RESUMO
Boron nitride nanotubes (BNNTs) have experienced considerable growth in recent years due to their unique intrinsic properties, in particular for the fabrication of polymer nanocomposites. Dispersion of pure BNNTs in nanocomposites is often difficult due to their poor compatibility with most polymer matrices. An approach involving the creation of hydroxyl groups on their surface could improve their dispersion. While some harsh oxidation processes have been reported so far, a mild oxidation of BNNTs using air as the oxidant is reported here. This new catalytic reaction leads to slightly oxidized BNNTs, which were characterized by scanning electron microscope, x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. Polycarbonate nanocomposites were then fabricated using pristine and oxidized BNNTs as nanofillers. The measured thermal conductivity increased linearly with the mildly oxidized BNNTs content. It reached a five-fold increase up to 1.19 W m.K-1at 15% vol. content which is significantly improved over nanocomposites fabricated with severely oxidized BNNTs, while the electrically insulating character remained unchanged.
RESUMO
The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. The toolbox includes newly developed algorithms and interactive tools for image pre-processing and segmentation, estimation of significant single-neuron single-trial signals, mapping event-related neuronal responses, detection of activity-correlated neuronal clusters, exploration of population dynamics, and analysis of clusters' features against surrogate control datasets. The modules are integrated in a modular and versatile processing pipeline, adaptable to different needs. The clustering module is capable of detecting flexible, dynamically activated neuronal assemblies, consistent with the distributed population coding of the brain. We demonstrate the suitability of the toolbox for a variety of calcium imaging datasets. The toolbox open-source code, a step-by-step tutorial and a case study dataset are available at https://github.com/zebrain-lab/Toolbox-Romano-et-al.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Interpretação de Imagem Assistida por Computador/métodos , Neurônios/fisiologia , Software , Imagens com Corantes Sensíveis à Voltagem/métodos , Cálcio/metabolismo , Rastreamento de Células/métodos , Conectoma/métodos , Imagem Molecular/métodos , Neurônios/citologia , Linguagens de Programação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.
Assuntos
Linhagem da Célula , Células-Tronco Multipotentes/citologia , Crista Neural/citologia , Pele/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Adulto , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Face , Feminino , Imunofluorescência , Folículo Piloso/citologia , Folículo Piloso/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Masculino , Melanócitos/citologia , Melanócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células-Tronco Multipotentes/fisiologia , Crista Neural/fisiologia , Neuroglia/citologia , Neuroglia/fisiologia , Fatores de Transcrição SOXE , Fatores de Transcrição/metabolismoRESUMO
The neural crest is generally believed to be the embryonic source of skeletogenic mesenchyme (ectomesenchyme) in the vertebrate head and other derivatives, including pigment cells and neurons and glia of the peripheral nervous system. Although classical transplantation experiments leading to this conclusion assumed that embryonic neural folds were homogeneous epithelia, we reported that embryonic cranial neural folds contain spatially and phenotypically distinct domains, including a lateral nonneural domain with cells that coexpress E-cadherin and PDGFRalpha and a thickened mediodorsal neuroepithelial domain where these proteins are reduced or absent. We now show that Wnt1-Cre is expressed in the lateral nonneural epithelium of rostral neural folds and that cells coexpressing Cre-recombinase and PDGFRalpha delaminate precociously from some of this nonneural epithelium. We also show that ectomesenchymal cells exhibit beta-galactosidase activity in embryos heterozygous for an Ecad-lacZ reporter knock- in allele. We conclude that a lateral nonneural domain of the neural fold epithelium, which we call "metablast," is a source of ectomesenchyme distinct from the neural crest. We suggest that closer analysis of the origin of ectomesenchyme might help to understand (i) the molecular-genetic regulation of development of both neural crest and ectomesenchyme lineages; (ii) the early developmental origin of skeletogenic and connective tissue mesenchyme in the vertebrate head; and (iii) the presumed origin of head and branchial arch skeletal and connective tissue structures during vertebrate evolution.
