RESUMEN
Neural crest cells originate in the dorsal neural tube but subsequently undergo an epithelial-to-mesenchymal transition (EMT), delaminate, and migrate to diverse locations in the embryo where they contribute to a variety of derivatives. Cadherins are a family of cell-cell adhesion molecules expressed in a broad range of embryonic tissues, including the neural tube. In particular, cadherin6B (Cad6B) is expressed in the dorsal neural tube prior to neural crest emigration but is then repressed by the transcription factor Snail2, expressed by premigratory and early migrating cranial neural crest cells. To examine the role of Cad6B during neural crest EMT, we have perturbed Cad6B protein levels in the cranial neural crest-forming region and have examined subsequent effects on emigration and migration. The results show that knock-down of Cad6B leads to premature neural crest cell emigration, whereas Cad6B overexpression disrupts migration. Our data reveal a novel role for Cad6B in controlling the proper timing of neural crest emigration and delamination from the neural tube of the avian embryo.
Asunto(s)
Proteínas Aviares/metabolismo , Cadherinas/metabolismo , Cresta Neural/embriología , Animales , Proteínas Aviares/genética , Cadherinas/genética , Diferenciación Celular , Embrión de Pollo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epitelio/embriología , Epitelio/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Mesodermo/embriología , Mesodermo/fisiología , Cresta Neural/citología , Cresta Neural/metabolismo , Factores de Transcripción SOXE , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The neurofibromatosis 2 (NF2) tumor suppressor protein merlin, or schwannomin, functions as a negative growth regulator such that inactivating mutations in Nf2 predispose humans to tumors. In addition, merlin has a critical role during embryonic development. Nf2-deficient mice die early during embryogenesis, with defects in gastrulation and extraembryonic tissues. To investigate the function of Nf2/merlin during embryonic development, we first identified the homologous Nf2 gene in chicken (cNf2) and examined the distribution of chicken merlin (c-merlin) during myogenesis. cNf2 encoded a full-length mRNA of 1,770 nucleotides and a protein of 589 residues. C-merlin shared high sequence homology and common protein motifs with vertebrate and Drosophila merlins. In addition, cNF2 functions as a negative growth regulator similar to human and Drosophila merlin in vitro. In vivo, c-merlin was expressed diffusely in the forming dermomyotome but down-regulated in migratory muscle precursors in the forelimb. As muscle formed in the limb, c-merlin expression was up-regulated. As an initial examination of c-merlin function during myogenesis, c-merlin was ectopically expressed in muscle precursors and the effects on muscle development were examined. We show that ectopic merlin expression reduces the proliferation of muscle precursors as well as their ability to migrate effectively in limb mesoderm. Collectively, these results demonstrate that c-merlin is developmentally regulated in migrating and differentiating myogenic cells, where it functions as a negative regulator of both muscle growth and motility.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Neurofibromina 2/biosíntesis , Neurofibromina 2/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Bromodesoxiuridina/farmacología , Diferenciación Celular , División Celular , Línea Celular , Movimiento Celular , Pollos , Colorantes/farmacología , ADN Complementario/metabolismo , Regulación hacia Abajo , Drosophila , Electroporación , Extremidades/embriología , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Músculos/citología , Músculos/embriología , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , Regulación hacia ArribaRESUMEN
The chick homologue of the helix-loop-helix gene Id3 was isolated, and its expression pattern was analyzed during early stages of chick development. Chick Id3 is dynamically expressed in the olfactory, lens, and otic placodes. It is also observed in the epiphysis, nephric primordium, stomodeum, dermomyotome, distal branchial arches, dorsolateral hindbrain, foregut endoderm, dorsal spinal cord, and somites.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Oído/embriología , Cristalino/embriología , Proteínas de Neoplasias , Bulbo Olfatorio/embriología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , ADN Complementario/metabolismo , Ectodermo/metabolismo , Hibridación in Situ , Proteínas Inhibidoras de la Diferenciación , Datos de Secuencia Molecular , Cresta Neural/embriología , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución TisularRESUMEN
