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.

CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.

The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.

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.

Loss of Lkb1 cooperates with BrafV600E and ultraviolet radiation, increasing melanoma multiplicity and neural-like dedifferentiation.

The mechanisms that work alongside BRAFV600E oncogene in melanoma development, in addition to ultraviolet (UV) radiation (UVR), are of great interest. Analysis of human melanoma tumors [data from The Cancer Genome Atlas (TCGA)] revealed that 50% or more of the samples expressed no or low amounts of serine/threonine protein kinase STK11 (also known as LKB1) protein. Here, we report that, in a mouse model, concomitant neonatal BrafV600E activation and Lkb1 tumor suppressor ablation in melanocytes led to full melanoma development. A single postnatal dose of UVB radiation had no effect on melanoma onset in Lkb1-depleted mice compared with BrafV600E-irradiated mice, but increased tumor multiplicity. In concordance with these findings and previous reports, Lkb1-null irradiated mice exhibited deficient DNA damage repair (DDR). Histologically, tumors lacking Lkb1 were enriched in neural-like tumor morphology. Genetic profiling and gene set enrichment analyses of tumor sample mutated genes indicated that loss of Lkb1 promoted the selection of altered genes associated with neural differentiation processes. Thus, these results suggest that the loss of Lkb1 cooperates with BrafV600E and UVR, impairing the DDR and increasing melanoma multiplicity and neural-like dedifferentiation.

Neural crest and sons: role of neural crest cells and Schwann cell precursors in development and gland embryogenesis.

In this review, we consider the multipotency of neural crest cells (NCCs), Schwann cell precursors (SCPs), and their role in embryogenesis base on genetic tracing and knock out model animals and single cell transcriptomic analysis. In particular, we summarize and analyze data on the contribution of NCCs and SCPs to the gland development and functions.

Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation.

Neural Crest cells (NC) are a multipotent cell population that give rise to a multitude of cell types including Schwann cells (SC) in the peripheral nervous system (PNS). Immature SC interact with neuronal axons via the neuregulin 1 (NRG1) ligand present on the neuronal surface and ultimately form the myelin sheath. Multiple attempts to derive functional SC from pluripotent stem cells have met challenges with respect to expression of mature markers and axonal sorting. Here, they hypothesized that sustained signaling from immobilized NRG1 (iNRG1) might enhance the differentiation of NC derived from glabrous neonatal epidermis towards a SC phenotype. Using this strategy, NC derived SC expressed mature markers to similar levels as compared to explanted rat sciatic SC. Signaling studies revealed that sustained NRG1 signaling led to yes-associated protein 1 (YAP) activation and nuclear translocation. Furthermore, NC derived SC on iNRG1 exhibited mature SC function as they aligned with rat dorsal root ganglia (DRG) neurons in an in vitro coculture model; and most notably, aligned on neuronal axons upon implantation in a chick embryo model in vivo. Taken together their work demonstrated the importance of signaling dynamics in SC differentiation, aiming towards development of drug testing platforms for de-myelinating disorders.

Primary Hepatic Neuroendocrine Carcinoma: A Rare Entity.

Neuroendocrine tumors are typically low-malignancy growths arising from neuroectodermal cells of neural crest origin. Neuroendocrine carcinoma, on the other hand, represents a high-malignancy form of these tumors. While rare in the liver, they often indicate metastasis when present. We present a unique case of incidentally discovered primary hepatic neuroendocrine carcinoma. Initially, the patient's management was based on misleading radiological findings. However, histopathology confirmed the diagnosis, with subsequent imaging ruling out an extrahepatic source. Despite this, the patient opted against surgical intervention, resulting in a fatal outcome. This case underscores the critical importance of prompt diagnosis and intervention to avert adverse outcomes.

Predicting in vivo therapeutic efficacy of CelTrac1000-labeled hair follicle epidermal neural crest stem cells in models of repairing rat facial nerve defects via second near-infrared fluorescence imaging.

