RESUMEN
The dynamics of multipotent neural crest cell differentiation and invasion as cells travel throughout the vertebrate embryo remain unclear. Here, we preserve spatial information to derive the transcriptional states of migrating neural crest cells and the cellular landscape of the first four chick cranial to cardiac branchial arches (BA1-4) using label-free, unsorted single-cell RNA sequencing. The faithful capture of branchial arch-specific genes led to identification of novel markers of migrating neural crest cells and 266 invasion genes common to all BA1-4 streams. Perturbation analysis of a small subset of invasion genes and time-lapse imaging identified their functional role to regulate neural crest cell behaviors. Comparison of the neural crest invasion signature to other cell invasion phenomena revealed a shared set of 45 genes, a subset of which showed direct relevance to human neuroblastoma cell lines analyzed after exposure to the in vivo chick embryonic neural crest microenvironment. Our data define an important spatio-temporal reference resource to address patterning of the vertebrate head and neck, and previously unidentified cell invasion genes with the potential for broad impact.
Asunto(s)
Región Branquial/crecimiento & desarrollo , Cabeza/crecimiento & desarrollo , Cuello/crecimiento & desarrollo , Cresta Neural/crecimiento & desarrollo , Animales , Tipificación del Cuerpo/genética , Región Branquial/embriología , Diferenciación Celular/genética , Movimiento Celular/genética , Microambiente Celular/genética , Embrión de Pollo , Embrión de Mamíferos , Embrión no Mamífero , Desarrollo Embrionario/genética , Cabeza/embriología , Humanos , Mesodermo/crecimiento & desarrollo , Células Madre Multipotentes/citología , Cuello/embriología , Cresta Neural/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Organogénesis/genética , Microambiente Tumoral/genética , Vertebrados/genética , Vertebrados/crecimiento & desarrolloRESUMEN
BACKGROUND: Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell migration remain unclear. RESULTS: Computational simulations predicted that confinement is required only along the initial one-third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin-12, a transmembrane scavenger receptor C-type lectin) and Trail (tumor necrosis factor-related apoptosis-inducing ligand, CD253) which we show expressed in chick cranial NCC-free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain- or loss-of-function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three-dimensional confocal microscopy, respectively, resulting in disrupted collective migration. CONCLUSIONS: These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.
Asunto(s)
Movimiento Celular , Pollos , Cresta Neural , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Pollos/genética , Pollos/fisiología , Simulación por Computador , Cresta Neural/citología , Cresta Neural/fisiología , CráneoRESUMEN
BACKGROUND: The molecular identification of neural progenitor cell populations that connect to establish the sympathetic nervous system (SNS) remains unclear. This is due to technical limitations in the acquisition and spatial mapping of molecular information to tissue architecture. RESULTS: To address this, we applied Slide-seq spatial transcriptomics to intact fresh frozen chick trunk tissue transversely cryo-sectioned at the developmental stage prior to SNS formation. In parallel, we performed age- and location-matched single cell (sc) RNA-seq and 10× Genomics Visium to inform our analysis. Downstream bioinformatic analyses led to the unique molecular identification of neural progenitor cells within the peripheral sympathetic ganglia (SG) and spinal cord preganglionic neurons (PGNs). We then successfully applied the HiPlex RNAscope fluorescence in situ hybridization and multispectral confocal microscopy to visualize 12 gene targets in stage-, age- and location-matched chick trunk tissue sections. CONCLUSIONS: Together, these data demonstrate a robust strategy to acquire and integrate single cell and spatial transcriptomic information, resulting in improved resolution of molecular heterogeneities in complex neural tissue architectures. Successful application of this strategy to the developing SNS provides a roadmap for functional studies of neural connectivity and platform to address complex questions in neural development and regeneration.
Asunto(s)
Sistema Nervioso Simpático , Transcriptoma , Animales , ARN Mensajero , Hibridación Fluorescente in Situ , Ganglios Simpáticos , PollosRESUMEN
Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.
