RESUMEN
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.
Asunto(s)
Comunicación Celular , Vesículas Extracelulares , MicroARNs , Cresta Neural , Ganglio del Trigémino , Cresta Neural/metabolismo , Cresta Neural/embriología , Cresta Neural/citología , Animales , MicroARNs/metabolismo , MicroARNs/genética , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/embriología , Ganglio del Trigémino/citología , Vesículas Extracelulares/metabolismo , Embrión de Pollo , Comunicación Celular/genética , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico-pathologically distinct subtypes in sun-exposed and non-sun-exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue-resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment-producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans-differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell-like properties such as pseudo-epithelial-to-mesenchymal (EMT-like) transition and expression of stem cell-related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans-differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance.
Asunto(s)
Células Madre Pluripotentes Inducidas , Melanoma , Neoplasias Cutáneas , Animales , Ratones , Humanos , Melanoma/patología , Neoplasias Cutáneas/patología , Plasticidad de la Célula , Melanocitos/metabolismo , Diferenciación Celular , Resistencia a Medicamentos , Células Madre Pluripotentes Inducidas/metabolismo , Cresta Neural/metabolismoRESUMEN
The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Cresta Neural/citología , Organogénesis , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Diferenciación Celular , Anomalías Craneofaciales/etiología , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Cresta Neural/metabolismo , Trastornos de la Pigmentación/etiología , Trastornos de la Pigmentación/metabolismo , Trastornos de la Pigmentación/patología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
miR-203 is a tumor-suppressor microRNA with known functions in cancer metastasis. Here, we explore its normal developmental role in the context of neural crest development. During the epithelial-to-mesenchymal transition of neural crest cells to emigrate from the neural tube, miR-203 displays a reciprocal expression pattern with key regulators of neural crest delamination, Phf12 and Snail2, and interacts with their 3'UTRs. We show that ectopic maintenance of miR-203 inhibits neural crest migration in chick, whereas its functional inhibition using a 'sponge' vector or morpholinos promotes premature neural crest delamination. Bisulfite sequencing further shows that epigenetic repression of miR-203 is mediated by the de novo DNA methyltransferase DNMT3B, the recruitment of which to regulatory regions on the miR-203 locus is directed by SNAIL2 in a negative-feedback loop. These findings reveal an important role for miR-203 in an epigenetic-microRNA regulatory network that influences the timing of neural crest delamination.
Asunto(s)
Cresta Neural/citología , Cresta Neural/metabolismo , Factores de Transcripción/metabolismo , Animales , Embrión de Pollo , Epigenómica , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Factores de Transcripción/genéticaRESUMEN
Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.
Asunto(s)
Movimiento Celular , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Cresta Neural/citología , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Adhesiones Focales/efectos de los fármacos , Modelos Biológicos , Morfolinos/farmacología , Cresta Neural/metabolismo , Fenotipo , Transducción de Señal/efectos de los fármacos , Xenopus/embriología , Familia-src Quinasas/metabolismoRESUMEN
Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.
Asunto(s)
Deficiencia de Ácido Fólico/fisiopatología , Cresta Neural/metabolismo , Factores de Transcripción SOXB1/fisiología , Animales , Embrión de Pollo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/genética , Represión Epigenética/genética , Represión Epigenética/fisiología , Epigenómica , Receptor 1 de Folato , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/fisiopatologíaRESUMEN
The neural crest (NC) is one of the most fascinating structures during embryonic development. Unique to vertebrate embryos, these cells give rise to important components of the craniofacial skeleton, such as the jaws and skull, as well as melanocytes and ganglia of the peripheral nervous system. Worldwide, several groups have been studying NC development and specifically in the Latin America (LA) they have been growing in numbers since the 1990s. It is important for the world to recognize the contributions of LA researchers on the knowledge of NC development, as it can stimulate networking and improvement in the field. We developed a database of LA publications on NC development using ORCID and PUBMED as search engines. We thoroughly describe all of the contributions from LA, collected in five major topics on NC development mechanisms: i) induction and specification; ii) migration; iii) differentiation; iv) adult NC; and, v) neurocristopathies. Further analysis was done to correlate each LA country with topics and animal models, and to access collaboration between LA countries. We observed that some LA countries have made important contributions to the comprehension of NC development. Interestingly, some LA countries have a topic and an animal model as their strength; in addition, collaboration between LA countries is almost inexistent. This review will help LA NC research to be acknowledged, and to facilitate networking between students and researchers worldwide.