AIMS: To detect the therapeutic efficacy of CelTrac1000-labeled hair follicle epidermal neural crest stem cells (EPI-NCSCs) on repairing facial nerve defects by second near-infrared (NIR-II) fluorescence imaging. MATERIALS AND METHODS: Firstly, CelTrac1000-labeled EPI-NCSCs were microinjected into the acellular nerve allografts (ANAs) to bridge a 10-mm-long gap in the buccal branch of facial nerve in adult rats. Then, Celtrac1000-labeled EPI-NCSCs were detected by NIR-II fluorescence imaging system to visualize the behavior of the transplanted cells in vivo. Additionally, the effect of the transplanted EPI-NCSCs on repairing facial nerve defect was examined. KEY FINDINGS: Through 14 weeks of dynamic observation, the transplanted EPI-NCSCs survived in the ANAs in vivo after surgery. Meanwhile, the region of the NIR-II fluorescence signals was gradually limited to be consistent with the direction of the regenerative nerve segment. Furthermore, the results of functional and morphological analysis showed that the transplanted EPI-NCSCs could promote the recovery of facial paralysis and neural regeneration after injury. SIGNIFICANCE: Our research provides a novel way to track the transplanted cells in preclinical studies of cell therapy for facial paralysis, and demonstrates the therapeutic potential of EPI-NCSCs combined with ANAs in bridging rat facial nerve defects.

A novel perspective of calvarial development: the cranial morphogenesis and differentiation regulated by dura mater.

There are lasting concerns on calvarial development because cranium not only accommodates the growing brain, but also safeguards it from exogenous strikes. In the past decades, most studies attributed the dynamic expansion and remodeling of cranium to the proliferation of osteoprecursors in cranial primordium, and the proliferation of osteoprogenitors at the osteogenic front of cranial suture mesenchyme. Further investigations identified series genes expressed in suture mesenchymal cells as the markers of the progenitors, precursors and postnatal stem cells in cranium. However, similar to many other organs, it is suggested that the reciprocal interactions among different tissues also play essential roles in calvarial development. Actually, there are increasing evidence indicating that dura mater (DM) is indispensable for the calvarial morphogenesis and osteogenesis by secreting multiple growth factors, cytokines and extracellular matrix (ECM). Thus, in this review, we first briefly introduce the development of cranium, suture and DM, and then, comprehensively summarize the latest studies exploring the involvement of ECM in DM and cranium development. Eventually, we discussed the reciprocal interactions between calvarium and DM in calvarial development. Actually, our review provides a novel perspective for cranium development by integrating previous classical researches with a spotlight on the mutual interplay between the developing DM and cranium.

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.

The origin of ovarian Leydig cells: a possibly solved enigma?

Over the years, the origin of ovarian Leydig cells has been, and still is, a topic subject to deep debate. Seven years ago, we proposed that this origin resided in intraneural elements that came from a possible reservoir of neural crest cells, a reservoir that may be located in the ganglia of the celiac plexus. We believe we have found the evidence necessary to prove this hypothesis.

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.

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.

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.

Epidermal Neural Crest Stem Cell Conditioned Medium Enhances Spinal Cord Injury Recovery via PI3K/AKT-Mediated Neuronal Apoptosis Suppression.