Assuntos
Mesoderma/crescimento & desenvolvimento , Crista Neural/anatomia & histologia , Crista Neural/fisiologia , Crânio/embriologia , Animais , Caderinas/genética , Embrião de Mamíferos , Epitélio/embriologia , Integrases/biossíntese , Integrases/genética , Camundongos , Camundongos Transgênicos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Wnt1/biossíntese , Proteína Wnt1/genética , beta-Galactosidase/genéticaRESUMO
Organisms use their sensory systems to acquire information from their environment and integrate this information to produce relevant behaviors. Nevertheless, how sensory information is converted into adequate motor patterns in the brain remains an open question. Here, we addressed this question using two-photon and light-sheet calcium imaging in intact, behaving zebrafish larvae. We monitored neural activity elicited by auditory stimuli while simultaneously recording tail movements. We observed a spatial organization of neural activity according to four different response profiles (frequency tuning curves), suggesting a low-dimensional representation of frequency information, maintained throughout the development of the larvae. Low frequencies (150-450 Hz) were locally processed in the hindbrain and elicited motor behaviors. In contrast, higher frequencies (900-1,000 Hz) rarely induced motor behaviors and were also represented in the midbrain. Finally, we found that the sensorimotor transformations in the zebrafish auditory system are a continuous and gradual process that involves the temporal integration of the sensory response in order to generate a motor behavior.
Assuntos
Vias Auditivas/fisiologia , Percepção Auditiva , Encéfalo/fisiologia , Peixe-Zebra/fisiologia , Animais , Vias Auditivas/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The brain is spontaneously active, even in the absence of sensory stimulation. The functionally mature zebrafish optic tectum shows spontaneous activity patterns reflecting a functional connectivity adapted for the circuit's functional role and predictive of behavior. However, neither the emergence of these patterns during development nor the role of retinal inputs in their maturation has been characterized. Using two-photon calcium imaging, we analyzed spontaneous activity in intact and enucleated zebrafish larvae throughout tectum development. At the onset of retinotectal connections, intact larvae showed major changes in the spatiotemporal structure of spontaneous activity. Although the absence of retinal inputs had a significant impact on the development of the temporal structure, the tectum was still capable of developing a spatial structure associated with the circuit's functional roles and predictive of behavior. We conclude that neither visual experience nor intrinsic retinal activity is essential for the emergence of a spatially structured functional circuit.
Assuntos
Retina/fisiologia , Colículos Superiores/fisiologia , Percepção Visual , Animais , Sinalização do Cálcio , Estimulação Luminosa , Retina/crescimento & desenvolvimento , Retina/metabolismo , Colículos Superiores/crescimento & desenvolvimento , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/fisiologia , Peixe-ZebraRESUMO
Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Recently, a zebrafish carrying a mecp2-null mutation has been developed with the resulting phenotypes exhibiting defective sensory and thigmotactic responses, and abnormal motor behavior reminiscent of the human disease. Here, we performed a proteomic analysis to examine protein expression changes in mecp2-null vs. wild-type larvae and adult zebrafish. We found a total of 20 proteins differentially expressed between wild-type and mutant zebrafish, suggesting skeletal and cardiac muscle functional defects, a stunted glycolysis and depleted energy availability. This molecular evidence is directly linked to the mecp2-null zebrafish observed phenotype. In addition, we identified changes in expression of proteins critical for a proper redox balance, suggesting an enhanced oxidative stress, a phenomenon also documented in human patients and RTT murine models. The molecular alterations observed in the mecp2-null zebrafish expand our knowledge on the molecular cascade of events that lead to the RTT phenotype. BIOLOGICAL SIGNIFICANCE: We performed a proteomic study of a non-mammalian vertebrate model (zebrafish, Danio rerio) for Rett syndrome (RTT) at larval and adult stages of development. Our results reveal major protein expression changes pointing out to defects in energy metabolism, redox status imbalance, and muscle function, both skeletal and cardiac. Our molecular analysis grants the mecp2-null zebrafish as a valuable RTT model, triggering new research approaches for a better understanding of the RTT pathogenesis and phenotype expression. This non-mammalian vertebrate model of RTT strongly suggests a broad impact of Mecp2 dysfunction.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Metabolismo Energético/genética , Larva/química , Músculos/fisiologia , Mutação , Estresse Oxidativo/genética , Fenótipo , Proteínas/análise , Proteínas/fisiologia , Proteômica/métodos , Peixe-ZebraRESUMO
Following moving visual stimuli (conditioning stimuli, CS), many organisms perceive, in the absence of physical stimuli, illusory motion in the opposite direction. This phenomenon is known as the motion aftereffect (MAE). Here, we use MAE as a tool to study the neuronal basis of visual motion perception in zebrafish larvae. Using zebrafish eye movements as an indicator of visual motion perception, we find that larvae perceive MAE. Blocking eye movements using optogenetics during CS presentation did not affect MAE, but tectal ablation significantly weakened it. Using two-photon calcium imaging of behaving GCaMP3 larvae, we find post-stimulation sustained rhythmic activity among direction-selective tectal neurons associated with the perception of MAE. In addition, tectal neurons tuned to the CS direction habituated, but neurons in the retina did not. Finally, a model based on competition between direction-selective neurons reproduced MAE, suggesting a neuronal circuit capable of generating perception of visual motion.
