Rebecca McLennan

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.

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.

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.

Neural Crest Invasion is a Spatially-Ordered Progression Into the Head with Higher Cell Proliferation at the Migratory Front as Revealed by the Photoactivatable Protein, KikGR

Neural crest cell (NCC) invasion is a complex sculpting of individual cells into organized migratory streams that lead to organ development along the vertebrate axis. Key to our understanding of how molecular mechanisms modulate the NCC migratory pattern is information about cell behaviors, yet it has been challenging to selectively mark and analyze migratory NCCs in a living embryo. Here, we apply an innovative in vivo strategy to investigate chick NCC behaviors within the rhombomere 4 (r4) migratory stream by combining photoactivation of KikGR and confocal time-lapse analysis of H2B-mRFP1 transfected NCCs. We find that the spatial order of r4 NCC emergence translates into a distal-to-proximal invasion of the 2nd branchial arch. Lead and trailing NCCs display similar average cell speeds and directionalities. Surprisingly, we find that lead NCCs proliferate along the migratory route and grow to outnumber trailing NCCs by nearly 3 to 1. A simple, cell-based computational model reproduces the r4 NCC migratory pattern and predicts the invasion order can be disrupted by slower, less directional lead cells or by environmental noise. Our results suggest a model in which NCC behaviors maintain a spatially-ordered invasion of the branchial arches with differences in cell proliferation between the migratory front and trailing NCCs.

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.

Neuropilin-1 interacts with the second branchial arch microenvironment to mediate chick neural crest cell dynamics

Cranial neural crest cells (NCCs) require neuropilin signaling to reach and invade the branchial arches. Here, we use an in vivo chick model to investigate whether the neuropilin‐1 knockdown phenotype is specific to the second branchial arch (ba2), changes in NCC behaviors and phenotypic consequences, and whether neuropilins work together to facilitate entry into and invasion of ba2. We find that cranial NCCs with reduced neuropilin‐1 expression displayed shorter protrusions and decreased cell body and nuclear length‐to‐width ratios characteristic of a loss in polarity and motility, after specific interaction with ba2. Directed NCC migration was rescued by transplantation of transfected NCCs into rhombomere 4 of younger hosts. Lastly, reduction of neuropilin‐2 expression by shRNA either solely or with reduction of neuropilin‐1 expression did not lead to a stronger head phenotype. Thus, NCCs, independent of rhombomere origin, require neuropilin‐1, but not neuropilin‐2 to maintain polarity and directed migration into ba2. Developmental Dynamics 239:1664–1673, 2010.

Neural crest migration: trailblazing ahead

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

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.

Multispectral fingerprinting for improved in vivo cell dynamics analysis

BackgroundTracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks.ResultsWe found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets.ConclusionsOur results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cells position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.

Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors.