Colec12 and Trail signaling confine cranial neural crest cell trajectories and promote collective cell migration.

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.

The embryonic trunk neural crest microenvironment regulates the plasticity and invasion of human neuroblastoma via TrkB signaling.

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.

Modelling Cell Invasion: A Review of What JD Murray and the Embryo Can Teach Us.

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.

Single-cell reconstruction with spatial context of migrating neural crest cells and their microenvironments during vertebrate head and neck formation.

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.

Modelling collective cell migration: neural crest as a model paradigm.

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.

Predicting neuroblastoma using developmental signals and a logic-based model.

Genomic information from human patient samples of pediatric neuroblastoma cancers and known outcomes have led to specific gene lists put forward as high risk for disease progression. However, the reliance on gene expression correlations rather than mechanistic insight has shown limited potential and suggests a critical need for molecular network models that better predict neuroblastoma progression. In this study, we construct and simulate a molecular network of developmental genes and downstream signals in a 6-gene input logic model that predicts a favorable/unfavorable outcome based on the outcome of the four cell states including cell differentiation, proliferation, apoptosis, and angiogenesis. We simulate the mis-expression of the tyrosine receptor kinases, trkA and trkB, two prognostic indicators of neuroblastoma, and find differences in the number and probability distribution of steady state outcomes. We validate the mechanistic model assumptions using RNAseq of the SHSY5Y human neuroblastoma cell line to define the input states and confirm the predicted outcome with antibody staining. Lastly, we apply input gene signatures from 77 published human patient samples and show that our model makes more accurate disease outcome predictions for early stage disease than any current neuroblastoma gene list. These findings highlight the predictive strength of a logic-based model based on developmental genes and offer a better understanding of the molecular network interactions during neuroblastoma disease progression.

The convergent roles of CD271/p75 in neural crest-derived melanoma plasticity.

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.

NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor.

Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type.

DAN (NBL1) promotes collective neural crest migration by restraining uncontrolled invasion.

Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling.

VEGF signals induce trailblazer cell identity that drives neural crest migration.

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.

Neural crest migration: trailblazing ahead.

Embryonic cell migration patterns are amazingly complex in the timing and spatial distribution of cells throughout the vertebrate landscape. However, advances in in vivo visualization, cell interrogation, and computational modeling are extracting critical features that underlie the mechanistic nature of these patterns. The focus of this review highlights recent advances in the study of the highly invasive neural crest cells and their migratory patterns during embryonic development. We discuss these advances within three major themes and include a description of computational models that have emerged to more rapidly integrate and test hypothetical mechanisms of neural crest migration. We conclude with technological advances that promise to reveal new insights and help translate results to human neural crest-related birth defects and metastatic cancer.

Dynamic interactions between cancer cells and the embryonic microenvironment regulate cell invasion and reveal EphB6 as a metastasis suppressor.

UNLABELLED: Metastatic dissemination drives the high mortality associated with melanoma. However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasive melanoma cell behavior, and whether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of intravasation. IMPLICATIONS: This article links cellular metastasis to behaviors observed in the ancestrally related embryonic neural crest and demonstrates the powerful influence of the embryonic microenvironment in regulating cell migratory behavior.

The neural crest and cancer: a developmental spin on melanoma.

Neural crest (NC) cells undergo an epithelial to mesenchymal transition (EMT) in order to exit from the dorsal neural tube. Similarly, ancestrally related melanoma cells employ an EMT-like event during the initial stages of metastasis to dissociate from surrounding keratinocytes. Whether or not the molecular pathogenesis and cellular dynamics of melanoma metastasis resemble the embryonic NC invasion program is unclear. Here, we highlight advances in our understanding of tumor cell behaviors and plasticity, focusing on the relationship between melanoma and the NC invasion programs. We summarize recent discoveries of NC cell guidance and emerging in vivo imaging strategies that permit single cell resolution of fluorescently labeled tumor cells, with a focus on our recently developed in vivo chick embryo transplant model. Crucial to the molecular pathogenesis of metastasis, we highlight advances in gene profiling of small cell numbers, including our novel ability to gather gene expression information during distinct stages of melanoma invasion. Lastly, we present preliminary details of a comparison of specific genetic pathways associated with the early phases of melanoma invasion and known NC induction and migration signals. Our results suggest that malignant melanoma cells hijack portions of the NC program to promote plasticity and facilitate metastasis. In summary, there is considerable power in combining an in vivo model system with molecular analysis of gene expression, within the context of established developmental signaling pathways, to identify and study the molecular mechanisms of metastasis.

Multiscale mechanisms of cell migration during development: theory and experiment.

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.

Melanoma revives an embryonic migration program to promote plasticity and invasion.

Cancer cells must regulate plasticity and invasion to survive and metastasize. However, the identification of targetable mechanisms to inhibit metastasis has been slow. Signaling programs that drive stem and progenitor cells during normal development offer an inroad to discover mechanisms common to metastasis. Using a chick embryo transplant model, we have compared molecular signaling programs of melanoma and their embryonic progenitors, the neural crest. We report that malignant melanoma cells hijack portions of the embryonic neural crest invasion program. Genes associated with neural crest induction, delamination, and migration are dynamically regulated by melanoma cells exposed to an embryonic neural crest microenvironment. Specifically, we demonstrate that metastatic melanoma cells exploit neural crest-related receptor tyrosine kinases to increase plasticity and facilitate invasion while primary melanocytes may actively suppress these responses under the same microenvironmental conditions. We conclude that aberrant regulation of neural crest developmental genes promotes plasticity and invasiveness in malignant melanoma.

CycleTrak: a novel system for the semi-automated analysis of cell cycle dynamics.