Assuntos
Encéfalo/fisiologia , Percepção de Movimento/fisiologia , Percepção Visual/fisiologia , Peixe-Zebra/fisiologia , Animais , Condicionamento Psicológico , Movimentos Oculares/fisiologia , Pós-Efeito de Figura/fisiologia , Habituação Psicofisiológica , Larva/fisiologia , Modelos Biológicos , Modelos Neurológicos , Movimento , Neurônios/fisiologia , Optogenética , Colículos Superiores/fisiologia , CaudaRESUMO
Spontaneous neuronal activity is spatiotemporally structured, influencing brain computations. Nevertheless, the neuronal interactions underlying these spontaneous activity patterns, and their biological relevance, remain elusive. Here, we addressed these questions using two-photon calcium imaging of intact zebrafish larvae to monitor the neuron-to-neuron spontaneous activity fine structure in the tectum, a region involved in visual spatial detection. Spontaneous activity was organized in topographically compact assemblies, grouping functionally similar neurons rather than merely neighboring ones, reflecting the tectal retinotopic map despite being independent of retinal drive. Assemblies represent all-or-none-like sub-networks shaped by competitive dynamics, mechanisms advantageous for visual detection in noisy natural environments. Notably, assemblies were tuned to the same angular sizes and spatial positions as prey-detection performance in behavioral assays, and their spontaneous activation predicted directional tail movements. Therefore, structured spontaneous activity represents "preferred" network states, tuned to behaviorally relevant features, emerging from the circuit's intrinsic non-linear dynamics, adapted for its functional role.
Assuntos
Adaptação Fisiológica/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Estimulação Luminosa/métodos , Vias Visuais/fisiologia , Animais , Animais Geneticamente Modificados , Colículos Superiores/fisiologia , Peixe-ZebraRESUMO
Mutations in the gene encoding the MECP2 underlies Rett syndrome, a neurodevelopmental disorder in young females. Although reduced pain sensitivity in Rett syndrome patients and in partial MeCP2 deficient mice had been reported, these previous studies focused predominantly on motor impairments. Therefore, it is still unknown how MeCP2 is involved in these sensory defects. In addition, the human disease manifestations where males with mutations in MECP2 gene normally do not survive and females show typical neurological symptoms only after 18 months of age, is profoundly different in MeCP2-deficient mouse where all animals survived, and males but not females displayed Rett syndrome phenotypes at an early age. Thus, the mecp2-deficient zebrafish serves as an additional animal model to aid in deciphering the role and mechanisms of Mecp2 in neurodevelopment. Here, we used two independent methods of silencing expression of Mecp2 in zebrafish to uncover a novel role of Mecp2 in trigeminal ganglion sensory neurons during the embryonic development. mecp2-null mutation and morpholino-mediated silencing of Mecp2 in the zebrafish embryos resulted in defects in peripheral innervation of trigeminal sensory neurons and consequently affecting the sensory function. These defects were demonstrated to be dependent on the expression of Sema5b and Robo2. The expression of both proteins together could better overcome the defects caused by Mecp2 deficiency as compared to the expression of either Sema5b or Robo2 alone. Sema5b and Robo2 were downregulated upon Mecp2 silencing or in mecp2-null embryos, and Chromatin immunoprecipitation (ChIP) assay using antibody against Mecp2 was able to pull down specific regions of both Sema5b and Robo2 promoters, showing interaction between Mecp2 and the promoters of both genes. In addition, cell-specific expression of Mecp2 can overcome the innervation and sensory response defects in Mecp2 morphants indicating that these MeCP2-mediated defects are cell-autonomous. The sensory deficits caused by Mecp2 deficiency mirror the diminished sensory response observed in Rett syndrome patients. This suggests that zebrafish could be an unconventional but useful model for this disorder manifesting defects that are not easily studied in full using rodent models.
