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.

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.

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.

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.

Neural crest migration is driven by a few trailblazer cells with a unique molecular signature narrowly confined to the invasive front.

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.

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.

Quantitative single cell gene expression profiling in the avian embryo.

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.

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.

Sea lamprey enlightens the origin of the coupling of retinoic acid signaling to vertebrate hindbrain segmentation.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.

Positional information modulates transient regeneration-activated cell states during vertebrate appendage regeneration.

Injury is common in the life of organisms. Because the extent of damage cannot be predicted, injured organisms must determine how much tissue needs to be restored. Although it is known that amputation position affects the regeneration speed of appendages, mechanisms conveying positional information remain unclear. We investigated tissue dynamics in regenerating caudal fins of the African killifish (Nothobranchius furzeri) and found position-specific, differential spatial distribution modulation, persistence, and magnitude of proliferation. Single-cell RNA sequencing revealed a transient regeneration-activated cell state (TRACS) in the basal epidermis that is amplified to match a given amputation position and expresses components and modifiers of the extracellular matrix (ECM). Notably, CRISPR-Cas9-mediated deletion of the ECM modifier sequestosome 1 (sqstm1) increased the regenerative capacity of distal injuries, suggesting that regeneration growth rate can be uncoupled from amputation position. We propose that basal epidermis TRACS transduce positional information to the regenerating blastema by remodeling the ECM.

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.

Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation.

Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.

Developmental cues and persistent neurogenic potential within an in vitro neural niche.

BACKGROUND: Neurogenesis, the production of neural cell-types from neural stem cells (NSCs), occurs during development as well as within select regions of the adult brain. NSCs in the adult subependymal zone (SEZ) exist in a well-categorized niche microenvironment established by surrounding cells and their molecular products. The components of this niche maintain the NSCs and their definitive properties, including the ability to self-renew and multipotency (neuronal and glial differentiation). RESULTS: We describe a model in vitro NSC niche, derived from embryonic stem cells, that produces many of the cells and products of the developing subventricular zone (SVZ) and adult SEZ NSC niche. We demonstrate a possible role for apoptosis and for components of the extracellular matrix in the maintenance of the NSC population within our niche cultures. We characterize expression of genes relevant to NSC self-renewal and the process of neurogenesis and compare these findings to gene expression produced by an established neural-induction protocol employing retinoic acid. CONCLUSIONS: The in vitro NSC niche shows an identity that is distinct from the neurally induced embryonic cells that were used to derive it. Molecular and cellular components found in our in vitro NSC niche include NSCs, neural progeny, and ECM components and their receptors. Establishment of the in vitro NSC niche occurs in conjunction with apoptosis. Applications of this culture system range from studies of signaling events fundamental to niche formation and maintenance as well as development of unique NSC transplant platforms to treat disease or injury.

Detection of calcium transients in embryonic stem cells and their differentiated progeny.

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.

Treatment of lysosomal storage disorders: focus on the neuronal ceroid-lipofuscinoses.

Recent advances in our understanding of lysosomal storage disorders (LSDs) may lead to new therapies to treat the neuronal ceroid-lipofuscinoses (NCLs). In this review, enzyme replacement therapy, gene therapy, cell-mediated therapy and pharmaceutical treatments are considered across the LSDs and extended to therapies for the NCLs. It is likely that a combination of approaches will produce the most beneficial clinical outcome for treatment of pathologies displayed by the NCLs.

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.

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.

Elements of a neural stem cell niche derived from embryonic stem cells.

Recent studies show that adult neural tissues can harbor stem cells within unique niches. In the mammalian central nervous system, neural stem cell (NSC) niches have been identified in the dentate gyrus and the subventricular zone (SVZ). Stem cells in the well-characterized SVZ exist in a microenvironment established by surrounding cells and tissue components, including transit-amplifying cells, neuroblasts, ependymal cells, blood vessels, and a basal lamina. Within this microenvironment, stem cell properties, including proliferation and differentiation, are maintained. Current NSC culture techniques often include the addition of molecular components found within the in vivo niche, such as mitogenic growth factors. Some protocols use bio-scaffolds to mimic the physical growth environment of living tissue. We describe a novel NSC culture system, derived from embryonic stem (ES) cells, that displays elements of an NSC niche in the absence of exogenously applied mitogens or complex physical scaffolding. Mouse ES cells were neuralized with retinoic acid and plated on an entactin-collagen-laminin-coated glass surface at high density (250,000 cells/cm(2)). Six to eight days after plating, complex multicellular structures consisting of heterogeneous cell types developed spontaneously. NSC and progenitor cell proliferation and differentiation continued within these structures. The identity of cellular and molecular components within the cultures was documented using RT-PCR, immunocytochemistry, and neurosphere-forming assays. We show that ES cells can be induced to form structures that exhibit key properties of a developing NSC niche. We believe this system can serve as a useful model for studies of neurogenesis and stem cell maintenance in the NSC niche as well as for applications in stem cell transplantation.

Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method.

During collective cell migration individual cells display diverse behaviors that complicate our understanding of group cell decisions of direction and cohesion. In vivo gene and protein expression analyses would shed light on the underlying molecular choreography. However, this information has been limited due to difficulties to integrate single cell detection methods and the simultaneous readout of multiple signals deep within the embryo. Here, we optimize and integrate multiplex fluorescence in situ hybridization by RNAscope, immunohistochemistry, and tissue clearing to visualize transcript and protein localization within single cells deep within intact chick embryos. Using standard confocal microscopy, we visualize the mRNA expression of up to 3 genes simultaneously within protein labeled HNK1-positive migrating cranial neural crest cells within 2day old cleared chick embryos. Gene expression differences measured between adjacent cells or within subregions are quantified using spot counting and polyline kymograph methods, respectively. This optimization and integration of methods provide an improved 3D in vivo molecular interrogation of collective cell migration and foundation to broaden into a wider range of embryo and adult model systems.