RESUMEN
During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.
Asunto(s)
Embrión de Mamíferos , Cresta Neural , Movimiento Celular/fisiología , Sistema Nervioso PeriféricoRESUMEN
The content and activity of extracellular vesicles purified from cell culture media or bodily fluids have been studied extensively; however, the physiological relevance of exosomes within normal biological systems is poorly characterized, particularly during development. Although exosomes released by invasive metastatic cells alter migration of neighboring cells in culture, it is unclear whether cancer cells misappropriate exosomes released by healthy differentiated cells or reactivate dormant developmental programs that include exosome cell-cell communication. Using chick cranial neural fold cultures, we show that migratory neural crest cells, a developmentally critical cell type and model for metastasis, release and deposit CD63-positive 30-100â nm particles into the extracellular environment. Neural crest cells contain ceramide-rich multivesicular bodies and produce larger vesicles positive for migrasome markers as well. We conclude that neural crest cells produce extracellular vesicles including exosomes and migrasomes. When Rab27a plasma membrane docking is inhibited, neural crest cells become less polarized and rounded, leading to a loss of directional migration and reduced speed. These results indicate that neural crest cell exosome release is critical for migration.
Asunto(s)
Exosomas , Vesículas Extracelulares , Movimiento Celular , Exosomas/metabolismo , Cresta Neural/fisiologíaRESUMEN
SETD2 is a histone H3 lysine 36 (H3K36) tri-methylase that is upregulated in response to neural crest induction. Because the H3K36 di-methylase NSD3 and cytoplasmic non-histone protein methylation are necessary for neural crest development, we investigated the expression and requirement for SETD2 in the neural crest. SetD2 is expressed throughout the chick blastoderm beginning at gastrulation. Subsequently, SetD2 mRNA becomes restricted to the neural plate, where it is strongly and dynamically expressed as neural tissue is regionalized and cell fate decisions are made. This includes expression in premigratory neural crest cells, which is downregulated prior to migration. Likely due to the early onset of its expression, SETD2 morpholino knockdown does not significantly alter premigratory Sox10 expression or neural crest migration; however, both are disrupted by a methyltransferase mutant SETD2 construct. These results suggest that SETD2 activity is essential for early neural crest development, further demonstrating that lysine methylation is an important mechanism regulating the neural crest.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Cresta Neural/crecimiento & desarrollo , Neurogénesis/genética , Factores de Transcripción SOXE/genética , Animales , Movimiento Celular/genética , Embrión de Pollo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/genética , Morfolinos/genética , Cresta Neural/metabolismo , Procesamiento Proteico-Postraduccional/genética , ARN Mensajero/genéticaRESUMEN
The lysine methyltransferase NSD3 is required for the expression of key neural crest transcription factors and the migration of neural crest cells. Nevertheless, a complete view of the genes dependent upon NSD3 for expression and the developmental processes impacted by NSD3 in the neural crest was lacking. We used RNA sequencing (RNA-seq) to profile transcripts differentially expressed after NSD3 knockdown in chick premigratory neural crest cells, identifying 674 genes. Gene Ontology and gene set enrichment analyses further support a requirement for NSD3 during neural crest development and show that NSD3 knockdown also upregulates ribosome biogenesis. To validate our results, we selected three genes not previously associated with neural crest development, Astrotactin 1 (Astn1), Dispatched 3 (Disp3), and Tropomyosin 1 (Tpm1). Using whole mount in situ hybridization, we show that premigratory neural crest cells express these genes and that NSD3 knockdown downregulates (Astn1 and Disp3) and upregulates (Tpm1) their expression, consistent with RNA-seq results. Altogether, this study identifies novel putative regulators of neural crest development and provides insight into the transcriptional consequences of NSD3 in the neural crest, with implications for cancer.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cresta Neural/fisiología , Animales , Embrión de Pollo , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , N-Metiltransferasa de Histona-Lisina/genética , Hibridación in Situ/métodos , Cresta Neural/embriología , Cresta Neural/metabolismo , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/metabolismoRESUMEN
The ability to combine embryological manipulations with gene function analysis in an amniote embryo makes the chick a valuable system for the vertebrate developmental biologist. This chapter describes methods for those unfamiliar with the chick system wishing to initiate experiments in their lab. After outlining methods to prepare chick embryos, protocols are provided for introducing beads or cells expressing secreted factors, and for culturing tissue explants as a means of assessing development in vitro. Approaches to achieve gain of function and loss of function (morpholino oligonucleotides) in chick are outlined, and methods for introducing these reagents by electroporation are detailed.
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Desarrollo Embrionario/genética , Ingeniería Genética , Animales , Embrión de Pollo , Electroporación , Técnicas de Cultivo de Embriones , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Genes Reporteros , Transducción de Señal , TransgenesRESUMEN
The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.