Asunto(s)
Cresta Neural/citología , Cresta Neural/metabolismo , Animales , Tipificación del Cuerpo , Diferenciación Celular , Movimiento Celular , Humanos , América Latina , Modelos BiológicosRESUMEN
The neural crest (NC) is a transient embryonic cell population that migrates extensively during development. Ric-8A, a guanine nucleotide exchange factor (GEF) for different Gα subunits regulates cranial NC (CNC) cell migration in Xenopus through a mechanism that still remains to be elucidated. To properly migrate, CNC cells establish an axis of polarization and undergo morphological changes to generate protrusions at the leading edge and retraction of the cell rear. Here, we aim to study the role of Ric-8A in cell polarity during CNC cell migration by examining whether its signaling affects the localization of GTPase activity in Xenopus CNC using GTPase-based probes in live cells and aPKC and Par3 as polarity markers. We show that the levels of Ric-8A are critical during migration and affect the localization of polarity markers and the subcellular localization of GTPase activity, suggesting that Ric-8A, probably through heterotrimeric G-protein signaling, regulates cell polarity during CNC migration.
Asunto(s)
Movimiento Celular/fisiología , Polaridad Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cresta Neural/metabolismo , Cresta Neural/fisiología , Animales , Transducción de Señal/fisiología , XenopusRESUMEN
Human dental tissues are sources of neural crest origin multipotent stem cells whose regenerative potential is a focus of extensive studies. Rational programming of clinical applications requires a more detailed knowledge of the characters inherited from neural crest. Investigation of neural crest cells generated from human pluripotent stem cells provided opportunity for their comparison with the postnatal dental cells. The purpose of this study was to investigate the role of the culture conditions in the expression by dental cells of neural crest characters. The results of the study demonstrate that specific neural crest cells requirements, serum-free, active WNT signaling and inactive SMAD 2/3, are needed for the activity of the neural crest characters in dental cells. Specifically, the decreasing concentration of fetal bovine serum (FBS) from regularly used for dental cells 10% to 2% and below, or using serum-free medium, led to emergence of a subset of epithelial-like cells expressing the two key neural crest markers, p75 and HNK-1. Further, the serum-free medium supplemented with neural crest signaling requirements (WNT inducer BIO and TGF-ß inhibitor REPSOX), induced epithelial-like phenotype, upregulated the p75, Sox10 and E-Cadherin and downregulated the mesenchymal genes (SNAIL1, ZEB1, TWIST). An expansion medium containing 2% FBS allowed to obtain an epithelial/mesenchymal SHED population showing high proliferation, clonogenic, multi-lineage differentiation capacities. Future experiments will be required to determine the effects of these features on regenerative potential of this novel SHED population.
Asunto(s)
Diferenciación Celular/genética , Células Madre Mesenquimatosas/citología , Cresta Neural/citología , Diente Primario/citología , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular/genética , Medio de Cultivo Libre de Suero , Pulpa Dental/citología , Regulación del Desarrollo de la Expresión Génica , Humanos , Cresta Neural/metabolismo , Células Madre Pluripotentes , Transducción de Señal/genética , Diente Primario/metabolismoRESUMEN
Collective cell migration is essential in many fundamental aspects of normal development, like morphogenesis, organ formation, wound healing, and immune responses, as well as in the etiology of severe pathologies, like cancer metastasis. In spite of the huge amount of data accumulated on cell migration, such a complex process involves many molecular actors, some of which still remain to be functionally characterized. One of these signals is the heterotrimeric G-protein pathway that has been studied mainly in gastrulation movements. Recently we have reported that Ric-8A, a GEF for Gα proteins, plays an important role in neural crest migration in Xenopus development. Xenopus neural crest cells, a highly migratory embryonic cell population induced at the border of the neural plate that migrates extensively in order to differentiate in other tissues during development, have become a good model to understand the dynamics that regulate cell migration. In this review, we aim to provide sufficient evidence supporting how useful Xenopus model with its different tools, such as explants and transplants, paired with improved in vivo imaging techniques, will allow us to tackle the multiple signaling mechanisms involved in neural crest cell migration.
Asunto(s)
Movimiento Celular/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Morfogénesis/genética , Xenopus laevis/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Placa Neural/crecimiento & desarrollo , Placa Neural/metabolismo , Transducción de Señal/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development.
