RESUMEN
A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.
Asunto(s)
Proliferación Celular , Mesodermo , Modelos Biológicos , Morfogénesis , Tubo Neural , Animales , Mesodermo/embriología , Mesodermo/citología , Tubo Neural/embriología , Tubo Neural/citología , Codorniz/embriología , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , ViscosidadRESUMEN
In this issue of Developmental Cell, Bolondi et al. systematically assesses neuro-mesodermal progenitor (NMP) dynamics by combining a mouse stem-cell-based embryo model with molecular recording of single cells, shedding light on the dynamics of neural tube and paraxial mesoderm formation during mammalian development.
Asunto(s)
Mesodermo , Animales , Ratones , Mesodermo/citología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Tubo Neural/citología , Tubo Neural/embriología , Diferenciación Celular/fisiología , Células Madre/citología , Células Madre/metabolismo , Tipificación del CuerpoRESUMEN
During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.
Asunto(s)
Placa Neural , Tubo Neural , Xenopus laevis , Animales , Tubo Neural/embriología , Tubo Neural/citología , Tubo Neural/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Placa Neural/citología , Xenopus laevis/embriología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Microscopía de Fuerza Atómica , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriologíaRESUMEN
During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.
Asunto(s)
Proteína Morfogenética Ósea 4 , Tubo Neural , Notocorda , Organoides , Animales , Ratones , Organoides/metabolismo , Organoides/citología , Tubo Neural/metabolismo , Tubo Neural/citología , Notocorda/metabolismo , Notocorda/citología , Proteína Morfogenética Ósea 4/metabolismo , Transducción de Señal , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Morfogenéticas Óseas/metabolismoRESUMEN
The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.
Asunto(s)
Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Tubo Neural , Transducción de Señal , Tubo Neural/embriología , Tubo Neural/metabolismo , Tubo Neural/citología , Animales , Tipificación del Cuerpo/genética , Humanos , Redes Reguladoras de Genes , Médula Espinal/embriología , Médula Espinal/citología , Médula Espinal/metabolismo , Diferenciación Celular , Movimiento CelularRESUMEN
The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.
Asunto(s)
Tipificación del Cuerpo , Microfluídica , Tubo Neural , Humanos , Técnicas de Cultivo Tridimensional de Células , Diferenciación Celular , Cresta Neural/citología , Cresta Neural/embriología , Tubo Neural/citología , Tubo Neural/embriología , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Prosencéfalo/embriología , Médula Espinal/citología , Médula Espinal/embriologíaRESUMEN
During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.
Asunto(s)
Estructuras de la Membrana Celular , Miosinas , Tubo Neural , Transducción de Señal , Animales , Ratones , Transporte Biológico , Estructuras de la Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismoRESUMEN
Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.
Asunto(s)
Glucosa , Cresta Neural , Codorniz , Codorniz/crecimiento & desarrollo , Codorniz/metabolismo , Animales , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Glucosa/metabolismo , Tubo Neural/citología , Células Cultivadas , Técnicas In Vitro , Fosforilación Oxidativa , Redes y Vías Metabólicas , Adhesión CelularRESUMEN
Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.
Asunto(s)
Gastrulación , Macaca fascicularis , Organogénesis , Análisis de la Célula Individual , Animales , Humanos , Ratones , Gastrulación/genética , Macaca fascicularis/embriología , Macaca fascicularis/genética , Organogénesis/genética , Cuerpos Embrioides , Perfilación de la Expresión Génica , Línea Primitiva/citología , Línea Primitiva/embriología , Tubo Neural/citología , Tubo Neural/embriología , Cresta Neural/citología , Cresta Neural/embriología , Vía de Señalización Hippo , Mesodermo/citología , Mesodermo/embriología , Células MadreRESUMEN
Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.
