► Neural crest (NC) cells undergo EMT to begin migration. ► The neural crest (NC) is an excellent embryonic model for studying EMT under normal conditions. ► NC EMT includes several steps from ...maintenance of multipotency to depolarization and emigration. ► NC EMT may involve multiple parallel triggering mechanisms.
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.
Unique to vertebrates, the neural crest (NC) is an embryonic stem cell population that contributes to a greatly expanding list of derivatives ranging from neurons and glia of the peripheral nervous ...system, facial cartilage and bone, pigment cells of the skin to secretory cells of the endocrine system. Here, we focus on what is specifically known about establishment and maintenance of NC stemness and ultimate fate commitment mechanisms, which could help explain its exceptionally high stem cell potential that exceeds the “rules set during gastrulation.” In fact, recent discoveries have shed light on the existence of NC cells that coexpress commonly accepted pluripotency factors like Nanog, Oct4/PouV, and Klf4. The coexpression of pluripotency factors together with the exceptional array of diverse NC derivatives encouraged us to propose a new term “pleistopotent” (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post‐gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC‐derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos.
Neural crest stem cells possess exceptionally high stem cell potential post‐gastrulation. These cells give rise to a diverse range of derivatives of ectodermal, mesodermal‐like, and endodermal‐like origin, either directly from the initial embryonic neural crest cells or later in development through an intermediate stem cell stage such as the Schwann Cell Precursors and postnatal neural crest‐derived stem cell reservoirs.
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.
The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains ...poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like derivatives is obtained. Here, we monitor transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. We show maintenance of pluripotency-like signature (Nanog, Oct4/PouV, Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-like signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-like signature is found at multiple axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.
Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with ...phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more “normal” neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.
The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and ...many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders.
The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures ...are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.
Incomplete functional recovery after peripheral nerve injury (PNI) often results in devastating physical disabilities in human patients. Despite improved progress in surgical and non-surgical ...approaches, achieving complete functional recovery following PNI remains a challenge. This study demonstrates that phentolamine may hold a significant promise in treating nerve injuries and denervation induced muscle atrophy following PNI. In a sciatic nerve crush injury mouse model, we found that phentolamine treatment enhanced motor and functional recovery, protected axon myelination, and attenuated injury-induced muscle atrophy in mice at 14 days post-injury (dpi) compared to saline treatment. In the soleus of phentolamine treated animals, we observed the downregulation of phosphorylated signal transducer and activator of transcription factor 3 (p-STAT3) as well as muscle atrophy-related genes Myogenin, muscle ring finger 1 (MuRF-1), and Forkhead box O proteins (FoxO1, FoxO3). Our results show that both nerve and muscle recovery are integral components of phentolamine treatment-induced global functional recovery in mice at 14 dpi. Moreover, phentolamine treatment improved locomotor functional recovery in the mice after spinal cord crush (SCC) injury. The fact that phentolamine is an FDA approved non-selective alpha-adrenergic blocker, clinically prescribed for oral anesthesia reversal, hypertension, and erectile dysfunction makes this drug a promising candidate for repurposing in restoring behavioral recovery following PNI and SCC injuries, axonal neuropathy, and muscle wasting disorders.
There is a growing need for single-cell level data analysis in correlation with the advancements of microscopy techniques. Morphology-based statistics gathered from individual cells are essential for ...detection and quantification of even subtle changes within the complex tissues, yet the information available from high-resolution imaging is oftentimes sub-optimally utilized due to the lack of proper computational analysis software. Here we present ShapeMetrics, a 3D cell segmentation pipeline that we have developed to identify, analyze, and quantify single cells in an image. This MATLAB-based script enables users to extract morphological parameters, such as ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. We have specifically invested in creating a user-friendly pipeline, aimed for biologists with a limited computational background. Our pipeline is presented with detailed stepwise instructions, starting from the establishment of machine learning-based prediction files of immuno-labeled cell membranes followed by the application of 3D cell segmentation and parameter extraction script, leading to the morphometric analysis and spatial visualization of cell clusters defined by their morphometric features.
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.
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•Long-term maintenance of premigratory chick neural crest cells as crestospheres•A self-renewing population of multipotent neuroepithelial neural crest stem cells•Crestospheres differentiate into neural crest derivatives in vitro and in vivo•Long-term maintenance of human ESC-derived crestospheres for several weeks
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