Jason A. Morrison

Melanoma revives an embryonic migration program to promote plasticity and invasion

Cancer cells must regulate plasticity and invasion to survive and metastasize. However, the identification of targetable mechanisms to inhibit metastasis has been slow. Signaling programs that drive stem and progenitor cells during normal development offer an inroad to discover mechanisms common to metastasis. Using a chick embryo transplant model, we have compared molecular signaling programs of melanoma and their embryonic progenitors, the neural crest. We report that malignant melanoma cells hijack portions of the embryonic neural crest invasion program. Genes associated with neural crest induction, delamination, and migration are dynamically regulated by melanoma cells exposed to an embryonic neural crest microenvironment. Specifically, we demonstrate that metastatic melanoma cells exploit neural crest‐related receptor tyrosine kinases to increase plasticity and facilitate invasion while primary melanocytes may actively suppress these responses under the same microenvironmental conditions. We conclude that aberrant regulation of neural crest developmental genes promotes plasticity and invasiveness in malignant melanoma.

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.

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.

Gene Profiling in the Avian Embryo Using Laser Capture Microdissection and RT-qPCR

The dynamic nature of the developing embryo makes it challenging to understand complex morphogenetic events using information from large-scale gene expression patterns. What would be more insightful is molecular profiling of small numbers of cells selectively surveyed at specific developmental stages. However, detecting gene expression profile information from small numbers of cells (<10) in homogenous tissue has remained a major challenge. Here, we describe the use of laser capture microdissection (LCM), immunohistochemistry (IHC), and RT-qPCR to extract gene profile information in distinct embryo tissue more precisely than is possible with any other method. We use the chick embryo model system and combine electroporation and dual-label IHC to specifically identify cells for harvest by LCM without significant degradation of total RNA. We describe the development of a pre-amplification protocol for small subpopulations of cells to produce sensitive RT-qPCR results. The gene-specific pre-amplification efficiently and linearly amplifies only gene transcripts of interest from the harvested material without the need for RNA isolation. By combining the above techniques with microfluidic RT-qPCR, we robustly analyze the expression of ∼300 genes from as few as 10 cells harvested by LCM. Together, this protocol presents a confident isolation and means of sensitive expression analysis of small cell numbers from tissues and overcomes a technical hurdle that limits gene profiling.

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.

Quantitative single cell gene expression profiling in the avian embryo.

Single cell gene profiling has been successfully applied to cultured cells. However, isolation and preservation of a cells 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. Developmental Dynamics 244:774–784, 2015.

Multi-Position Photoactivation and Multi-Time Acquisition for Large-Scale Cell Tracing in Avian Embryos

Vertebrate development is a complex orchestration of cell and tissue movements. Tracing individual cell positions can rapidly become a large-scale problem because cell numbers often grow exponentially in the early embryo. A typical approach consists of fluorescently marking small numbers of cells within a large number of embryos, followed by comprehensive three-dimensional static or time-lapse imaging to map cell positions. However, for large-scale cell tracing, such as during organogenesis, the time, effort, and expense of this approach can be limiting. The multi-position acquisition method can be used to capture more than one location on a microscope stage and allow for multi-specimen imaging. When combined with photoactivation cell labeling, a tool for selective cell marking using laser excitation, multi-position imaging offers a powerful tool for rapid data acquisition. This protocol describes the technique and demonstrates its use to map cell movements in the chick spinal cord, using slice culture explants. The details of multiple slice culture preparation, multi-position photoactivation, and multi-time acquisition are described. These events are coordinated by setting up blocks of microscope instructions that execute sequentially. This method significantly decreases the time, effort, microscopy, and embryo costs by a factor of the number of specimens imaged per session, typically six.

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.

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.

NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor

ABSTRACT 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. Summary: We identify and test the function of nerve growth factor to reprogram human metastatic melanoma cells to a less aggressive phenotype. This article has an associated First Person interview with the first author of the paper as part of the supplementary information.