RESUMO
The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.
Assuntos
Encéfalo/fisiologia , Sinalização do Cálcio/fisiologia , Iluminação/métodos , Neurônios/química , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Larva , Microscopia Confocal/métodos , Fatores de Tempo , Peixe-ZebraRESUMO
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and one of the most common causes of mental retardation in affected girls. Other symptoms include a rapid regression of motor and cognitive skills after an apparently early normal development. Sporadic mutations in the transcription factor MECP2 has been shown to be present in more than 90% of the patients and several models of MeCP2-deficient mice have been created to understand the role of this gene. These models have pointed toward alterations in the maintenance of the central nervous system rather than its development, in line with the late onset of the disease in humans. However, the exact functions of MeCP2 remain difficult to delineate and the animal models have yielded contradictory results. Here, we present the first mecp2-null allele mutation zebrafish model. Surprisingly and in contrast to MeCP2-null mouse models, mecp2-null zebrafish are viable and fertile. They present nonetheless clear behavioral alterations during their early development, including spontaneous and sensory-evoked motor anomalies, as well as defective thigmotaxis.
Assuntos
Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/fisiologia , Modelos Animais , Atividade Motora/fisiologia , Fatores Etários , Animais , Animais Geneticamente Modificados , Feminino , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Mutação/genética , Gravidez , Peixe-ZebraRESUMO
Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch-evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA-type glutamate receptor activation. Transection experiments suggest that the circuits required for touch-evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA-type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation.
Assuntos
Reação de Fuga/fisiologia , Ácido Glutâmico/metabolismo , Receptores de AMPA/metabolismo , Reflexo/fisiologia , Medula Espinal/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Maleato de Dizocilpina/farmacologia , Eletrofisiologia , Embrião não Mamífero , Reação de Fuga/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Imuno-Histoquímica , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Reflexo/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/embriologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Natação/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/fisiologia , Peixe-ZebraRESUMO
The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.
Assuntos
Moléculas de Adesão Celular/genética , Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Moléculas de Adesão Celular/classificação , Sistema Nervoso Central/crescimento & desenvolvimento , Hibridização In Situ , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Homologia de Sequência de Aminoácidos , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover, beta1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network organisation. Organotypic cultures of gut explants reveal that beta1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that beta1 integrins are required for the villi innervation in the small intestine. Our findings highlight the crucial roles played by beta1 integrins at various steps of enteric nervous system development.
