Cell-type profiling of the sympathetic nervous system using spatial transcriptomics and spatial mapping of mRNA.

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.

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.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

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.

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.

TrkB/BDNF signalling patterns the sympathetic nervous system.

The sympathetic nervous system is essential for maintaining mammalian homeostasis. How this intricately connected network, composed of preganglionic neurons that reside in the spinal cord and post-ganglionic neurons that comprise a chain of vertebral sympathetic ganglia, arises developmentally is incompletely understood. This problem is especially complex given the vertebral chain of sympathetic ganglia derive secondarily from the dorsal migration of 'primary' sympathetic ganglia that are initially located several hundred microns ventrally from their future pre-synaptic partners. Here we report that the dorsal migration of discrete ganglia is not a simple migration of individual cells but a much more carefully choreographed process that is mediated by extensive interactions of pre-and post-ganglionic neurons. Dorsal migration does not occur in the absence of contact with preganglionic axons, and this is mediated by BDNF/TrkB signalling. Thus BDNF released by preganglionic axons acts chemotactically on TrkB-positive sympathetic neurons, to pattern the developing peripheral nervous system.

CXCR4 controls ventral migration of sympathetic precursor cells.

The molecular mechanisms that sort migrating neural crest cells (NCCs) along a shared pathway into two functionally discrete structures, the dorsal root ganglia and sympathetic ganglia (SGs), are unknown. We report here that this patterning is attributable in part to differential expression of the chemokine receptor, CXCR4. We show that (1) a distinct subset of ventrally migrating NCCs express CXCR4 and this subset is destined to form the neural core of the sympathetic ganglia, and (2) the CXCR4 ligand, SDF-1, is a chemoattractant for NCCs in vivo and is expressed adjacent to the future SGs. Reduction of CXCR4 expression in NCCs disrupts their migration toward the future SGs, whereas overexpression of CXCR4 in non-SG-destined NCCs induces them to migrate aberrantly toward the SGs. These data are the first to demonstrate a major role for chemotaxis in the patterning of NCC migration and demonstrate the neural crest is composed of molecularly heterogeneous cell populations.

Cranial neural crest migration: new rules for an old road.

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration. Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube in a rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes to precise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifully choreographed and programmed invasion, the underlying orchestration of molecular events is not well known. For example, it is still unclear how single CNCCs react to signals that direct their choice of direction and how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In this review, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus on events from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel through different microenvironments and into the branchial arches. We describe the patterning of discrete cell migratory streams that emerge from the hindbrain, rhombomere (r) segments r1-r7, and the signals that coordinate directed migration. We propose a model that attempts to unify many complex events that establish the CNCC migratory pattern, and based on this model we integrate information between cranial and trunk neural crest development.

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.

Watching the assembly of an organ a single cell at a time using confocal multi-position photoactivation and multi-time acquisition.

Tracing cell movements in vivo yields important clues to organogenesis, yet it has been challenging to accurately and reproducibly fluorescently mark single and small groups of cells to build a picture of tissue assembly. In the early embryo, the small size (hundreds of cells) of progenitor cell regions has made it easier to identify and selectively mark superficially located cells by glass needle injection. However,during early organogenesis,subregions of interest may be several millions of cells in volume located deeper within the embryo requiring an alternative approach. Here, we combined (confocal and 2-photon) photoactivation cell labeling and multi-position, multi-time imaging to trace single cell and small subgroups of cells in the developing brain and spinal cord. We compared the photostability and photoefficiency of a photoswitchable fluorescent protein, PSCFP2, with a novel nuclear localized H2B-PSCFP2 protein. We showed that both fluorescent proteins have similar photophysical properties and H2B-PSCFP2 is more effective in single cell identification in dense tissue. To accurately and reproducibly fluorescently trace subregions of cells in a 3D tissue volume, we developed a protocol for multi-position photoactivation and multi-time acquisition in the chick spinal cord in up to eight tissue sections. We applied our techniques to address the formation of the sympathetic ganglia,a major component of the autonomic nervous system,and showed there are phenotypic differences between early and later emerging neural crest cells and their positions in the developing ganglia. Thus, targeted fluorescent cell marking by confocal or 2-photon multi-position photoactivation and multi-time acquisition offer a more efficient, less invasive technique to trace cell movements in large regions of interest and move us closer towards mapping the cellular events of organogenesis.

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.

Photoactivation cell labeling for cell tracing in avian development.

INTRODUCTIONTracing cell movements in a living embryo or embryo slice culture remains a challenging problem due to difficulties in cell accessibility and in accurate delivery of fluorescent labels into an individual cell or subgroup of cells. Here, we describe a photoactivation cell-labeling technique in avian embryos that allows for selective marking of individual cells or groups of cells at precise times and spatial locations normally not accessible using previous techniques. The current protocol is also less invasive than previously described methods. We provide details of fluorescent protein delivery into cells of interest utilizing microinjection and in ovo electroporation, as well as the optical parameters needed for photoactivation and imaging of both single- and dual-color photoactivatable fluorescent proteins (PAFPs). We present applications to label single cells and small subgroups of cells throughout the head and trunk of the developing vertebrate embryo, using the avian neural crest as our model cell population.

Sagittal Explant Culture for 3D Confocal Time-Lapse Analysis of Chick Peripheral Nervous System Formation.

INTRODUCTIONThe peripheral nervous system (PNS) regulates key events within the body including breathing, heart rate, and pain and temperature sensation. In the trunk of the developing embryo, neural crest (NC) cells that follow a ventro-medial pathway form the dorsal root ganglia (DRG) and sympathetic ganglia (SG) of the PNS. The cellular and molecular mechanisms that mediate the formation of the PNS are not completely understood due to the lack of a model system to monitor NC cell migratory behaviors deep within the embryo. Here, we describe a unique sagittal explant culture technique that allows for visualization of DRG and SG formation in the relatively intact chick embryo to assess potential molecules and signals influencing the formation of the PNS. By making a midline cut down the chick antero-posterior axis, each half of the embryo is an explant that can be imaged either from the medial perspective, to reveal events occurring deep within the embryo (SG formation), or from the lateral surface, to image events occurring in the dorso-lateral plane (DRG formation).

Construction of a Heated Incubation Chamber around a Microscope Stage for Time-Lapse Imaging.

INTRODUCTIONIntravital imaging of embryogenesis has the potential to provide valuable information on cell proliferation, cell shape changes, and cell migratory behaviors. However, most embryo model systems require a temperature-controlled environment. Several expensive commercially available temperature control devices have emerged, including microscope stages surrounded by custom-fit Plexiglas boxes, heated plates for culture dishes, and objective warmers for water-immersion lenses, that strictly control temperature and, in some cases, help control local gas mixtures. This protocol describes an easy-to-assemble, cost-effective, custom-made cardboard box and incubator, adaptable to each user's specifications and microscope set-up. The cardboard box fits around the microscope, primarily the stage area, to assist in maintaining a prescribed temperature near the microscope stage. Warmed air, blown into the box enclosure from an incubator, circulates around the stage. The heated incubation box maintains a set temperature with minimal fluctuations and has been tested and utilized for studies of chick, mouse, and zebrafish embryogenesis.