What is bad in cancer is good in the embryo: Importance of EMT in neural crest development

Although the epithelial to mesenchymal transition (EMT) is famous for its role in cancer metastasis, it also is a normal developmental event in which epithelial cells are converted into migratory mesenchymal cells. A prime example of EMT during development occurs when neural crest (NC) cells emigrate from the neural tube thus providing an excellent model to study the principles of EMT in a nonmalignant environment. NC cells start life as neuroepithelial cells intermixed with precursors of the central nervous system. After EMT, they delaminate and begin migrating, often to distant sites in the embryo. While proliferating and maintaining multipotency and cell survival the transitioning neural crest cells lose apicobasal polarity and the basement membrane is broken down. This review discusses how these events are coordinated and regulated, by series of events involving signaling factors, gene regulatory interactions, as well as epigenetic and post-transcriptional modifications. Even though the series of events involved in NC EMT are well known, the sequence in which these steps take place remains a subject of debate, raising the intriguing possibility that, rather than being a single event, neural crest EMT may involve multiple parallel mechanisms

cMyc Regulates the Size of the Premigratory Neural Crest Stem Cell Pool

The neural crest is a transient embryonic population that originates within the central nervous system (CNS) and then migrates into the periphery and differentiates into multiple cell types. The mechanisms that govern neural crest stem-like characteristics and self-renewal ability are poorly understood. Here, we show that the proto-oncogene cMyc is a critical factor in the chick dorsal neural tube, where it regulates the size of the premigratory neural crest stem cell pool. Loss of cMyc dramatically decreases the number of emigrating neural crest cells due to reduced self-renewal capacity, increased cell death, and shorter duration of the emigration process. Interestingly, rather than via E-Box binding, cMyc acts in the dorsal neural tube by interacting with another transcription factor, Miz1, to promote self-renewal. The finding that cMyc operates in a non-canonical manner in the premigratory neural crest highlights the importance of examining its role at specific time points and in an in vivo context

Biphasic influence of Miz1 on neural crest development by regulating cell survival and apical adhesion complex formation in the developing neural tube

Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle– and cell adhesion–related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest–selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein

cMyc Regulates the Size of the Premigratory Neural Crest Stem Cell Pool

The neural crest is a transient embryonic population that originates within the central nervous system (CNS) and then migrates into the periphery and differentiates into multiple cell types. The mechanisms that govern neural crest stem-like characteristics and self-renewal ability are poorly understood. Here, we show that the proto-oncogene cMyc is a critical factor in the chick dorsal neural tube, where it regulates the size of the premigratory neural crest stem cell pool. Loss of cMyc dramatically decreases the number of emigrating neural crest cells due to reduced self-renewal capacity, increased cell death, and shorter duration of the emigration process. Interestingly, rather than via E-Box binding, cMyc acts in the dorsal neural tube by interacting with another transcription factor, Miz1, to promote self-renewal. The finding that cMyc operates in a non-canonical manner in the premigratory neural crest highlights the importance of examining its role at specific time points and in an in vivo context

Identification of a neural crest stem cell niche by Spatial Genomic Analysis

The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates. Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous population. By coupling multiplex single molecule fluorescence in situ hybridization with machine learning algorithm based cell segmentation, we examine expression of 35 genes at single cell resolution in vivo. Unbiased hierarchical clustering reveals five spatially distinct subpopulations within the chick dorsal neural tube. Here we identify a neural crest stem cell niche that centers around the dorsal midline with high expression of neural crest genes, pluripotency factors, and lineage markers. Interestingly, neural and neural crest stem cells express distinct pluripotency signatures. This Spatial Genomic Analysis toolkit provides a straightforward approach to study quantitative multiplex gene expression in numerous biological systems, while offering insights into gene regulatory networks via synexpression analysis

ShapeMetrics: a userfriendly pipeline for 3D cell segmentation and spatial tissue analysis

The demand for single-cell level data is constantly increasing within life sciences. In order to meet this demand, robust cell segmentation methods that can tackle challenging in vivo tissues with complex morphology are required. However, currently available cell segmentation and volumetric analysis methods perform poorly on 3D images. Here, we generated ShapeMetrics, a MATLAB-based script that segments cells in 3D and, by performing unbiased clustering using a heatmap, separates the cells into subgroups according to their volumetric and morphological differences. The cells can be accurately segregated according to different biologically meaningful features such as cell ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. Our machine learning based script enables dissection of a large amount of novel data from microscope images in addition to the traditional information based on fluorescent biomarkers. Furthermore, the cells in different subgroups can be spatially mapped back to their original locations in the tissue image to help elucidate their roles in their respective morphological contexts. In order to facilitate the transition from bulk analysis to single-cell level accuracy, we emphasize the user-friendliness of our method by providing detailed step-by-step instructions through the pipeline hence aiming to reach users with less experience in computational biology.Peer reviewe

Crestospheres: Long-Term Maintenance of Multipotent, Premigratory Neural Crest Stem Cells

Premigratory neural crest cells comprise a transient, embryonic population that arises within the CNS, but subsequently migrates away and differentiates into many derivatives. Previously, premigratory neural crest could not be maintained in a multipotent, adhesive state without spontaneous differentiation. Here, we report conditions that enable maintenance of neuroepithelial “crestospheres” that self-renew and retain multipotency for weeks. Moreover, under differentiation conditions, these cells can form multiple derivatives in vitro and in vivo after transplantation into chick embryos. Similarly, human embryonic stem cells directed to a neural crest fate can be maintained as crestospheres and subsequently differentiated into several derivatives. By devising conditions that maintain the premigratory state in vitro, these results demonstrate that neuroepithelial neural crest precursors are capable of long-term self-renewal. This approach will help uncover mechanisms underlying their developmental potential, differentiation and, together with the induced pluripotent stem cell techniques, the pathology of human neurocristopathies

Maintaining multipotent trunk neural crest stem cells as self-renewing crestospheres

Neural crest cells have broad migratory and differentiative ability that differs according to their axial level of origin. However, their transient nature has limited understanding of their stem cell and self-renewal properties. While an in vitro culture method has made it possible to maintain cranial neural crest cells as self-renewing multipotent crestospheres (Kerosuo et al., 2015), these same conditions failed to preserve trunk neural crest in a stem-like state. Here we optimize culture conditions for maintenance of avian trunk crestospheres, comprised of both neural crest stem and progenitor cells. Our trunk-derived crestospheres are multipotent and display self-renewal capacity over several weeks. Trunk crestospheres display elevated expression of neural crest cell markers as compared to those characteristic of ventrolateral neural tube or mesodermal fates. Moreover, trunk crestospheres express increased levels of trunk neural crest-enriched markers as compared to cranial crestospheres. Finally, we use lentiviral transduction as a tool to manipulate gene expression in trunk crestospheres. Taken together, this method enables long-term in vitro maintenance and manipulation of multipotent trunk neural crest cells in a premigratory stem or early progenitor state. Trunk crestospheres are a valuable resource for probing mechanisms underlying neural crest sternness and lineage decisions as well as accompanying diseases.Peer reviewe