Assuntos
Sistema Nervoso Entérico/embriologia , Integrina beta1/genética , Crista Neural/citologia , Crista Neural/embriologia , Fenótipo , Animais , Modelos Animais de Doenças , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiologia , Doença de Hirschsprung/embriologia , Imuno-Histoquímica , Integrases , Camundongos , Modelos Genéticos , Mutação , Técnicas de Cultura de Órgãos , Proteínas ViraisRESUMO
Sympathetic axons embedded in a few arterioles and vasa vasora were recently shown to store tissue plasminogen activator (t-PA) in vesicles. But the extension of such t-PA axons to arteries and arterioles throughout the organism has not been verified. Confirmation of this anatomy would identify a second significant source of vessel wall t-PA. To visualize fine embedded axons independent of endothelium, we created a transgenic mouse whose expressions of the t-PA promoter and enhanced green fluorescent protein are confined to sympathetic neurons and other neural crest derivatives. Confocal images reveal the extension of t-PA axons to arterioles serving heart, brain, kidney, lung, mesentery, and skin; plus aortic, carotid, and mesenteric artery walls. Ganglion neurons and adrenal chromaffin cells also show strong expressions. These new sightings confirm the existence of a system of t-PA axons that is prominent in arterioles, and compatible with the release of neural t-PA into their walls.
Assuntos
Artérias/inervação , Arteríolas/inervação , Axônios/metabolismo , Sistema Nervoso Simpático/metabolismo , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Artérias/metabolismo , Artérias/ultraestrutura , Arteríolas/metabolismo , Arteríolas/ultraestrutura , Linhagem Celular , Feminino , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
RNA in situ hybridization on early chicken embryos revealed that the beta3 integrin gene started to be expressed after Hamburger and Hamilton (HH) stage 6 in the presumptive epidermis adjacent to the neural plate, before closure of the neural tube. The beta3 integrin gene was also strongly expressed in cephalic neural crest cells at the same stage in which they begin their migration but disappeared progressively in these cells along the route they take to the branchial arches. The gene was weakly expressed in the differentiating cranial neural crest cells. The alphaVbeta3 integrin protein complex was also mainly detected in the migratory cephalic neural crest cells. However, during early mouse embryogenesis and in contrast to the chick, the beta3 integrin gene was expressed in the foregut diverticulum and in the heart and not in the cephalic neural crest cells. Therefore, the difference in the beta3 integrin expression suggests that mouse and chicken cranial neural crest cells may have distinct integrin requirements during their ontogenesis.
Assuntos
Integrina beta3/genética , Crista Neural/embriologia , Crista Neural/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Crista Neural/citologia , RNA Mensageiro/análiseRESUMO
The ontogeny of neural crest cells (NCC) involves a number of orchestrated variety of derivatives, including components of the peripheral nervous system and melanocytes. Thus, it represents an excellent model system to investigate mechanisms controlling epithelial-mesenchymal transitions, cell migration and differentiation, as well as cell proliferation and death. We have established a new transgenic line expressing the Cre recombinase under the control of the human tissue plasminogen activator promoter (Ht-PA). The activity of the reporter in the Ht-PA-Cre/R26R embryos is observed as early as Theiler stage 12 in the cephalic mesenchyme. Later, the targeted cells include all the known derivatives of cranial, vagal, and trunk NCC, including craniofacial structures and cranial ganglia, cardiac and endocrine derivatives, melanocytes, peripheral, and enteric nervous system. At the vagal level, the location of presumptive enteric NCC differs from their avian counterparts in their ability to invade the mesenchyme lateral to the neural tube. In contrast to the Wnt1-Cre line, the Ht-PA-Cre line does not target the central nervous system and therefore renders it more specific for NCC. Our Ht-PA-Cre mice represent a novel model to specifically target conditional mutations in migratory NCC.
Assuntos
Integrases/fisiologia , Crista Neural/citologia , Regiões Promotoras Genéticas , Ativador de Plasminogênio Tecidual/genética , Proteínas Virais/fisiologia , Animais , Movimento Celular , Humanos , Camundongos , Camundongos Transgênicos , beta-Galactosidase/genéticaRESUMO
Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. We have generated mice with a neural crest cell-specific deletion of the beta1-integrin gene to analyse the role of beta1-integrins in neural crest cell migration and differentiation. This targeted mutation caused death within a month of birth. The loss of beta1-integrins from the embryo delayed the migration of Schwann cells along axons and induced multiple defects in spinal nerve arborisation and morphology. There was an almost complete absence of Schwann cells and sensory axon segregation and defective maturation in neuromuscular synaptogenesis. Thus, beta1-integrins are important for the control of embryonic and postnatal peripheral nervous system development.