas do Tecido Nervoso/genética , Placa Neural/citologia , Placa Neural/embriologia , Tubo Neural/citologia , Defeitos do Tubo Neural/genética , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Células Neuroepiteliais/ultraestrutura , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismoRESUMO
The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.
Assuntos
Córtex Cerebral/embriologia , Células Ependimogliais/citologia , Células-Tronco Neurais/citologia , Células Neuroepiteliais/citologia , Neurogênese , Animais , Córtex Cerebral/citologia , Humanos , Análise de Célula ÚnicaRESUMO
The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine ß-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.
Assuntos
Lesões Encefálicas/genética , Regeneração do Cérebro/genética , Cistationina beta-Sintase/genética , Proteínas de Peixes/genética , Glutamato-Amônia Ligase/genética , Fator de Transcrição PAX2/genética , Telencéfalo/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Diferenciação Celular , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Sulfeto de Hidrogênio/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurogênese/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oncorhynchus keta , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Telencéfalo/lesões , Telencéfalo/patologiaRESUMO
Neutrophils are generally considered as short-lived, homogenous, and terminally differentiated phagocytes that play crucial roles in conquering infection, although they occasionally cause severe collateral tissue damage or chronic inflammation. Recent reports have indicated that neutrophils also play a protective role in inflammation resolution and tissue repair. However, how terminally differentiated neutrophils have diverse functions remains unclear. Here, we show that neutrophils undergo conversion into Ly6G+ SiglecF+ double-positive cells expressing neurosupportive genes in the olfactory neuroepithelium (OE) under an inflammatory state. Through comprehensive flow cytometric analysis of murine nose, we identified Ly6G+ SiglecF+ double-positive cells that reside only in the OE under steady-state conditions. Double-positive cells were neutrophil-derived cells and increased by more than 10-fold during inflammation or tissue injury. We found that neutrophils infiltrate into the nose to express proinflammatory genes in the acute phase of inflammatory state, and they gradually change their surface markers and gene expression, expressing some neurogenesis-related genes in addition to inflammation related genes in the later phase. As the OE is known to have exceptionally high regeneration capacity as a nervous system, these findings suggest that neutrophils have the potential to contribute neurogenesis after conversion in peripheral nervous tissues, providing a challenge on a classic view of neutrophils as terminally differentiated leukocytes.
Assuntos
Antígenos Ly/metabolismo , Células Neuroepiteliais/citologia , Neurônios/citologia , Neutrófilos/imunologia , Bulbo Olfatório/citologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Contagem de Células , Proliferação de Células , Forma Celular , Eosinófilos/metabolismo , Feminino , Regulação da Expressão Gênica , Inflamação/patologia , Camundongos Endogâmicos C57BL , Neurogênese/genética , Nariz/patologiaRESUMO
Neuroepithelial cells act as neural stem cells by renewing themselves during embryonic development. These cells are tightly interconnected and make contact with the basement membrane of the neuroepithelium. Under such circumstances, Ca2+ fluorescence recording is a successful method to study physiological properties of the neuroepithelial stem cell. This chapter describes detailed techniques of Ca2+ fluorescence recording from neuroepithelial stem cells.
Assuntos
Cálcio/análise , Fluorescência , Células Neuroepiteliais/química , Animais , Galinhas , Células Neuroepiteliais/citologiaRESUMO
Peripheral chemosensitivity in fishes is thought to be mediated by serotonin-enriched neuroepithelial cells (NECs) that are localized to the gills of adults and the integument of larvae. In adult zebrafish (Danio rerio), branchial NECs are presumed to mediate the cardiorespiratory reflexes associated with hypoxia or hypercapnia, whereas in larvae, there is indirect evidence linking cutaneous NECs to hypoxic hyperventilation and hypercapnic tachycardia. No study yet has examined the ventilatory response of larval zebrafish to hypercapnia, and regardless of developmental stage, the signaling pathways involved in CO2 sensing remain unclear. In the mouse, a background potassium channel (TASK-2) contributes to the sensitivity of chemoreceptor cells to CO2. Zebrafish possess two TASK-2 channel paralogs, TASK-2 and TASK-2b, encoded by kcnk5a and kcnk5b, respectively. The present study aimed to determine whether TASK-2 channels are expressed in NECs of larval zebrafish and whether they are involved in CO2 sensing. Using immunohistochemical approaches, TASK-2 protein was observed on the surface of NECs in larvae. Exposure of larvae to hypercapnia caused cardiac and breathing frequencies to increase, and these responses were blunted in fish experiencing TASK-2 and/or TASK-2b knockdown. The results of these experiments suggest that TASK-2 channels are involved in CO2 sensing by NECs and contribute to the initiation of reflex cardiorespiratory responses during exposure of larvae to hypercapnia.
