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.

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.

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.

Visualizing mesoderm and neural crest cell dynamics during chick head morphogenesis.

Vertebrate head morphogenesis involves carefully-orchestrated tissue growth and cell movements of the mesoderm and neural crest to form the distinct craniofacial pattern. To better understand structural birth defects, it is important that we characterize the dynamics of these processes and learn how they rely on each other. Here we examine this question during chick head morphogenesis using time-lapse imaging, computational modeling, and experiments. We find that head mesodermal cells in culture move in random directions as individuals and move faster in the presence of neural crest cells. In vivo, mesodermal cells migrate in a directed manner and maintain neighbor relationships; neural crest cells travel through the mesoderm at a faster speed. The mesoderm grows with a non-uniform spatio-temporal profile determined by BrdU labeling during the period of faster and more-directed neural crest collective migration through this domain. We use computer simulations to probe the robustness of neural crest stream formation by varying the spatio-temporal growth profile of the mesoderm. We follow this with experimental manipulations that either stop mesoderm growth or prevent neural crest migration and observe changes in the non-manipulated cell population, implying a dynamic feedback between tissue growth and neural crest cell signaling to confer robustness to the system. Overall, we present a novel descriptive analysis of mesoderm and neural crest cell dynamics that reveals the coordination and co-dependence of these two cell populations during head morphogenesis.

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.

An interdisciplinary approach to investigate collective cell migration in neural crest.

The neural crest serves as a powerful and tractable model paradigm for understanding collective cell migration. The neural crest cell populations are well-known for their long-distance collective migration and contribution to diverse cell lineages during vertebrate development. If neural crest cells fail to reach a target or populate an incorrect location, then improper cell differentiation or uncontrolled cell proliferation can result. A wide range of interdisciplinary studies has been carried out to understand the response of neural crest cells to different stimuli and their ability to migrate to distant targets. In this critical commentary, we illustrate how an interdisciplinary collaboration involving experimental and mathematical modeling has led to a deeper understanding of cranial neural crest cell migration. We identify open questions and propose possible ways to start answering some of the challenges arising.

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.

The neural crest cell cycle is related to phases of migration in the head.

Embryonic cells that migrate long distances must critically balance cell division in order to maintain stream dynamics and population of peripheral targets. Yet details of individual cell division events and how cell cycle is related to phases of migration remain unclear. Here, we examined these questions using the chick cranial neural crest (NC). In vivo time-lapse imaging revealed that a typical migrating NC cell division event lasted ~1 hour and included four stereotypical steps. Cell tracking showed that dividing NC cells maintained position relative to non-dividing neighbors. NC cell division orientation and the time and distance to first division after neural tube exit were stochastic. To address how cell cycle is related to phases of migration, we used FACs analysis to identify significant spatiotemporal differences in NC cell cycle profiles. Two-photon photoconversion of single and small numbers of mKikGR-labeled NC cells confirmed that lead NC cells exhibited a nearly fourfold faster doubling time after populating the branchial arches. By contrast, Ki-67 staining showed that one out of every five later emerging NC cells exited the cell cycle after reaching proximal head targets. The relatively quiescent mitotic activity during NC cell migration to the branchial arches was altered when premigratory cells were reduced in number by tissue ablation. Together, our results provide the first comprehensive details of the pattern and dynamics of cell division events during cranial NC cell migration.

Angiopoietin 2 signaling plays a critical role in neural crest cell migration.

BACKGROUND: Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood. RESULTS: Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells. CONCLUSIONS: Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

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.

Evidence for dynamic rearrangements but lack of fate or position restrictions in premigratory avian trunk neural crest.

Neural crest (NC) cells emerge from the dorsal trunk neural tube (NT) and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the NT have similar or differential ability to contribute to NC cells and their derivatives. To this end, we precisely labeled NT precursors at specific dorsoventral levels of the chick NT using fluorescent dyes and a photoconvertible fluorescent protein. NT and NC cell dynamics were then examined in vivo and in slice culture using two-photon and confocal time-lapse imaging. The results show that NC precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the NT, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the NT to contribute to NC derivatives, with the exception of sympathetic ganglia, which appeared to be 'filled' by the first population to emigrate. Rather than restricted developmental potential, however, this is probably due to a matter of timing.

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.

Autoantibodies to ACE2 and immune molecules are associated with COVID-19 disease severity.

BACKGROUND: Increased inflammation caused by SARS-CoV-2 infection can lead to severe coronavirus disease 2019 (COVID-19) and long-term disease manifestations. The mechanisms of this variable long-term immune activation are poorly defined. One feature of this increased inflammation is elevated levels of proinflammatory cytokines and chemokines. Autoantibodies targeting immune factors such as cytokines, as well as the viral host cell receptor, angiotensin-converting enzyme 2 (ACE2), have been observed after SARS-CoV-2 infection. Autoantibodies to immune factors and ACE2 could interfere with normal immune regulation and lead to increased inflammation, severe COVID-19, and long-term complications. METHODS: Here, we deeply profiled the features of ACE2, cytokine, and chemokine autoantibodies in samples from patients recovering from severe COVID-19. We measured the levels of immunoglobulin subclasses (IgG, IgA, IgM) in the peripheral blood against ACE2 and 23 cytokines and other immune molecules. We then utilized an ACE2 peptide microarray to map the linear epitopes targeted by ACE2 autoantibodies. RESULTS: We demonstrate that ACE2 autoantibody levels are increased in individuals with severe COVID-19 compared with those with mild infection or no prior infection. We identify epitopes near the catalytic domain of ACE2 targeted by these antibodies. Levels of autoantibodies targeting ACE2 and other immune factors could serve as determinants of COVID-19 disease severity, and represent a natural immunoregulatory mechanism in response to viral infection. CONCLUSIONS: These results demonstrate that SARS-CoV-2 infection can increase autoantibody levels to ACE2 and other immune factors. The levels of these autoantibodies are associated with COVID-19 disease severity.