Asunto(s)
Morfogénesis , Tubo Neural/anatomía & histología , Tubo Neural/embriología , Técnicas de Cultivo de Órganos/métodos , Ectodermo/citología , Ectodermo/embriología , Humanos , Modelos Biológicos , Placa Neural/citología , Placa Neural/embriología , Tubo Neural/citología , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/patología , Regeneración , Células Madre/citologíaRESUMEN
To ensure the formation of a properly patterned embryo, multiple processes must operate harmoniously at sequential phases of development. This is implemented by mutual interactions between cells and tissues that together regulate the segregation and specification of cells, their growth and morphogenesis. The formation of the spinal cord and paraxial mesoderm derivatives exquisitely illustrate these processes. Following early gastrulation, while the vertebrate body elongates, a population of bipotent neuromesodermal progenitors resident in the posterior region of the embryo generate both neural and mesodermal lineages. At later stages, the somitic mesoderm regulates aspects of neural patterning and differentiation of both central and peripheral neural progenitors. Reciprocally, neural precursors influence the paraxial mesoderm to regulate somite-derived myogenesis and additional processes by distinct mechanisms. Central to this crosstalk is the activity of the axial notochord, which, via sonic hedgehog signaling, plays pivotal roles in neural, skeletal muscle and cartilage ontogeny. Here, we discuss the cellular and molecular basis underlying this complex developmental plan, with a focus on the logic of sonic hedgehog activities in the coordination of the neural-mesodermal axis.
Asunto(s)
Diferenciación Celular , Mesodermo/citología , Tubo Neural/citología , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Mesodermo/embriología , Mesodermo/metabolismo , Tubo Neural/embriología , Tubo Neural/metabolismoRESUMEN
The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1-8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system.
Asunto(s)
Encéfalo/citología , Encéfalo/embriología , Análisis de la Célula Individual , Transcriptoma , Animales , Animales Recién Nacidos/genética , Encéfalo/anatomía & histología , Femenino , Gastrulación/genética , Masculino , Ratones , Tubo Neural/anatomía & histología , Tubo Neural/citología , Tubo Neural/embriologíaRESUMEN
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.
Asunto(s)
Tipificación del Cuerpo/genética , Cuerpos Embrioides/metabolismo , Desarrollo Embrionario/genética , Células Madre Embrionarias de Ratones/metabolismo , Transducción de Señal/genética , Animales , Ectodermo/citología , Ectodermo/crecimiento & desarrollo , Ectodermo/metabolismo , Embrión de Mamíferos , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Gástrula/citología , Gástrula/crecimiento & desarrollo , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Tubo Neural/citología , Tubo Neural/crecimiento & desarrollo , Tubo Neural/metabolismo , Notocorda/citología , Notocorda/crecimiento & desarrollo , Notocorda/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismoRESUMEN
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.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Defectos del Tubo Neural/embriología , Tubo Neural/embriología , Animales , Polaridad Celular , Forma de la Célula , Proteínas del Citoesqueleto , Expresión Génica , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Morfogénesis , Mutación , Proteínas del Tejido Nervioso/genética , Placa Neural/citología , Placa Neural/embriología , Tubo Neural/citología , Defectos del Tubo Neural/genética , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Células Neuroepiteliales/ultraestructura , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.
Asunto(s)
Tubo Neural/citología , Organoides/fisiología , Ingeniería de Tejidos/métodos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Línea Celular , Matriz Extracelular/fisiología , Humanos , Hidrogeles/química , Mecanotransducción Celular/fisiología , Células Madre Pluripotentes , Polietilenglicoles/química , RNA-Seq , Medicina Regenerativa/métodos , Análisis de la Célula Individual , Ingeniería de Tejidos/instrumentaciónRESUMEN
The origin, migratory pathways and adult derivatives of neural crest cells (NCCs) are well known. However, less is known about how these cells migrate. In this study, in a laboratory based in a low-resource setting, a hanging drop culture assay was utilised to study the movement of individual avian trunk neural crest cells. Mode of migration by means of lamellipodia and filopodia was studied in live cell cultures with a laser scanning confocal microscope and Airyscan module. Both distance migrated and speed of migration were calculated. NCCs migrated in a chain soon after emerging from the explanted neural tube, but were more dispersed and had random movements when they reached the periphery of the culture. While the distances travelled by these NCCs were less and the cells were slower on gelatine than on other extracellular matrices reported in the literature, the assay afforded detailed observation of actin filament distribution and cytoplasmic protrusions. The study has provided unique evidence of individual NCC movements in vitro, in a simple hanging drop assay optimized for the study of NCCs. The assay could be used for further analysis of the behaviour of NCCs on different extracellular matrices or with targeted action.
Asunto(s)
Movimiento Celular , Rastreo Celular , Cresta Neural/citología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Comunicación Celular , Ensayos de Migración Celular , Células Cultivadas , Pollos , Fluorescencia , Imagenología Tridimensional , Tubo Neural/citología , Seudópodos/metabolismo , Imagen de Lapso de TiempoRESUMEN
Neurally differentiating human pluripotent stem cells (hPSCs) possess the ability to self-organize into structures reminiscent of the developing fetal brain. In 2- and 3D cultures, this phenomenon initiates with formation of polarized areas of neural stem cells (NSCs), known as rosettes that resemble cross-sectional slices of the embryonic neural tube, i.e., the central nervous system (CNS) anlage. Thus, neural rosettes serve as an excellent starting point for bioengineering tissue models of all CNS tissues. Here, we provide detailed methods for bioengineering controlled induction of hPSC-derived neural assemblies with a biomimetic, singular neural rosette cytoarchitecture.
Asunto(s)
Células Madre Embrionarias Humanas/fisiología , Células-Madre Neurales/fisiología , Tubo Neural/fisiología , Neurogénesis , Neuronas/fisiología , Ingeniería de Tejidos , Materiales Biomiméticos , Técnicas de Cultivo de Célula , Línea Celular , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunohistoquímica , Microscopía , Morfogénesis , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismo , Neuronas/metabolismo , Esferoides CelularesRESUMEN
Cellular differentiation is a tightly regulated process under the control of intricate signaling and transcription factors interaction network working in coordination. These interactions make the systems dynamic, robust and stable but also difficult to dissect. In the spinal cord, recent work has shown that a network of FGF, WNT and Retinoic Acid (RA) signaling factors regulate neural maturation by directing the activity of a transcription factor network that contains CDX at its core. Here we have used partial and ordinary (Hill) differential equation based models to understand the spatiotemporal dynamics of the FGF/WNT/RA and the CDX/transcription factor networks, alone and in combination. We show that in both networks, the strength of interaction among network partners impacts the dynamics, behavior and output of the system. In the signaling network, interaction strength determine the position and size of discrete regions of cell differentiation and small changes in the strength of the interactions among networking partners can result in a signal overriding, balancing or oscillating with another signal. We also show that the spatiotemporal information generated by the signaling network can be conveyed to the CDX/transcription network to produces a transition zone that separates regions of high cell potency from regions of cell differentiation, in agreement with most in vivo observations. Importantly, one emerging property of the networks is their robustness to extrinsic disturbances, which allows the system to retain or canalize NP cells in developmental trajectories. This analysis provides a model for the interaction conditions underlying spinal cord cell maturation during embryonic axial elongation.
Asunto(s)
Modelos Teóricos , Neurogénesis , Médula Espinal/metabolismo , Animales , Embrión de Pollo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Tubo Neural/citología , Tubo Neural/embriología , Tubo Neural/metabolismo , Médula Espinal/citología , Médula Espinal/embriología , Tretinoina/metabolismo , Vía de Señalización WntRESUMEN
The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.
Asunto(s)
Transdiferenciación Celular/genética , Transformación Celular Neoplásica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/genética , Animales , Biopsia , Embrión de Pollo , Niño , Ciclina D1/genética , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Invasividad Neoplásica/genética , Células-Madre Neurales/patología , Tubo Neural/citología , Proteínas de Fusión Oncogénica/genética , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/patología , Fase S/genéticaRESUMEN
Mammalian embryos exhibit a transition from head morphogenesis to trunk elongation to meet the demand of axial elongation. The caudal neural tube (NT) is formed with neural progenitors (NPCs) derived from neuromesodermal progenitors localized at the tail tip. However, the molecular and cellular basis of elongating NT morphogenesis is yet elusive. Here, we provide evidence that caudal NPCs exhibit strong adhesion affinity that is gradually decreased along the anteroposterior (AP) axis in mouse embryonic spinal cord and human cellular models. Strong cell-cell adhesion causes collective migration, allowing AP alignment of NPCs depending on their birthdate. We further validated that this axial adhesion gradient is associated with the extracellular matrix and is under the control of graded Wnt signaling emanating from tail buds and antagonistic retinoic acid (RA) signaling. These results suggest that progressive reduction of NPC adhesion along the AP axis is under the control of Wnt-RA molecular networks, which is essential for a proper elongation of the spinal cord.