Asunto(s)
Acuaporina 1/fisiología , Movimiento Celular/fisiología , Cresta Neural/citología , Seudópodos/fisiología , Animales , Región Branquial/citología , Región Branquial/embriología , Membrana Celular/fisiología , Microambiente Celular , Embrión de Pollo , Biología Computacional , Adhesiones Focales , Cresta Neural/embriología , Receptor EphB1/metabolismo , Receptor EphB3/metabolismoRESUMEN
Mistakes in trunk neural crest (NC) cell migration may lead to birth defects of the sympathetic nervous system (SNS) and neuroblastoma (NB) cancer. Receptor tyrosine kinase B (TrkB) and its ligand BDNF critically regulate NC cell migration during normal SNS development and elevated expression of TrkB is correlated with high-risk NB patients. However, in the absence of a model with in vivo interrogation of human NB cell and gene expression dynamics, the mechanistic role of TrkB in NB disease progression remains unclear. Here, we study the functional relationship between TrkB, cell invasion and plasticity of human NB cells by taking advantage of our validated in vivo chick embryo transplant model. We find that LAN5 (high TrkB) and SHSY5Y (moderate TrkB) human NB cells aggressively invade host embryos and populate typical NC targets, however loss of TrkB function significantly reduces cell invasion. In contrast, NB1643 (low TrkB) cells remain near the transplant site, but over-expression of TrkB leads to significant cell invasion. Invasive NB cells show enhanced expression of genes indicative of the most invasive host NC cells. In contrast, transplanted human NB cells down-regulate known NB tumor initiating and stem cell markers. Human NB cells that remain within the dorsal neural tube transplant also show enhanced expression of cell differentiation genes, resulting in an improved disease outcome as predicted by a computational algorithm. These in vivo data support TrkB as an important biomarker and target to control NB aggressiveness and identify the chick embryonic trunk neural crest microenvironment as a source of signals to drive NB to a less aggressive state, likely acting at the dorsal neural tube.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Invasividad Neoplásica/genética , Cresta Neural/embriología , Receptor trkB/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Plasticidad de la Célula/genética , Transformación Celular Neoplásica/metabolismo , Embrión de Pollo , Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Cresta Neural/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor trkB/genética , Transducción de Señal/genética , Microambiente Tumoral/genéticaRESUMEN
Vertebrate head morphogenesis involves carefully-orchestrated tissue growth and cell movements of the mesoderm and neural crest to form the distinct craniofacial pattern. To better understand structural birth defects, it is important that we characterize the dynamics of these processes and learn how they rely on each other. Here we examine this question during chick head morphogenesis using time-lapse imaging, computational modeling, and experiments. We find that head mesodermal cells in culture move in random directions as individuals and move faster in the presence of neural crest cells. In vivo, mesodermal cells migrate in a directed manner and maintain neighbor relationships; neural crest cells travel through the mesoderm at a faster speed. The mesoderm grows with a non-uniform spatio-temporal profile determined by BrdU labeling during the period of faster and more-directed neural crest collective migration through this domain. We use computer simulations to probe the robustness of neural crest stream formation by varying the spatio-temporal growth profile of the mesoderm. We follow this with experimental manipulations that either stop mesoderm growth or prevent neural crest migration and observe changes in the non-manipulated cell population, implying a dynamic feedback between tissue growth and neural crest cell signaling to confer robustness to the system. Overall, we present a novel descriptive analysis of mesoderm and neural crest cell dynamics that reveals the coordination and co-dependence of these two cell populations during head morphogenesis.
Asunto(s)
Embrión de Pollo/citología , Cabeza/embriología , Mesodermo/citología , Cresta Neural/citología , Tubo Neural/citología , Animales , División Celular , Movimiento Celular , Células Cultivadas , Pollos , Simulación por Computador , Coturnix/embriología , Ectodermo/citología , Modelos Biológicos , Morfogénesis , Imagen de Lapso de TiempoRESUMEN
Cell invasion and cell plasticity are critical to human development but are also striking features of cancer metastasis. By distributing a multipotent cell type from a place of birth to distal locations, the vertebrate embryo builds organs. In comparison, metastatic tumor cells often acquire a de-differentiated phenotype and migrate away from a primary site to inhabit new microenvironments, disrupting normal organ function. Countless observations of both embryonic cell migration and tumor metastasis have demonstrated complex cell signaling and interactive behaviors that have long confounded scientist and clinician alike. James D. Murray realized the important role of mathematics in biology and developed a unique strategy to address complex biological questions such as these. His work offers a practical template for constructing clear, logical, direct and verifiable models that help to explain complex cell behaviors and direct new experiments. His pioneering work at the interface of development and cancer made significant contributions to glioblastoma cancer and embryonic pattern formation using often simple models with tremendous predictive potential. Here, we provide a brief overview of advances in cell invasion and cell plasticity using the embryonic neural crest and its ancestral relationship to aggressive cancers that put into current context the timeless aspects of his work.
Asunto(s)
Modelos Biológicos , Invasividad Neoplásica , Neoplasias , Humanos , Neoplasias/fisiopatología , Cresta Neural/citologíaRESUMEN
The neural crest serves as a powerful and tractable model paradigm for understanding collective cell migration. The neural crest cell populations are well-known for their long-distance collective migration and contribution to diverse cell lineages during vertebrate development. If neural crest cells fail to reach a target or populate an incorrect location, then improper cell differentiation or uncontrolled cell proliferation can result. A wide range of interdisciplinary studies has been carried out to understand the response of neural crest cells to different stimuli and their ability to migrate to distant targets. In this critical commentary, we illustrate how an interdisciplinary collaboration involving experimental and mathematical modeling has led to a deeper understanding of cranial neural crest cell migration. We identify open questions and propose possible ways to start answering some of the challenges arising.
Asunto(s)
Movimiento Celular/fisiología , Cresta Neural/citología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/genética , Humanos , Estudios Interdisciplinarios , Modelos Teóricos , Cresta Neural/metabolismo , Transducción de Señal/fisiologíaRESUMEN
A huge variety of mathematical models have been used to investigate collective cell migration. The aim of this brief review is twofold: to present a number of modelling approaches that incorporate the key factors affecting cell migration, including cell-cell and cell-tissue interactions, as well as domain growth, and to showcase their application to model the migration of neural crest cells. We discuss the complementary strengths of microscale and macroscale models, and identify why it can be important to understand how these modelling approaches are related. We consider neural crest cell migration as a model paradigm to illustrate how the application of different mathematical modelling techniques, combined with experimental results, can provide new biological insights. We conclude by highlighting a number of future challenges for the mathematical modelling of neural crest cell migration.
Asunto(s)
Movimiento Celular/fisiología , Modelos Biológicos , Cresta Neural/crecimiento & desarrollo , Animales , Comunicación Celular/fisiología , Línea Celular Tumoral , Humanos , Cresta Neural/citología , Xenopus , Pez CebraRESUMEN
The embryonic microenvironment is an important source of signals that promote multipotent cells to adopt a specific fate and direct cells along distinct migratory pathways. Yet, the ability of the embryonic microenvironment to retain multipotent progenitors or reprogram de-differentiated cells is less clear. Mistakes in cell differentiation or migration often result in developmental defects and tumorigenesis, including aggressive cancers that share many characteristics with embryonic progenitor cells. This is a striking feature of the vertebrate neural crest, a multipotent and highly migratory cell population first identified by His (1868) with the potential to metamorphose into aggressive melanoma cancer. In this perspective, we address the roles of CD271/p75 in tumor initiation, phenotype switching and reprogramming of metastatic melanoma and discuss the convergence of these roles in melanoma plasticity.