Antibodies are small proteins that are produced by your immune system to protect you when an unwanted foreign invader such as bacteria, viruses and toxins enters your body. When these antibodies target proteins on our own cells instead of the invader, we call them autoantibodies. Autoantibodies that target host immune molecules, as well as ACE2, a receptor molecule that interacts with the SARS-CoV-2 virus, have been observed after COVID-19. We found that patients who had severe COVID-19 displayed higher levels of these autoantibodies compared to those who had mild infection or were uninfected. These findings suggest that these autoantibody levels could serve as indicators of COVID-19 severity.

SARS-CoV-2 envelope protein regulates innate immune tolerance.

Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.

Mapping structural variants to rare disease genes using long-read whole genome sequencing and trait-relevant polygenic scores.

Recent studies have revealed the pervasive landscape of rare structural variants (rSVs) present in human genomes. rSVs can have extreme effects on the expression of proximal genes and, in a rare disease context, have been implicated in patient cases where no diagnostic single nucleotide variant (SNV) was found. Approaches for integrating rSVs to date have focused on targeted approaches in known Mendelian rare disease genes. This approach is intractable for rare diseases with many causal loci or patients with complex, multi-phenotype syndromes. We hypothesized that integrating trait-relevant polygenic scores (PGS) would provide a substantial reduction in the number of candidate disease genes in which to assess rSV effects. We further implemented a method for ranking PGS genes to define a set of core/key genes where a rSV has the potential to exert relatively larger effects on disease risk. Among a subset of patients enrolled in the Genomic Answers for Kids (GA4K) rare disease program (N=497), we used PacBio HiFi long-read whole genome sequencing (lrWGS) to identify rSVs intersecting genes in trait-relevant PGSs. Illustrating our approach in Autism (N=54 cases), we identified 22, 019 deletions, 2,041 duplications, 87,826 insertions, and 214 inversions overlapping putative core/key PGS genes. Additionally, by integrating genomic constraint annotations from gnomAD, we observed that rare duplications overlapping putative core/key PGS genes were frequently in higher constraint regions compared to controls (P = 1×10-03). This difference was not observed in the lowest-ranked gene set (P = 0.15). Overall, our study provides a framework for the annotation of long-read rSVs from lrWGS data and prioritization of disease-linked genomic regions for downstream functional validation of rSV impacts. To enable reuse by other researchers, we have made SV allele frequencies and gene associations freely available.

Developmental imaging: the avian embryo hatches to the challenge.

The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair.

Autoantibodies against Angiotensin-converting enzyme 2 and immune molecules are associated with COVID-19 disease severity.

Increased inflammation caused by SARS-CoV-2 infection can lead to severe coronavirus disease 2019 (COVID-19) and long-term disease manifestations referred to as post-acute sequalae of COVID (PASC). The mechanisms of this variable long-term immune activation are poorly defined. Autoantibodies targeting immune factors such as cytokines, as well as the viral host cell receptor, angiotensin-converting enzyme 2 (ACE2), have been observed after SARS-CoV-2 infection. Autoantibodies to immune factors and ACE2 could interfere with normal immune regulation and lead to increased inflammation, severe COVID-19, and long-term complications. Here, we deeply pro led the features of ACE2, cytokine, and chemokine autoantibodies in samples from patients recovering from severe COVID-19. We identified epitopes in the catalytic domain of ACE2 targeted by these antibodies, that could inhibit ACE2 function. Levels of autoantibodies targeting ACE2 and other immune factors could serve as determinants of COVID-19 disease severity, and represent a natural immunoregulatory mechanism in response to viral infection.

The cellular and immunological dynamics of early and transitional human milk.

Human milk is essential for infant nutrition and immunity, providing protection against infections and other immune-mediated diseases during the lactation period and beyond in later childhood. Milk contains a broad range of bioactive factors such as nutrients, hormones, enzymes, immunoglobulins, growth factors, cytokines, and antimicrobial factors, as well as heterogeneous populations of maternal cells. The soluble and cellular components of milk are dynamic over time to meet the needs of the growing infant. In this study, we utilize systems-approaches to define and characterize 62 analytes of the soluble component, including immunoglobulin isotypes, as well as the cellular component of human milk during the first two weeks postpartum from 36 mothers. We identify soluble immune and growth factors that are dynamic over time and could be utilized to classify milk into different phenotypic groups. We identify 24 distinct populations of both epithelial and immune cells by single-cell transcriptome analysis of 128,016 human milk cells. We found that macrophage populations have shifting inflammatory profiles during the first two weeks of lactation. This analysis provides key insights into the soluble and cellular components of human milk and serves as a substantial resource for future studies of human milk.