Assuntos
Dióxido de Carbono/metabolismo , Hipercapnia/metabolismo , Hipóxia/metabolismo , Células Neuroepiteliais/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Células Quimiorreceptoras/metabolismo , Brânquias/metabolismo , Hiperventilação/metabolismo , Células Neuroepiteliais/citologia , Oxigênio/metabolismo , Peixe-Zebra/fisiologiaRESUMO
The mammalian vestibular epithelia exhibit a remarkably stereotyped organization featuring cellular characteristics under planar cell polarity (PCP) control. PCP mechanisms are responsible for the organization of hair cell morphologic polarization vectors, and are thought to be responsible for the postsynaptic expression of the calcium-binding protein calretinin that defines the utricular striola and cristae central zone. However, recent analyses revealed that subtle differences in the topographic expression of oncomodulin, another calcium-binding protein, reflects heterogeneous factors driving the subtle variations in expression. Calbindin represents a third calcium-binding protein that has been previously described to be expressed in both hair cells and afferent calyces in proximity to the utricular striola and crista central zone. The objective of the present investigation was to determine calbindin's topographic pattern of expression to further elucidate the extent to which PCP mechanisms might exert control over the organization of vestibular neuroepithelia. The findings revealed that calbindin exhibited an expression pattern strikingly similar to oncomodulin. However, within calyces of the central zone calbindin was colocalized with calretinin. These results indicate that organizational features of vestibular epithelia are governed by a suite of factors that include PCP mechanisms as well others yet to be defined.
Assuntos
Calbindina 1/biossíntese , Calbindina 2/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Neuroepiteliais/metabolismo , Vestíbulo do Labirinto/metabolismo , Animais , Calbindina 1/metabolismo , Calbindina 2/metabolismo , Polaridade Celular/fisiologia , Células Ciliadas Auditivas/citologia , Camundongos Endogâmicos C57BL , Células Neuroepiteliais/citologia , Vestíbulo do Labirinto/citologiaRESUMO
Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.
Assuntos
Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neuroepiteliais/citologia , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Plasticidade Celular , Citoplasma/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Neuroepiteliais/metabolismo , FenótipoRESUMO
We aimed to evaluate the therapeutic effects of exosomes, which were collected from human neuroepithelial stem cells (HNESCs) treated by miR-29b mimics, on the treatment of spinal cord injury (SCI). Computational analysis, real-time PCR, Western blot analysis and TUNEL assay, a BBB score system, the Nissl staining and IHC assay were conducted to explore the molecular signalling pathway underlying the function of exosomes in SCI. Exosomes isolated from cells treated with HNESC exhibited the strongest inhibitory effect on cell apoptosis while exhibiting the highest level of miR-29b expression and the lowest levels of PTEN and caspase-3 expression. Moreover, PTEN and caspase-3 were identified as the direct target genes of miR-29b. The exosomes isolated from the groups of HNESC and HNESC + miR-29b mimics exhibited in vivo therapeutic effects by restoring the BBB score and apoptosis index of post-SCI neuron cells to those of normal neuron cells, with the exosomes collected from the group of HNESC + miR-29b mimics showing the strongest effect. We suggested that the exosomes derived from the group of HNESC + miR-29b mimics exerted therapeutic effects on SCI by down-regulating the expression of PTEN/caspase-3 and subsequently suppressing the apoptosis of neuron cells.
Assuntos
Exossomos/transplante , MicroRNAs/genética , Traumatismos da Medula Espinal/terapia , Células-Tronco/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Exossomos/genética , Exossomos/metabolismo , Regulação da Expressão Gênica , Humanos , Locomoção/fisiologia , Masculino , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurônios/metabolismo , Neurônios/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Células-Tronco/citologiaRESUMO
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.
Assuntos
Ácido Fólico/uso terapêutico , Defeitos do Tubo Neural/prevenção & controle , Aminoácido Oxirredutases/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Feminino , Deficiência de Ácido Fólico/prevenção & controle , Humanos , Modelos Biológicos , Complexos Multienzimáticos/efeitos dos fármacos , Tubo Neural/embriologia , Tubo Neural/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/efeitos dos fármacos , Células Neuroepiteliais/metabolismo , Ácidos Nucleicos/metabolismo , Necessidades Nutricionais , Gravidez , Tetra-Hidrofolatos/metabolismo , Transferases/efeitos dos fármacosRESUMO
Pseudostratified epithelia (PSE) are a common type of columnar epithelia found in a wealth of embryonic and adult tissues such as ectodermal placodes, the trachea, the ureter, the gut and the neuroepithelium. PSE are characterized by the choreographed displacement of cells' nuclei along the apicobasal axis according to phases of their cell cycle. Such movements, called interkinetic movements (INM), have been proposed to influence tissue expansion and shape and suggested as culprit in several congenital diseases such as CAKUT (Congenital anomalies of kidney and urinary tract) and esophageal atresia. INM rely on cytoskeleton dynamics just as adhesion, contractility and mitosis do. Therefore, long term impairment of INM without affecting proliferation and adhesion is currently technically unachievable. Here we bypassed this hurdle by generating a 2D agent-based model of a proliferating PSE and compared its output to the growth of the chick neuroepithelium to assess the interplay between INM and these other important cell processes during growth of a PSE. We found that INM directly generates apical expansion and apical nuclear crowding. In addition, our data strongly suggest that apicobasal elongation of cells is not an emerging property of a proliferative PSE but rather requires a specific elongation program. We then discuss how such program might functionally link INM, tissue growth and differentiation.
