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.
Key Findings
We applied Slide‐seq spatial transcriptomics to intact fresh frozen chick trunk tissue.
In parallel, we performed age‐ and location‐matched single cell (sc) RNA‐seq and 10x Genomics Visium to inform our Slide‐seq analysis.
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 expanded 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.
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.
Key Findings
Model simulations predict neural crest cell confinement along the entire length of the migratory domain is not required to maintain discrete streams over long distances.
Colec12 and Trail, we previously identified, confine neural crest cell trajectories off of protein stripes in in vitro stripe assays.
Gain‐ or loss‐of‐function of any single factor or in combinations significantly disrupt the in vivo cranial neural crest migratory pattern.
The neural crest is a migratory population of cells that produces many diverse structures within the embryo. Trunk neural crest cells give rise to such structures as the dorsal root ganglia (DRG) and ...sympathetic ganglia (SG), which form in a metameric pattern along the anterior-posterior axis of the embryo. While static analyses have provided invaluable information concerning the development of these structures, time-lapse imaging of neural crest cells navigating through their normal environment could potentially reveal previously unidentified cellular and molecular interactions integral to DRG and SG development. In this study, we follow fluorescently labeled trunk neural crest cells using a novel sagittal explant and time-lapse confocal microscopy. We show that along their dorsoventral migratory route, trunk neural crest cells are highly motile and interact extensively with neighboring cells and the environment, with many cells migrating in chain-like formations. Surprisingly, the segregated pattern of crest cell streams through the rostral somite is not maintained once these cells arrive alongside the dorsal aorta. Instead, neural crest cells disperse along the ventral outer border of the somite, interacting extensively with each other and their environment via dynamic extension and retraction of filopodia. Discrete sympathetic ganglia arise as a consequence of intermixing and selective reorganization of neural crest cells at the target site. The diverse cell migratory behaviors and active reorganization at the target suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes.
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.
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.
•Trunk neural crest cells are highly migratory and diverse in cell fate.•The low affinity NGF receptor, CD271/p75 is a marker for melanoma stem cells.•CD271 promotes melanoma cell invasion and mediates phenotype switching.•Metastatic melanoma may be reprogrammed by NGF to a benign cell type.•Targeting CD271/NGF signaling may limit neural crest-derived cancer invasion.
Human metastatic melanoma cells express a dedifferentiated, plastic phenotype, which may serve as a selective advantage, because melanoma cells invade various microenvironments. Over the last three ...decades, there has been an increased focus on the role of the tumor microenvironment in cancer progression, with the goal of reversing the metastatic phenotype. Here, using an embryonic chick model, we explore the possibility of reverting the metastatic melanoma phenotype to its cell type of origin, the neural-crest-derived melanocyte. GFP-labeled adult human metastatic melanoma cells were transplanted in ovo adjacent to host chick premigratory neural crest cells and analyzed 48 and 96 h after egg reincubation. Interestingly, the transplanted melanoma cells do not form tumors. Instead, we find that transplanted melanoma cells invade surrounding chick tissues in a programmed manner, distributing along host neural-crest-cell migratory pathways. The invading melanoma cells display neural-crest-cell-like morphologies and populate host peripheral structures, including the branchial arches, dorsal root and sympathetic ganglia. Analysis of a melanocyte-specific phenotype marker (MART-1) and a neuronal marker (Tuj1) revealed a subpopulation of melanoma cells that invade the chick periphery and express MART-1 and Tuj1. Our results demonstrate the ability of adult human metastatic melanoma cells to respond to chick embryonic environmental cues, a subset of which may undergo a reprogramming of their metastatic phenotype. This model has the potential to provide insights into the regulation of tumor cell plasticity by an embryonic milieu, which may hold significant therapeutic promise.
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.
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.
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.
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•Human neuroblastoma cells analyzed in chick embryo transplant model.•Knockdown and over-expression of TrkB dramatically affects invasion and plasticity.•Reduced tumor-initiation and stem cell expression in cells that remain at transplant.•Simulations of TrkB network model show improved disease outcome of transplanted cells.
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