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ADN (Citosina-5-)-Metiltransferasas/genética , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/enzimología , Neurogénesis/genética , Animales , Ganglios Basales/embriología , Ganglios Basales/enzimología , Región Branquial/embriología , Región Branquial/enzimología , Diferenciación Celular , Movimiento Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Embrión de Mamíferos , Corazón/embriología , Integrasas/genética , Integrasas/metabolismo , Mesodermo/embriología , Mesodermo/enzimología , Ratones , Ratones Transgénicos , Mutación , Cresta Neural/embriología , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Cráneo/embriología , Cráneo/enzimología , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , ADN Metiltransferasa 3BRESUMEN
Although a network of transcription factors that specifies neural crest identity in the ectoderm has been defined, expression of neural crest transcription factors does not guarantee eventual migration as a neural crest cell. While much work has gone into determining regulatory relationships within the transcription factor network, the ability of protein modifications like phosphorylation to modulate the function of neural crest regulatory factors and determine when and where they are active also has crucial implications. Paladin, which was previously classified as a phosphatase based on sequence similarity, is expressed in chick neural crest precursors and is maintained throughout their epithelial to mesenchymal transition and migration. Loss of Paladin delays the expression of transcription factors Snail2 and Sox10 in premigratory neural crest cells, but does not affect accumulation of FoxD3, Cad6B or RhoB, indicating that Paladin differentially modulates the expression of genes previously thought to be coregulated within the neural crest gene regulatory network. Both gain and loss of Paladin function result in disrupted neural crest migration, reinforcing the importance of precisely regulated phosphorylation for neural crest migration. Mutation of critical, catalytic cysteine residues within Paladin's predicted phosphatase active site motifs did not abolish the function of Paladin in the neural crest. Collectively, these data indicate that Paladin is an antiphosphatase that modulates the activity of specific neural crest regulatory factors during neural crest development. Our work identifies a novel regulator of phosphorylation status that provides an additional layer of regulation in the neural crest.
Asunto(s)
Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Proteínas Tirosina Fosfatasas/genética , Secuencias de Aminoácidos , Animales , Dominio Catalítico , Diferenciación Celular , Embrión de Pollo , Cresta Neural/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Neural crest cells, the migratory precursors of numerous cell types including the vertebrate peripheral nervous system, arise in the dorsal neural tube and follow prescribed routes into the embryonic periphery. While the timing and location of neural crest migratory pathways has been well documented in the trunk, a comprehensive collection of signals that guides neural crest migration along these paths has only recently been established. In this review, we outline the molecular cascade of events during trunk neural crest development. After describing the sequential routes taken by trunk neural crest cells, we consider the guidance cues that pattern these neural crest trajectories. We pay particular attention to segmental neural crest development and the steps and signals that generate a metameric peripheral nervous system, attempting to reconcile conflicting observations in chick and mouse. Finally, we compare cranial and trunk neural crest development in order to highlight common themes.
Asunto(s)
Cresta Neural/embriología , Cresta Neural/fisiología , Animales , Ratones , Neurogénesis , Neuronas/fisiología , Transducción de Señal/fisiología , VertebradosRESUMEN
The segmented trunk peripheral nervous system is generated by ventrally migrating neural crest cells that exclusively invade the anterior sclerotome and differentiate into metameric dorsal root and sympathetic ganglia. Meanwhile, ventral spinal motor axons also project through the somites in a segmental fashion. How peripheral nervous system segmentation is generated is unknown. We previously showed that neuropilin 2 (Nrp2)/semaphorin 3F (Sema3F) signaling is required for segmental neural crest migration, but not for metameric dorsal root gangliogenesis. We now expand these results to show that Nrp2 patterns initial motor axon outgrowth as well. Later, Nrp1/Sema3A signaling is essential for segmental dorsal root gangliogenesis and motor axonal fasciculation into ventral roots. Strikingly, Nrp/Sema signaling is not required for sympathetic ganglia segmentation. These data show that Nrp2 and Nrp1 work together to produce segmentation of sensory and motor nerves, and that dorsal peripheral nervous system metamerism is generated in a stepwise, Nrp-dependent process.
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Neuronas Motoras/fisiología , Neuropilina-1/fisiología , Neuropilina-2/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Axones/fisiología , Movimiento Celular/fisiología , Ganglios Espinales/citología , Ganglios Espinales/embriología , Ganglios Espinales/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Neuronas Motoras/citología , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/fisiología , Neurogénesis/fisiología , Semaforina-3A/metabolismo , Células Receptoras Sensoriales/citologíaRESUMEN
Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.
Asunto(s)
Adhesión Celular/fisiología , Proteínas de Microfilamentos/fisiología , Placa Neural/fisiología , Proteínas de Xenopus/fisiología , Xenopus/embriología , Animales , Comunicación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Células Epiteliales/citología , Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Proteínas de Microfilamentos/genética , Morfogénesis , Placa Neural/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Xenopus/genéticaRESUMEN
BACKGROUND: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. RESULTS: Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. CONCLUSION: Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.