Asunto(s)
Diferenciación Celular/genética , Desarrollo Embrionario/genética , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Xenopus/genética , Secuencia de Aminoácidos/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al GTP Heterotriméricas/genética , Hibridación in Situ , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Tubo Neural/crecimiento & desarrollo , Tubo Neural/metabolismo , Transducción de Señal , Somitos/crecimiento & desarrollo , Somitos/metabolismo , Xenopus/crecimiento & desarrolloRESUMEN
The vestigial gene (vg) was first characterized in Drosophila and several homologues were identified in vertebrates and called vestigial like 1-4 (vgll1-4). Vgll proteins interact with the transcription factors TEF-1 and MEF-2 through a conserved region called TONDU (TDU). Vgll4s are characterized by two tandem TDU domains which differentiate them from other members of the vestigial family. In Xenopus two genes were identified as vgll4. Our bioinformatic analysis demonstrated that these two genes are paralogues and must be named differently. We designated them as vgll4 and vgll4l. In situ hybridization analysis revealed that the expression of these two genes is rather different. At gastrula stage, both were expressed in the animal pole. However, at neurula stage, vgll4 was mainly expressed in the neural plate and neural folds, while vgll4l prevailed in the neural folds and epidermis. From the advanced neurula stage onward, expression of both genes was strongly enhanced in neural tissues, anterior neural plate, migrating neural crest, optic and otic vesicles. Nevertheless, there were some differences: vgll4 presented somite expression and vgll4l was localized at the skin and notochord. Our results demonstrate that Xenopus has two orthologues of the vgll4 gene, vgll4 and vgll4l with differential expression in Xenopus embryos and they may well have different roles during development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Animales , Embrión no Mamífero/metabolismo , Gástrula/embriología , Gástrula/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Somitos/embriología , Somitos/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF2) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF2, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins ßIII-tubulin and nestin, as well as upregulation of the pan neuronal marker ßIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF-FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF-FGF2 in neuronal differentiation protocols.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Epidermis/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Cresta Neural/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular , Regulación hacia Abajo/fisiología , Células Epiteliales/metabolismo , Proteína GAP-43/metabolismo , Folículo Piloso/metabolismo , Ratones , Células Madre Multipotentes/metabolismo , Miocitos del Músculo Liso/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.
Asunto(s)
Quimiotaxis , Mesencéfalo/citología , Cresta Neural/citología , Neurotrofina 3/metabolismo , Animales , Proliferación Celular , Embrión de Pollo , Pollos , Cresta Neural/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.
Asunto(s)
Movimiento Celular , Cresta Neural/citología , Imagen de Lapso de Tiempo/métodos , Grabación de Cinta de Video/métodos , Algoritmos , Animales , Animales Modificados Genéticamente , Depresores del Sistema Nervioso Central/farmacología , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Etanol/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Cresta Neural/embriología , Cresta Neural/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The neural crest (NC) is a transient embryonic structure induced at the border of the neural plate. NC cells extensively migrate towards diverse regions of the embryo, where they differentiate into various derivatives, including most of the craniofacial skeleton and the peripheral nervous system. The Ric-8A protein acts as a guanine nucleotide exchange factor for several Gα subunits, and thus behaves as an activator of signaling pathways mediated by heterotrimeric G proteins. Using in vivo transplantation assays, we demonstrate that Ric-8A levels are critical for the migration of cranial NC cells and their subsequent differentiation into craniofacial cartilage during Xenopus development. NC cells explanted from Ric-8A morphant embryos are unable to migrate directionally towards a source of the Sdf1 peptide, a potent chemoattractant for NC cells. Consistently, Ric-8A knock-down showed anomalous radial migratory behavior, displaying a strong reduction in cell spreading and focal adhesion formation. We further show that during in vivo and in vitro neural crest migration, Ric-8A localizes to the cell membrane, in agreement with its role as a G protein activator. We propose that Ric-8A plays essential roles during the migration of cranial NC cells, possibly by regulating cell adhesion and spreading.
Asunto(s)
Movimiento Celular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cresta Neural/citología , Proteínas de Xenopus/metabolismo , Animales , Adhesión Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Hibridación in Situ , Microscopía Confocal , Cresta Neural/embriología , Cresta Neural/metabolismo , Transducción de Señal/genética , Cráneo/embriología , Cráneo/inervación , Imagen de Lapso de Tiempo/métodos , Xenopus/embriología , Proteínas de Xenopus/genética , Xenopus laevis/embriologíaRESUMEN
MARCKS is a ubiquitous actin-binding protein, with special functions in the development of the central nervous system. We have previously described a neuronal-specific isoform, phosphorylated at serine 25 (S25p-MARCKS), which is present very early during neuronal differentiation in the chick retina. However, very little is known about MARCKS expression or functions in the peripheral nervous system (PNS). In the present work, we analyzed migrating PNS precursor cells in the chick embryo, particularly those originating from the neural crest, and found that they all express a high amount of MARCKS and that a subpopulation of them also contained S25p-MARCKS from early developmental stages. MARCKS protein was also found in dorsal root and trigeminal ganglia during embryo development. Not only is the protein present in these structures but it is also phosphorylated in differentiating neurons with a maximal signal on the ganglion periphery, where neurogenesis is occurring. In conclusion, MARCKS is present and phosphorylated at early stages during the differentiation of PNS cells and precursors, indicating that it might also be important for the differentiation of these tissues.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Cresta Neural/citología , Cresta Neural/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , Diferenciación Celular , Movimiento Celular , Embrión de Pollo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Cresta Neural/embriología , Fosforilación , Serina/metabolismoRESUMEN
The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. The derivatives of the NC at trunk level include neurons and glial cells of the peripheral nervous system. Despite the well-known influence of aflatoxins on the development of cancer, the issue of whether they also influence NC cells has not been yet addressed. In the present work, we have investigated the effects of aflatoxin B(1) on quail NC cells and the concomitant effects of the flavonoid hesperidin associated with this mycotoxin. We show for the first time that aflatoxin B(1) decreases the viability and the total number of glial and neuronal cells/field, although their proportions in relation to the total number of cells were not altered. Therefore, aflatoxin has no effect on NC differentiation. However, this compound was able to reduce NC proliferation and NC survival. Furthermore, the co-administration of hesperidin, a well-known polyphenolic protector of cell death, partially prevented the effect of aflatoxin B(1) . Taken together, our results demonstrate that aflatoxin B(1) is toxic to NC cells, an effect partially prevented by the flavonoid hesperidin. This study may contribute to the understanding of the effects of these compounds during early embryonic development and offer potentially more assertive diets and treatments for pregnant animals.
Asunto(s)
Aflatoxina B1/toxicidad , Flavonoides/farmacología , Hesperidina/farmacología , Cresta Neural/metabolismo , Venenos/toxicidad , Animales , Apoptosis , Muerte Celular , Células Cultivadas , Cresta Neural/efectos de los fármacos , Codorniz/embriologíaRESUMEN
Traditionally, the cartilaginous viscerocranium of vertebrates is considered as neural crest (NC)-derived. Morphological work carried out on amphibian embryos in the first half of the XX century suggested potentially mesodermal origin for some hyobranchial elements. Since then, the embryonic sources of the hyobranchial apparatus in amphibians has not been investigated due to lack of an appropriate long-term labelling system. We performed homotopic transplantations of neural folds along with the majority of cells of the presumptive NC, and/or fragments of the head lateral plate mesoderm (LPM) from transgenic GFP+ into white embryos. In these experiments, the NC-derived GFP+ cells contributed to all hyobranchial elements, except for basibranchial 2, whereas the grafting of GFP+ head mesoderm led to a reverse labelling result. The grafting of only the most ventral part of the head LPM resulted in marking of the basibranchial 2 and the heart myocardium, implying their origin from a common mesodermal region. This is the first evidence of contribution of LPM of the head to cranial elements in any vertebrate. If compared to fish, birds, and mammals, in which all branchial skeletal elements are NC-derived, the axolotl (probably this is true for all amphibians) demonstrates an evolutionary deviation, in which the head LPM replaces NC cells in a hyobranchial element. This implies that cells of different embryonic origin may have the same developmental program, leading to the formation of identical (homologous) elements of the skeleton.
Asunto(s)
Ambystoma mexicanum/embriología , Ambystoma mexicanum/fisiología , Animales , Animales Modificados Genéticamente , Aves , Huesos/embriología , Cartílago/embriología , Peces , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Cabeza/embriología , Corazón/embriología , Mesodermo/embriología , Mesodermo/metabolismo , Mesodermo/fisiología , Miocardio/patología , Cresta Neural/metabolismo , Cresta Neural/patologíaRESUMEN
Neural crest cells form within the neural tube and then undergo an epithelial to mesenchymal transition (EMT) to initiate migration to distant locations. The transcriptional repressor Snail2 has been implicated in neural crest EMT via an as of yet unknown mechanism. We report that the adaptor protein PHD12 is highly expressed before neural crest EMT. At cranial levels, loss of PHD12 phenocopies Snail2 knockdown, preventing transcriptional shutdown of the adhesion molecule Cad6b (Cadherin6b), thereby inhibiting neural crest emigration. Although not directly binding to each other, PHD12 and Snail2 both directly interact with Sin3A in vivo, which in turn complexes with histone deacetylase (HDAC). Chromatin immunoprecipitation revealed that PHD12 is recruited to the Cad6b promoter during neural crest EMT. Consistent with this, lysines on histone 3 at the Cad6b promoter are hyperacetylated before neural crest emigration, correlating with active transcription, but deacetylated during EMT, reflecting the repressive state. Knockdown of either PHD12 or Snail2 prevents Cad6b promoter deacetylation. Collectively, the results show that PHD12 interacts directly with Sin3A/HDAC, which in turn interacts with Snail2, forming a complex at the Cad6b promoter and thus revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.