Assuntos
Núcleo Celular/fisiologia , Epitélio/embriologia , Animais , Padronização Corporal/fisiologia , Contagem de Células , Ciclo Celular/fisiologia , Polaridade Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Biologia Computacional , Humanos , Modelos Biológicos , Movimento/fisiologia , Células Neuroepiteliais/citologia , Análise de Sistemas , Anormalidades Urogenitais/embriologia , Refluxo Vesicoureteral/embriologiaRESUMO
The formation of the vertebrate nervous system depends on the complex interplay of morphogen signaling pathways and cell cycle progression to establish distinct cell fates. The Sonic hedgehog (Shh) signaling pathway is well understood to promote ventral cell fates in the developing spinal cord. A key regulator of Shh signaling is its receptor Patched1 (Ptch1). However, because the Ptch1 null mutation is lethal early in mouse embryogenesis, its role in controlling cell cycle progression, neurogenesis, and axon guidance in the developing spinal cord is not fully understood. An allele of Ptch1 called Wiggable (Ptch1Wig), which was previously shown to enhance Shh signaling, was used to test its ability to regulate neurogenesis and proliferation in the developing spinal cord. Ptch1Wig/Wig mutants displayed enhanced ventral proneural gene activation, and aberrant proliferation of the neural tube and floor plate cells, the latter normally being a quiescent population. The expression of the cell cycle regulators p27Kip1 and p57Kip2 were expanded in Ptch1Wig/Wig mutant spinal cords, as was the number of mitotic and S-phase nuclei, suggesting enhanced cell cycle progression. However, Ptch1Wig/Wig mutants also showed enhanced apoptosis in the ventral embryonic spinal cord, which resulted in thinner spinal cords at later embryonic stages. Commissural axons largely failed to cross the floor plate of Ptch1Wig/Wig mutant embryos, suggesting enhanced Shh signaling in these mutants led to a dorsal expansion of the chemoattraction front. These findings are consistent with a role of Ptch1 in regulating neurogenesis and proliferation of neural progenitors, and in restricting the influence of Shh signaling in commissural axon guidance to the floor plate.
Assuntos
Orientação de Axônios , Diferenciação Celular , Embrião de Mamíferos/citologia , Proteínas Hedgehog/metabolismo , Neurogênese , Receptor Patched-1/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Animais , Ciclo Celular , Proliferação de Células , Camundongos , Mutação/genética , Tubo Neural/embriologia , Tubo Neural/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismoRESUMO
Massive, coordinated cellular changes accompany the transition of central nervous system (CNS) progenitors from forebrain neurectodermal cells to specified neuroepithelial cells. We have previously found that MYC regulates the changing ribosomal and proteostatic landscapes in mouse forebrain precursors at embryonic days E8.5 and E10.5 (before and after neural tube closure; NTC) (Chau et al., 2018). Here, we demonstrate parallel coordinated transcriptional changes in metabolic machinery during this same stage of forebrain specification. Progenitors showed striking mitochondrial structural changes transitioning from glycolytic cristae at E8.5, to more traditional mitochondria at E10.5. Accordingly, glucose use shifted in progenitors such that E8.5 progenitors relied on glycolysis, and after NTC increasingly used oxidative phosphorylation. This metabolic shift was matched by changes in surrounding amniotic and cerebrospinal fluid proteomes. Importantly, these mitochondrial morphological shifts depend on MYC downregulation. Together, our findings demonstrate that metabolic shifting accompanies dynamic organelle and proteostatic remodeling of progenitor cells during the earliest stages of forebrain development.
Assuntos
Mitocôndrias/metabolismo , Proteoma/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Epitélio/metabolismo , Feminino , Glicólise , Immunoblotting , Masculino , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Transmissão , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , RNA-Seq , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Brain development requires the generation of the right number, and type, of neurons and glial cells at the right time. The Drosophila optic lobe, like mammalian brains, develops from simple neuroepithelia; they first divide symmetrically to expand the progenitor pool and then differentiate into neuroblasts, which divide asymmetrically to generate neurons and glial cells. Here, we investigate the mechanisms that control neuroepithelial growth and differentiation in the optic lobe. We find that the Broad/Tramtrack/Bric a brac-zinc finger protein Broad, which is dynamically expressed in the optic lobe neuroepithelia, promotes the transition of neuroepithelial cells to medulla neuroblasts. Loss of Broad function causes neuroepithelial cells to remain highly proliferative and delays neuroepithelial cell differentiation into neuroblasts, which leads to defective lamina and medulla. Conversely, Broad overexpression induces neuroepithelial cells to prematurely transform into medulla neuroblasts. We find that the ecdysone receptor is required for neuroepithelial maintenance and growth, and that Broad expression in neuroepithelial cells is repressed by the ecdysone receptor. Our studies identify Broad as an important cell-intrinsic transcription factor that promotes the neuroepithelial-cell-to-neuroblast transition.