Asunto(s)
Adapaleno/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Melanoma/metabolismo , Factores de Transcripción/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Movimiento Celular , Transformación Celular Neoplásica/metabolismo , Células Madre Embrionarias/fisiología , Humanos , Melanocitos/citología , Melanoma/fisiopatología , Ratones , Células Madre Multipotentes , Cresta Neural/embriología , Cresta Neural/metabolismo , Cresta Neural/fisiología , Plasticidad Neuronal/fisiologíaRESUMEN
Trunk neural crest cells follow a common ventral migratory pathway but are distributed into two distinct locations to form discrete sympathetic and dorsal root ganglia along the vertebrate axis. Although fluorescent cell labeling and time-lapse studies have recorded complex trunk neural crest cell migratory behaviors, the signals that underlie this dynamic patterning remain unclear. The absence of molecular information has led to a number of mechanistic hypotheses for trunk neural crest cell migration. Here, we review recent data in support of three distinct mechanisms of trunk neural crest cell migration and develop and simulate a computational model based on chemotactic signaling. We show that by integrating the timing and spatial location of multiple chemotactic signals, trunk neural crest cells may be accurately positioned into two distinct targets that correspond to the sympathetic and dorsal root ganglia. In doing so, we honor the contributions of Wilhelm His to his identification of the neural crest and extend the observations of His and others to better understand a complex question in neural crest cell biology.
Asunto(s)
Movimiento Celular , Quimiotaxis , Modelos Biológicos , Cresta Neural/citología , Animales , Quimiocinas/fisiología , Células Endoteliales/citología , Humanos , Transducción de SeñalRESUMEN
Neural crest (NC) cell migration is crucial to the formation of peripheral tissues during vertebrate development. However, how NC cells respond to different microenvironments to maintain persistence of direction and cohesion in multicellular streams remains unclear. To address this, we profiled eight subregions of a typical cranial NC cell migratory stream. Hierarchical clustering showed significant differences in the expression profiles of the lead three subregions compared with newly emerged cells. Multiplexed imaging of mRNA expression using fluorescent hybridization chain reaction (HCR) quantitatively confirmed the expression profiles of lead cells. Computational modeling predicted that a small fraction of lead cells that detect directional information is optimal for successful stream migration. Single-cell profiling then revealed a unique molecular signature that is consistent and stable over time in a subset of lead cells within the most advanced portion of the migratory front, which we term trailblazers. Model simulations that forced a lead cell behavior in the trailing subpopulation predicted cell bunching near the migratory domain entrance. Misexpression of the trailblazer molecular signature by perturbation of two upstream transcription factors agreed with the in silico prediction and showed alterations to NC cell migration distance and stream shape. These data are the first to characterize the molecular diversity within an NC cell migratory stream and offer insights into how molecular patterns are transduced into cell behaviors.
Asunto(s)
Movimiento Celular , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Movimiento Celular/genética , Embrión de Pollo , Simulación por Computador , Técnicas de Silenciamiento del Gen , Cresta Neural/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
Embryonic cells that migrate long distances must critically balance cell division in order to maintain stream dynamics and population of peripheral targets. Yet details of individual cell division events and how cell cycle is related to phases of migration remain unclear. Here, we examined these questions using the chick cranial neural crest (NC). In vivo time-lapse imaging revealed that a typical migrating NC cell division event lasted ~1 hour and included four stereotypical steps. Cell tracking showed that dividing NC cells maintained position relative to non-dividing neighbors. NC cell division orientation and the time and distance to first division after neural tube exit were stochastic. To address how cell cycle is related to phases of migration, we used FACs analysis to identify significant spatiotemporal differences in NC cell cycle profiles. Two-photon photoconversion of single and small numbers of mKikGR-labeled NC cells confirmed that lead NC cells exhibited a nearly fourfold faster doubling time after populating the branchial arches. By contrast, Ki-67 staining showed that one out of every five later emerging NC cells exited the cell cycle after reaching proximal head targets. The relatively quiescent mitotic activity during NC cell migration to the branchial arches was altered when premigratory cells were reduced in number by tissue ablation. Together, our results provide the first comprehensive details of the pattern and dynamics of cell division events during cranial NC cell migration.