This study aimed to assess the impact of conditioned medium from epidermal neural crest stem cells (EPI-NCSCs-CM) on functional recovery following spinal cord injury (SCI), while also exploring the involvement of the PI3K-AKT signaling pathway in regulating neuronal apoptosis. EPI-NCSCs were isolated from 10-day-old Sprague-Dawley rats and cultured for 48 h to obtain EPI-NCSC-CM. SHSY-5Y cells were subjected with H2O2 treatment to induce apoptosis. Cell viability and survival rates were evaluated using the CCK-8 assay and calcein-AM/PI staining. SCI contusion model was established in adult Sprague-Dawley rats to assess functional recovery, utilizing the Basso, Beattie and Bresnahan (BBB) scoring system, inclined test, and footprint observation. Neurological restoration after SCI was analyzed through electrophysiological recordings. Histological analysis included hematoxylin and eosin (H&E) staining and Nissl staining to evaluate tissue organization. Apoptosis and oxidative stress levels were assessed using TUNEL staining and ROS detection methods. Additionally, western blotting was performed to examine the expression of apoptotic markers and proteins related to the PI3K/AKT signaling pathway. EPI-NCSC-CM significantly facilitated functional and histological recovery in SCI rats by inhibiting neuronal apoptosis through modulation of the PI3K/AKT pathway. Administration of EPI-NCSCs-CM alleviated H2O2-induced neurotoxicity in SHSY-5Y cells in vitro. The use of LY294002, a PI3K inhibitor, underscored the crucial role of the PI3K/AKT signaling pathway in regulating neuronal apoptosis. This study contributes to the ongoing exploration of molecular pathways involved in spinal cord injury (SCI) repair, focusing on the therapeutic potential of EPI-NCSC-CM. The research findings indicate that EPI-NCSC-CM exerts a neuroprotective effect by suppressing neuronal apoptosis through activation of the PI3K/AKT pathway in SCI rats. These results highlight the promising role of EPI-NCSC-CM as a potential treatment strategy for SCI, emphasizing the significance of the PI3K/AKT pathway in mediating its beneficial effects.

Exploring the origins of neurodevelopmental proteasomopathies associated with cardiac malformations: are neural crest cells central to certain pathological mechanisms?

Neurodevelopmental proteasomopathies constitute a recently defined class of rare Mendelian disorders, arising from genomic alterations in proteasome-related genes. These alterations result in the dysfunction of proteasomes, which are multi-subunit protein complexes essential for maintaining cellular protein homeostasis. The clinical phenotype of these diseases manifests as a syndromic association involving impaired neural development and multisystem abnormalities, notably craniofacial anomalies and malformations of the cardiac outflow tract (OFT). These observations suggest that proteasome loss-of-function variants primarily affect specific embryonic cell types which serve as origins for both craniofacial structures and the conotruncal portion of the heart. In this hypothesis article, we propose that neural crest cells (NCCs), a highly multipotent cell population, which generates craniofacial skeleton, mesenchyme as well as the OFT of the heart, in addition to many other derivatives, would exhibit a distinctive vulnerability to protein homeostasis perturbations. Herein, we introduce the diverse cellular compensatory pathways activated in response to protein homeostasis disruption and explore their potential implications for NCC physiology. Altogether, the paper advocates for investigating proteasome biology within NCCs and their early cranial and cardiac derivatives, offering a rationale for future exploration and laying the initial groundwork for therapeutic considerations.

Genetic Identity of Neural Crest Cell Differentiation in Tissue and Organ Development.

The neural crest (NC), also known as the "fourth germ layer", is an embryonic structure with important contributions to multiple tissue and organ systems. Neural crest cells (NCCs) are subjected to epithelial to mesenchymal transition and migrate throughout the embryo until they reach their destinations, where they differentiate into discrete cell types. Specific gene expression enables this precise NCCs delamination and colonization potency in distinct and diverse locations therein. This review aims to summarize the current experimental evidence from multiple species into the NCCs specifier genes that drive this embryo body axes segmentation. Additionally, it attempts to filter further into the genetic background that produces these individual cell subpopulations. Understanding the multifaceted genetic makeup that shapes NC-related embryonic structures will offer valuable insights to researchers studying organogenesis and disease phenotypes arising from dysmorphogenesis.

Aplasia Cutis Congenita Pathomechanisms Reveal Key Regulators of Skin and Skin Appendage Morphogenesis.

Aplasia cutis congenita (ACC) manifests at birth as a defect of the scalp skin. New findings answer 2 longstanding questions: why ACC forms and why it affects mainly the midline scalp skin. Dominant-negative mutations in the genes KCTD1 or KCTD15 cause ACC owing to loss of function of KCTD1/KCTD15 complexes in cranial neural crest cells (NCCs), which normally form midline cranial suture mesenchymal cells that express keratinocyte growth factors. Loss of KCTD1/KCTD15 function in NCCs impairs the formation of normal midline cranial sutures and, consequently, the overlying skin, resulting in ACC. Moreover, KCTD1/KCTD15 complexes in keratinocytes regulate skin appendage morphogenesis.