Cell proliferation is crucial to tissue growth and form during embryogenesis, yet dynamic tracking of cell cycle progression and cell position presents a challenging roadblock. We have developed a fluorescent cell cycle indicator and single cell analysis method, called CycleTrak, which allows for better spatiotemporal resolution and quantification of cell cycle phase and cell position than current methods. Our method was developed on the basis of the existing Fucci method. CycleTrak uses a single lentiviral vector that integrates mKO2-hCdt1 (30/120), and a nuclear-localized eGFP reporter. The single vector and nuclear localized fluorescence signals simplify delivery into cells and allow for rapid, automated cell tracking and cell cycle phase readout in single and subpopulations of cells. We validated CycleTrak performance in metastatic melanoma cells and identified novel cell cycle dynamics in vitro and in vivo after transplantation and 3D confocal time-lapse imaging in a living chick embryo.

Computational modelling of cell chain migration reveals mechanisms that sustain follow-the-leader behaviour.

Follow-the-leader chain migration is a striking cell migratory behaviour observed during vertebrate development, adult neurogenesis and cancer metastasis. Although cell-cell contact and extracellular matrix (ECM) cues have been proposed to promote this phenomenon, mechanisms that underlie chain migration persistence remain unclear. Here, we developed a quantitative agent-based modelling framework to test mechanistic hypotheses of chain migration persistence. We defined chain migration and its persistence based on evidence from the highly migratory neural crest model system, where cells within a chain extend and retract filopodia in short-lived cell contacts and move together as a collective. In our agent-based simulations, we began with a set of agents arranged as a chain and systematically probed the influence of model parameters to identify factors critical to the maintenance of the chain migration pattern. We discovered that chain migration persistence requires a high degree of directional bias in both lead and follower cells towards the target. Chain migration persistence was also promoted when lead cells maintained cell contact with followers, but not vice-versa. Finally, providing a path of least resistance in the ECM was not sufficient alone to drive chain persistence. Our results indicate that chain migration persistence depends on the interplay of directional cell movement and biased cell-cell contact.

Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo.

The neural crest is an excellent model to study embryonic cell migration, since cell behaviors can be studied in vivo with advanced optical imaging and molecular intervention. What is unclear is how molecular signals direct neural crest cell (NCC) migration through multiple microenvironments and into specific targets. Here, we tested the hypothesis that the invasion of cranial NCCs, specifically the rhombomere 4 (r4) migratory stream into branchial arch 2 (ba2), is due to chemoattraction through neuropilin-1-vascular endothelial growth factor (VEGF) interactions. We found that the spatio-temporal expression pattern of VEGF in the ectoderm correlated with the NCC migratory front. RT-PCR analysis of the r4 migratory stream showed that ba2 tissue expressed VEGF and r4 NCCs expressed VEGF receptor 2. When soluble VEGF receptor 1 (sVEGFR1) was injected distal to the r4 migratory front, to bind up endogenous VEGF, NCCs failed to completely invade ba2. Time-lapse imaging revealed that cranial NCCs were attracted to ba2 tissue or VEGF sources in vitro. VEGF-soaked beads or VEGF-expressing cells placed adjacent to the r4 migratory stream caused NCCs to divert from stereotypical pathways and move towards an ectopic VEGF source. Our results suggest a model in which NCC entry and invasion of ba2 is dependent on chemoattractive signaling through neuropilin-1-VEGF interactions.

The migration of autonomic precursor cells in the embryo.

The neural crest is an excellent model system to study cell fate and cell guidance signaling. Neural crest cells emerge from a common multipotent subpopulation and follow stereotypical migratory pathways to contribute to many diverse peripheral structures throughout the vertebrate embryo. The neural tube and diverse embryonic microenvironments from which the neural crest originate and migrate through are important sources of signals, yet it is still unclear how a common pool of neural crest stem and progenitor cells diversify and become distributed along specific stereotypical migratory paths. In the post-otic hindbrain and trunk, the neural crest emerge and contribute to the autonomic nervous system, and failure of proper cell navigation and differentiation often leads to congenital disorders that include dysautonomias, Hirschprung's disease, and neuroblastoma cancer. Recent exciting studies of neural crest cell behaviors have revealed the interplay of several molecular signaling pathways that guide and shape autonomic precursor cells to and into proper target structures, suggesting further work may help to better understand autonomic nervous system assembly, derived from a convergence of time-lapse imaging and molecular analyses. In this mini-review, we summarize recent fluorescent cell labeling strategies and cell behavior analyses that elucidate the role of molecular signals on the migration of autonomic precursor cells. We highlight advances in our understanding of the autonomic precursor cell behaviors and fate determination studied within the embryonic microenvironment.

Reprogramming multipotent tumor cells with the embryonic neural crest microenvironment.

The embryonic microenvironment is an important source of signals that program multipotent cells to adopt a particular fate and migratory path, yet its potential to reprogram and restrict multipotent tumor cell fate and invasion is unrealized. Aggressive tumor cells share many characteristics with multipotent, invasive embryonic progenitors, contributing to the paradigm of tumor cell plasticity. In the vertebrate embryo, multiple cell types originate from a highly invasive cell population called the neural crest. The neural crest and the embryonic microenvironments they migrate through represent an excellent model system to study cell diversification during embryogenesis and phenotype determination. Recent exciting studies of tumor cells transplanted into various embryo models, including the neural crest rich chick microenvironment, have revealed the potential to control and revert the metastatic phenotype, suggesting further work may help to identify new targets for therapeutic intervention derived from a convergence of tumorigenic and embryonic signals. In this mini-review, we summarize markers that are common to the neural crest and highly aggressive human melanoma cells. We highlight advances in our understanding of tumor cell behaviors and plasticity studied within the chick neural crest rich microenvironment. In so doing, we honor the tremendous contributions of Professor Elizabeth D. Hay toward this important interface of developmental and cancer biology.