Asunto(s)
Cresta Neural/citología , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Embrión de Pollo , Citometría de Flujo , Cresta Neural/metabolismoRESUMEN
BACKGROUND: Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood. RESULTS: Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells. CONCLUSIONS: Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.
Asunto(s)
Angiopoyetina 2/metabolismo , Cresta Neural/citología , Cresta Neural/metabolismo , Angiopoyetina 2/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Codorniz , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Embryonic neural crest cells travel in discrete streams to precise locations throughout the head and body. We previously showed that cranial neural crest cells respond chemotactically to vascular endothelial growth factor (VEGF) and that cells within the migratory front have distinct behaviors and gene expression. We proposed a cell-induced gradient model in which lead neural crest cells read out directional information from a chemoattractant profile and instruct trailers to follow. In this study, we show that migrating chick neural crest cells do not display distinct lead and trailer gene expression profiles in culture. However, exposure to VEGF in vitro results in the upregulation of a small subset of genes associated with an in vivo lead cell signature. Timed addition and removal of VEGF in culture reveals the changes in neural crest cell gene expression are rapid. A computational model incorporating an integrate-and-switch mechanism between cellular phenotypes predicts migration efficiency is influenced by the timescale of cell behavior switching. To test the model hypothesis that neural crest cellular phenotypes respond to changes in the VEGF chemoattractant profile, we presented ectopic sources of VEGF to the trailer neural crest cell subpopulation and show diverted cell trajectories and stream alterations consistent with model predictions. Gene profiling of trailer cells that diverted and encountered VEGF revealed upregulation of a subset of 'lead' genes. Injection of neuropilin1 (Np1)-Fc into the trailer subpopulation or electroporation of VEGF morpholino to reduce VEGF signaling failed to alter trailer neural crest cell trajectories, suggesting trailers do not require VEGF to maintain coordinated migration. These results indicate that VEGF is one of the signals that establishes lead cell identity and its chemoattractant profile is critical to neural crest cell migration.
Asunto(s)
Cresta Neural/citología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Movimiento Celular , Microambiente Celular , Quimiotaxis , Embrión de Pollo , Simulación por Computador , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Neural crest (NC) cells emerge from the dorsal trunk neural tube (NT) and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the NT have similar or differential ability to contribute to NC cells and their derivatives. To this end, we precisely labeled NT precursors at specific dorsoventral levels of the chick NT using fluorescent dyes and a photoconvertible fluorescent protein. NT and NC cell dynamics were then examined in vivo and in slice culture using two-photon and confocal time-lapse imaging. The results show that NC precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the NT, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the NT to contribute to NC derivatives, with the exception of sympathetic ganglia, which appeared to be 'filled' by the first population to emigrate. Rather than restricted developmental potential, however, this is probably due to a matter of timing.
Asunto(s)
Movimiento Celular/fisiología , Células Madre Embrionarias/citología , Cresta Neural/embriología , Tubo Neural/citología , Células Neuroepiteliales/fisiología , Animales , Embrión de Pollo , Biología Computacional , Células Madre Embrionarias/metabolismo , Colorantes Fluorescentes , Microscopía Confocal , Tubo Neural/embriología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Aggressive tumour cells share many characteristics with embryonic progenitors, contributing to the conundrum of tumour cell plasticity. Recent studies using embryonic models of human stem cells, the zebrafish and the chick have shown the reversion of the metastatic phenotype of aggressive melanoma cells, and revealed the convergence of embryonic and tumorigenic signalling pathways, which may help to identify new targets for therapeutic intervention. This Review will summarize the embryonic models used to reverse the metastatic melanoma phenotype, and highlight the prominent signalling pathways that have emerged as noteworthy targets for future consideration.