We present the sequence and expression pattern of chick Id4 and compare its distribution to that of other vertebrate Id genes. At early stages, Id4 expression is discrete, with transcript transiently expressed in subsets of migrating neural crest cells, the dorsal myocardium, the segmental plate mesoderm, and the tail bud. Later, expression is also observed in the telencephalic vesicles and corneal epithelium. Of all the Id genes, Id4 exhibits the most restricted pattern in the developing nervous system, with little expression in the presumptive neural crest or placodes. Id4 appears in the neural tube much later than other Id genes. However, all four Id genes display overlapping patterns in the branchial arches and tail bud.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Neoplasias , Proteínas Represoras , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , ADN Complementario/metabolismo , Humanos , Hibridación in Situ , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Proteínas Inhibidoras de la Diferenciación , Datos de Secuencia Molecular , Sistema Nervioso/embriología , Cresta Neural/citología , Filogenia , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Cola (estructura animal)/embriología , Telencéfalo/citología , Factores de Tiempo , Distribución TisularRESUMEN
We isolated the chick orthologue of the Id1 helix-loop-helix gene and analyzed its expression pattern during early chick embryo development by whole-mount in situ hybridization. The Id1 expression pattern is dynamic and confined to discrete locations including the neural plate border, prospective olfactory placode, hindbrain, mesenchyme of distal branchial arches and adjacent to placodes, and the distal mesoderm of the limb buds.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas Represoras , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Embrión de Pollo , Clonación Molecular , ADN Complementario/metabolismo , Extremidades/embriología , Secuencias Hélice-Asa-Hélice , Hibridación in Situ , Proteína 1 Inhibidora de la Diferenciación , Mesodermo/metabolismo , Datos de Secuencia Molecular , Neuronas/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Técnicas del Sistema de Dos HíbridosRESUMEN
Lunatic fringe is a vertebrate homologue of Drosophila fringe, which plays an important role in modulating Notch signaling. This study examines the distribution of chick lunatic fringe at sites of neural crest formation and explores its possible function by ectopic expression. Shortly after neural tube closure, lunatic fringe is expressed in most of the neural tube, with the exception of the dorsal midline containing presumptive neural crest. Thus, there is a fringe/non-fringe border at the site of neural crest production. Expression of excess lunatic fringe in the cranial neural tube and neural crest by retrovirally mediated gene transfer resulted in a significant increase ( approximately 60%) in the percentage of cranial neural crest cells 1 day after infection. This effect was mediated by an increase in cell division as assayed by BrdU incorporation. Infected embryos had an up-regulation of Delta-1 in the dorsal neural tube and redistribution of Notch-1 to the lumen of the neural tube, confirming that excess fringe modulates Notch signaling. These findings point to a novel role for lunatic fringe in regulating cell division and/or production of neural crest cells by the neural tube.
Asunto(s)
División Celular/fisiología , Glicosiltransferasas , Cresta Neural/citología , Proteínas/fisiología , Receptores de Superficie Celular , Cráneo/embriología , Factores de Transcripción , Animales , Proteínas Aviares , Embrión de Pollo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Receptor Notch1 , Retroviridae/genética , Transducción de Señal , Cráneo/citologíaRESUMEN
In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine beta-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling.
Asunto(s)
Antígenos de Diferenciación , Proteínas Aviares , Dopamina beta-Hidroxilasa , Proteínas del Tejido Nervioso , Cresta Neural/embriología , Oxigenasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Movimiento Celular , Embrión de Pollo , Mapeo Cromosómico , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Cresta Neural/cirugía , Oxigenasas/genética , Rombencéfalo/embriología , Homología de Secuencia de Aminoácido , Somitos , Distribución TisularRESUMEN
Cranial placodes are focal regions of thickened ectoderm in the head of vertebrate embryos that give rise to a wide variety of cell types, including elements of the paired sense organs and neurons in cranial sensory ganglia. They are essential for the formation of much of the cranial sensory nervous system. Although relatively neglected today, interest in placodes has recently been reawakened with the isolation of molecular markers for different stages in their development. This has enabled a more finely tuned approach to the understanding of placode induction and development and in some cases has resulted in the isolation of inducing molecules for particular placodes. Both morphological and molecular data support the existence of a preplacodal domain within the cranial neural plate border region. Nonetheless, multiple tissues and molecules (where known) are involved in placode induction, and each individual placode is induced at different times by a different combination of these tissues, consistent with their diverse fates. Spatiotemporal changes in competence are also important in placode induction. Here, we have tried to provide a comprehensive review that synthesises the highlights of a century of classical experimental research, together with more modern evidence for the tissues and molecules involved in the induction of each placode.
Asunto(s)
Ectodermo/fisiología , Inducción Embrionaria , Cabeza/embriología , Animales , Región Branquial/fisiología , Oído Interno/embriología , Corazón/embriología , Humanos , Cristalino/embriología , Cresta Neural/fisiología , Hipófisis/embriología , Factores de Transcripción/fisiología , Ganglio del Trigémino/embriologíaRESUMEN
Neurogenesis in Xenopus neural ectoderm involves multiple gene families, including basic helix-loop-helix transcription factors, which initiate and control primary neurogenesis. Equally important, though less well understood, are the downstream effectors of the activity of these transcription factors. We have investigated the role of a candidate downstream effector, Noelin-1, during Xenopus development. Noelin-1 is a secreted glycoprotein that likely forms large multiunit complexes. In avians, overexpression of Noelin-1 causes prolonged and excessive neural crest migration. Our studies in Xenopus reveal that this gene, while highly conserved in sequence, has a divergent function in primary neurogenesis. Xenopus Noelin-1 is expressed mainly by postmitotic neurogenic tissues in the developing central and peripheral nervous systems, first appearing after neural tube closure. Its expression is upregulated in ectopic locations upon overexpression of the neurogenic genes X-ngnr-1 and XNeuroD. Noelin-1 expression in animal caps induces expression of neural markers XBrn-3d and XNeuroD, and co-expression of secreted Noelin-1 with noggin amplifies noggin-induced expression of XBrn-3d and XNeuroD. Furthermore, in animal caps neuralized by expression of noggin, co-expression of Noelin-1 causes expression of neuronal differentiation markers several stages before neurogenesis normally occurs in this tissue. Finally, only secreted forms of the protein can activate sensory marker expression, while all forms of the protein can induce early neurogenesis. This suggests that the cellular localization of Noelin-1 may be important to its function. Thus, Noelin-1 represents a novel secreted factor involved in neurogenesis.
Asunto(s)
Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/embriología , Xenopus laevis/embriología , Xenopus laevis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/fisiología , Femenino , Ganglios Sensoriales/embriología , Ganglios Sensoriales/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevis/genéticaAsunto(s)
Embriología , Desarrollo Embrionario y Fetal/fisiología , Transducción de Señal , Animales , HumanosRESUMEN
During neurulation, a region of central ectoderm becomes thickened to form the neural plate which then folds upon itself to generate the neural tube, from which all neurons and glia cells of the central nervous system arise. Neural crest cells form at the border of the neural plate, where it abuts the prospective epidermis. The neural crest is a transient population of cells that undergo an epithelial-mesenchymal transition, become highly migratory and subsequently differentiate into most of the peripheral nervous systems as well as numerous other derivatives. The origin of neural crest cells at the epidermal-neural plate border suggests that an interaction between these two tissues may be involved in neural crest formation. By experimentally juxtaposing prospective epidermis with naive neural plate, we previously showed that an inductive interaction between these tissues can generate neural crest cells. Here, we further characterize the nature of this inductive interaction by co-culturing isolated neural plate and prospective epidermis on opposing sides of polycarbonate filters with differing pore sizes. We find that neural crest cells are generated even when epidermis and neural plate are separated by filters that do not allow cell contact. These results suggest that the epidermal inducer is a diffusible, secreted molecule. We discuss the developmental potential of neural crest precursors and lineage decisions that effect their differentiation into numerous derivatives.
Asunto(s)
Ectodermo/fisiología , Cresta Neural/fisiología , Transducción de Señal/fisiología , Animales , Embrión de Pollo , Colágeno , Ectodermo/citología , Geles , Hibridación in Situ , Melanocitos/metabolismo , Cresta Neural/citología , Cresta Neural/embriología , Células Madre/fisiologíaRESUMEN
The markers Xslug, