A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells.

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

Melanocyte lineage dynamics in development, growth and disease.

Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.

Motor innervation directs the correct development of the mouse sympathetic nervous system.

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the "fight-or-flight response". Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

Chemical Coaxing of Mesenchymal Stromal Cells by Drug Repositioning for Nestin Induction.

Mesenchymal stromal cells (MSCs) display heterogeneity in origin and functional role in tissue homeostasis. Subsets of MSCs derived from the neural crest express nestin and serve as niches in bone marrow, but the possibility of coaxing MSCs into nestin-expresing cells for enhanced supportive activity is unclear. In this study, as an approach to the chemical coaxing of MSC functions, we screened libraries of clinically approved chemicals to identify compounds capable of inducing nestin expression in MSCs. Out of 2000 clinical compounds, we chose vorinostat as a candidate to coax the MSCs into neural crest-like fates. When treated with vorinostat, MSCs exhibited a significant increase in the expression of genes involved in the pluripotency and epithelial-mesenchymal transition (EMT), as well as nestin and CD146, the markers for pericytes. In addition, these nestin-induced MSCs exhibited enhanced differentiation towards neuronal cells with the upregulation of neurogenic markers, including SRY-box transcription factor 2 (Sox2), SRY-box transcription factor 10 (Sox10) and microtubule associated protein 2 (Map2) in addition to nestin. Moreover, the coaxed MSCs exhibited enhanced supporting activity for hematopoietic progenitors without supporting leukemia cells. These results demonstrate the feasibility of the drug repositioning of MSCs to induce neural crest-like properties through the chemical coaxing of cell fates.

Molecular signaling directing neural plate border formation.

During embryonic development, the vertebrate embryonic epiblast is divided into two parts including neural and superficial ectoderm. The neural plate border (NPB) is a narrow transitional area which locates between these parts and contains multipotent progenitor cells. Despite its small size, the cellular heterogeneity in this region produces specific differentiated cells. Signaling pathways, transcription factors, and the expression/repression of certain genes are directly involved in these differentiation processes. Different factors such as the Wnt signaling cascade, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) signaling, and Notch, which are involved in various stages of the growth, proliferation, and differentiation of embryonic cells, are also involved in the determination and differentiation of neural plate border stem cells. Therefore, it is essential to consider the interactions and temporospatial coordination related to cells, tissues, and adjacent structures. This review examines our present knowledge of the formation of the neural plate border and emphasizes the requirement for interaction between different signaling pathways, including the BMP and Wnt cascades, the expression of its special target genes and their regulations, and the precise tissue crosstalk which defines the neural crest fate in the ectoderm at the early human embryonic stages.

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

miR-203 secreted in extracellular vesicles mediates the communication between neural crest and placode cells required for trigeminal ganglia formation.

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, by using chick embryos, we show that the microRNA (miR)-203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. By employing cell-specific electroporations for either miR-203 sponging or genomic editing using CRISPR/Cas9, we elucidated that neural crest cells serve as the source, while placode cells serve as the site of action for miR-203 in trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses an miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.

The people behind the papers - Paul Huber and Carole LaBonne.

The bromodomain and extra-terminal (BET) family of proteins reads epigenetic histone acetylation marks on the genome and regulates the transcriptional machinery. In their study, Carole LaBonne and colleagues reveal the role of BET protein activity in the maintenance of pluripotency and establishment of the neural crest in Xenopus laevis. To know more about their work, we spoke to the first author Paul Huber and the corresponding author Carole LaBonne, Developmental and Stem Cell Biologist at Northwestern University.

Post-transcriptional regulation as a conserved driver of neural crest and cancer-cell migration.

Cells have evolved mechanisms to migrate for diverse biological functions. A process frequently deployed during metazoan cell migration is the epithelial-mesenchymal transition (EMT). During EMT, adherent epithelial cells undergo coordinated cellular transitions to mesenchymalize and reduce their intercellular attachments. This is achieved via tightly regulated changes in gene expression, which modulates cell-cell and cell-matrix adhesion to allow movement. The acquisition of motility and invasive properties following EMT allows some mesenchymal cells to migrate through complex environments to form tissues during embryogenesis; however, these processes may also be leveraged by cancer cells, which often co-opt these endogenous programs to metastasize. Post-transcriptional regulation is now emerging as a major conserved mechanism by which cells modulate EMT and migration, which we discuss here in the context of vertebrate development and cancer.

FBLN2 is associated with Goldenhar syndrome and is essential for cranial neural crest cell development.

Goldenhar syndrome, a rare craniofacial malformation, is characterized by developmental anomalies in the first and second pharyngeal arches. Its etiology is considered to be heterogenous, including both genetic and environmental factors that remain largely unknown. To further elucidate the genetic cause in a five-generation Goldenhar syndrome pedigree and exploit the whole-exome sequencing (WES) data of this pedigree, we generated collapsed haplotype pattern markers based on WES and employed rare variant nonparametric linkage analysis. FBLN2 was identified as a candidate gene via analysis of WES data across the significant linkage region. A fbln2 knockout zebrafish line was established by CRISPR/Cas9 to examine the gene's role in craniofacial cartilage development. fbln2 was expressed specifically in the mandible during the zebrafish early development, while fbln2 knockout zebrafish exhibited craniofacial malformations with abnormal chondrocyte morphologies. Functional studies revealed that fbln2 knockout caused abnormal chondrogenic differentiation, apoptosis, and proliferation of cranial neural crest cells (CNCCs), and downregulated the bone morphogenic protein (BMP) signaling pathway in the zebrafish model. This study demonstrates the role of FBLN2 in CNCC development and BMP pathway regulation, and highlights FBLN2 as a candidate gene for Goldenhar syndrome, which may have implications for the selection of potential screening targets and the development of treatments for conditions like microtia-atresia.

Tlx3 mediates neuronal differentiation and proper condensation of the developing trigeminal ganglion.

The trigeminal ganglion, the largest of the vertebrate cranial ganglia, is comprised of sensory neurons that relay sensations of pain, touch, and temperature to the brain. These neurons are derived from two embryonic cell types, the neural crest and ectodermal placodes, whose interactions are critical for proper ganglion formation. While the T-cell leukemia homeobox 3 (Tlx3) gene is known to be expressed in placodally-derived sensory neurons and necessary for their differentiation, little was known about Tlx3 expression and/or function in the neural crest-derived component of the developing trigeminal ganglion. By combining lineage labeling with in situ hybridization in the chick embryo, we show that neural crest-derived cells that contribute to the cranial trigeminal ganglion express Tlx3 at a time point that coincides with the onset of ganglion condensation. Importantly, loss of Tlx3 function in vivo diminishes the overall size and abundance of neurons within the trigeminal ganglion. Conversely, ectopic expression of Tlx3 in migrating cranial neural crest results in their premature neuronal differentiation. Taken together, our results demonstrate a critical role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis.

Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice.

OBJECTIVES: This study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice. METHODS: The embryos of Wnt1-Cre;R26RmTmG and Pax2-Cre;R26RmTmG at embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26RAi9 and Wnt1-Cre;R26RAi9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26RmTmG and Pax2-cre;R26RmTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes. RESULTS: Wnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre-marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis. CONCLUSIONS: Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Efficient fabrication of 3D bioprinted functional sensory neurons using an inducible Neurogenin-2 human pluripotent stem cell line.

Three-dimensional (3D) tissue models have gained recognition for their improved ability to mimic the native cell microenvironment compared to traditional two-dimensional models. This progress has been driven by advances in tissue-engineering technologies such as 3D bioprinting, a promising method for fabricating biomimetic living tissues. While bioprinting has succeeded in generating various tissues to date, creating neural tissue models remains challenging. In this context, we present an accelerated approach to fabricate 3D sensory neuron (SN) structures using a transgenic human pluripotent stem cell (hPSC)-line that contains an inducible Neurogenin-2 (NGN2) expression cassette. The NGN2 hPSC line was first differentiated to neural crest cell (NCC) progenitors, then incorporated into a cytocompatible gelatin methacryloyl-based bioink for 3D bioprinting. Upregulated NGN2 expression in the bioprinted NCCs resulted in induced SN (iSN) populations that exhibited specific cell markers, with 3D analysis revealing widespread neurite outgrowth through the scaffold volume. Calcium imaging demonstrated functional activity of iSNs, including membrane excitability properties and voltage-gated sodium channel (NaV) activity. This efficient approach to generate 3D bioprinted iSN structures streamlines the development of neural tissue models, useful for the study of neurodevelopment and disease states and offering translational potential.

The chromatin regulator Ankrd11 controls cardiac neural crest cell-mediated outflow tract remodeling and heart function.

ANKRD11 (Ankyrin Repeat Domain 11) is a chromatin regulator and a causative gene for KBG syndrome, a rare developmental disorder characterized by multiple organ abnormalities, including cardiac defects. However, the role of ANKRD11 in heart development is unknown. The neural crest plays a leading role in embryonic heart development, and its dysfunction is implicated in congenital heart defects. We demonstrate that conditional knockout of Ankrd11 in the murine embryonic neural crest results in persistent truncus arteriosus, ventricular dilation, and impaired ventricular contractility. We further show these defects occur due to aberrant cardiac neural crest cell organization leading to outflow tract septation failure. Lastly, knockout of Ankrd11 in the neural crest leads to impaired expression of various transcription factors, chromatin remodelers and signaling pathways, including mTOR, BMP and TGF-ß in the cardiac neural crest cells. In this work, we identify Ankrd11 as a regulator of neural crest-mediated heart development and function.

Molecular Characterization of Nodose Ganglia Development Reveals a Novel Population of Phox2b+ Glial Progenitors in Mice.

The vagal ganglia, comprised of the superior (jugular) and inferior (nodose) ganglia of the vagus nerve, receive somatosensory information from the head and neck or viscerosensory information from the inner organs, respectively. Developmentally, the cranial neural crest gives rise to all vagal glial cells and to neurons of the jugular ganglia, while the epibranchial placode gives rise to neurons of the nodose ganglia. Crest-derived nodose glial progenitors can additionally generate autonomic neurons in the peripheral nervous system, but how these progenitors generate neurons is unknown. Here, we found that some Sox10+ neural crest-derived cells in, and surrounding, the nodose ganglion transiently expressed Phox2b, a master regulator of autonomic nervous system development, during early embryonic life. Our genetic lineage-tracing analysis in mice of either sex revealed that despite their common developmental origin and extreme spatial proximity, a substantial proportion of glial cells in the nodose, but not in the neighboring jugular ganglia, have a history of Phox2b expression. We used single-cell RNA-sequencing to demonstrate that these progenitors give rise to all major glial subtypes in the nodose ganglia, including Schwann cells, satellite glia, and glial precursors, and mapped their spatial distribution by in situ hybridization. Lastly, integration analysis revealed transcriptomic similarities between nodose and dorsal root ganglia glial subtypes and revealed immature nodose glial subtypes. Our work demonstrates that these crest-derived nodose glial progenitors transiently express Phox2b, give rise to the entire complement of nodose glial cells, and display a transcriptional program that may underlie their bipotent nature.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

Neural Crest.

This chapter discusses the role of cardiac neural crest cells in the formation of the septum that divides the cardiac arterial pole into separate systemic and pulmonary arteries. Further, cardiac neural crest cells directly support the normal development and patterning of derivatives of the caudal pharyngeal arches, including the great arteries, thymus, thyroid, and parathyroids. Recently, cardiac neural crest cells have also been shown to indirectly influence the development of the secondary heart field, another derivative of the caudal pharynx, by modulating signaling in the pharynx. The contribution and function of the cardiac neural crest cells has been learned in avian models; most of the genes associated with cardiac neural crest function have been identified using mouse models. Together these studies show that the neural crest cells may not only critical for normal cardiovascular development but also may be involved secondarily because they represent a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Cardiac neural crest cells span from the caudal pharynx into the outflow tract, and therefore may be susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations resulting from genetic and/or environmental insults necessarily requires better understanding the role of cardiac neural crest cells in cardiac development.

Molecular Pathways and Animal Models of Tetralogy of Fallot and Double Outlet Right Ventricle.

Tetralogy of Fallot and double-outlet right ventricle are outflow tract (OFT) alignment defects situated on a continuous disease spectrum. A myriad of upstream causes can impact on ventriculoarterial alignment that can be summarized as defects in either i) OFT elongation during looping morphogenesis or ii) OFT remodeling during cardiac septation. Embryological processes underlying these two developmental steps include deployment of second heart field cardiac progenitor cells, establishment and transmission of embryonic left/right information driving OFT rotation and OFT cushion and valve morphogenesis. The formation and remodeling of pulmonary trunk infundibular myocardium is a critical component of both steps. Defects in myocardial, endocardial, or neural crest cell lineages can result in alignment defects, reflecting the complex intercellular signaling events that coordinate arterial pole development. Importantly, however, OFT alignment is mechanistically distinct from neural crest-driven OFT septation, although neural crest cells impact indirectly on alignment through their role in modulating signaling during SHF development. As yet poorly understood nongenetic causes of alignment defects that impact the above processes include hemodynamic changes, maternal exposure to environmental teratogens, and stochastic events. The heterogeneity of causes converging on alignment defects characterizes the OFT as a hotspot of congenital heart defects.

Alcohol exposure suppresses ribosome biogenesis and causes nucleolar stress in cranial neural crest cells.

Prenatal alcohol exposure (PAE) causes cognitive impairment and a distinctive craniofacial dysmorphology, due in part to apoptotic losses of the pluripotent cranial neural crest cells (CNCs) that form facial bones and cartilage. We previously reported that PAE rapidly represses expression of >70 ribosomal proteins (padj = 10-E47). Ribosome dysbiogenesis causes nucleolar stress and activates p53-MDM2-mediated apoptosis. Using primary avian CNCs and the murine CNC line O9-1, we tested whether nucleolar stress and p53-MDM2 signaling mediates this apoptosis. We further tested whether haploinsufficiency in genes that govern ribosome biogenesis, using a blocking morpholino approach, synergizes with alcohol to worsen craniofacial outcomes in a zebrafish model. In both avian and murine CNCs, pharmacologically relevant alcohol exposure (20mM, 2hr) causes the dissolution of nucleolar structures and the loss of rRNA synthesis; this nucleolar stress persisted for 18-24hr. This was followed by reduced proliferation, stabilization of nuclear p53, and apoptosis that was prevented by overexpression of MDM2 or dominant-negative p53. In zebrafish embryos, low-dose alcohol or morpholinos directed against ribosomal proteins Rpl5a, Rpl11, and Rps3a, the Tcof homolog Nolc1, or mdm2 separately caused modest craniofacial malformations, whereas these blocking morpholinos synergized with low-dose alcohol to reduce and even eliminate facial elements. Similar results were obtained using a small molecule inhibitor of RNA Polymerase 1, CX5461, whereas p53-blocking morpholinos normalized craniofacial outcomes under high-dose alcohol. Transcriptome analysis affirmed that alcohol suppressed the expression of >150 genes essential for ribosome biogenesis. We conclude that alcohol causes the apoptosis of CNCs, at least in part, by suppressing ribosome biogenesis and invoking a nucleolar stress that initiates their p53-MDM2 mediated apoptosis. We further note that the facial deficits that typify PAE and some ribosomopathies share features including reduced philtrum, upper lip, and epicanthal distance, suggesting the facial deficits of PAE represent, in part, a ribosomopathy.