Asunto(s)
Embrión de Mamíferos/citología , Embrión no Mamífero , Melanoma/patología , Metástasis de la Neoplasia/patología , Animales , Comunicación Celular , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Embrión de Pollo , Células Madre Embrionarias , Humanos , Melanoma/metabolismo , Modelos Animales , Trasplante de Neoplasias , Cresta Neural , Proteína Nodal , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología , Pez Cebra/embriologíaRESUMEN
BACKGROUND: Single cell gene profiling has been successfully applied to cultured cells. However, isolation and preservation of a cell's native gene expression state from an intact embryo remain problematic. RESULTS: Here, we present a strategy for in vivo single cell profiling that optimizes cell identification, isolation and amplification of nucleic acids with nominal bias and sufficient material detection. We first tested several photoconvertible fluorescent proteins to selectively mark a cell(s) of interest in living chick embryos then accurately identify and isolate the same cell(s) in fixed tissue slices. We determined that the dual color mDendra2 provided the optimal signal/noise ratio for this purpose. We developed proper procedures to minimize cell death and preserve gene expression, and suggest nucleic acid amplification strategies for downstream analysis by microfluidic reverse transcriptase quantitative polymerase chain reaction or RNAseq. Lastly, we compared methods for single cell isolation and found that our fluorescence-activated cell sorting (FACS) protocol was able to preserve native transcripts and generate expression profiles with much higher efficiency than laser capture microdissection (LCM). CONCLUSIONS: Quantitative single cell gene expression profiling may be accurately applied to interrogate complex cell dynamics events during embryonic development by combining photoconversion cell labeling, FACS, proper handling of isolated cells, and amplification strategies.
Asunto(s)
Embrión de Pollo/citología , Pollos/genética , Perfilación de la Expresión Génica/métodos , ARN Mensajero/biosíntesis , Análisis de la Célula Individual/métodos , Animales , Supervivencia Celular , Embrión de Pollo/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/efectos de la radiación , Genes Reporteros , Dispositivos Laboratorio en un Chip , Captura por Microdisección con Láser , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/efectos de la radiación , Técnicas Analíticas Microfluídicas , Microinyecciones , Tubo Neural/citología , Fotoquímica , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ARN/métodos , Imagen de Lapso de Tiempo/métodosRESUMEN
Long-distance cell migration is an important feature of embryonic development, adult morphogenesis and cancer, yet the mechanisms that drive subpopulations of cells to distinct targets are poorly understood. Here, we use the embryonic neural crest (NC) in tandem with theoretical studies to evaluate model mechanisms of long-distance cell migration. We find that a simple chemotaxis model is insufficient to explain our experimental data. Instead, model simulations predict that NC cell migration requires leading cells to respond to long-range guidance signals and trailing cells to short-range cues in order to maintain a directed, multicellular stream. Experiments confirm differences in leading versus trailing NC cell subpopulations, manifested in unique cell orientation and gene expression patterns that respond to non-linear tissue growth of the migratory domain. Ablation experiments that delete the trailing NC cell subpopulation reveal that leading NC cells distribute all along the migratory pathway and develop a leading/trailing cellular orientation and gene expression profile that is predicted by model simulations. Transplantation experiments and model predictions that move trailing NC cells to the migratory front, or vice versa, reveal that cells adopt a gene expression profile and cell behaviors corresponding to the new position within the migratory stream. These results offer a mechanistic model in which leading cells create and respond to a cell-induced chemotactic gradient and transmit guidance information to trailing cells that use short-range signals to move in a directional manner.
Asunto(s)
Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , Modelos Biológicos , Animales , Movimiento Celular/genética , Quimiotaxis/fisiología , Embrión de Pollo , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Modelos Neurológicos , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/fisiologíaRESUMEN
The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair.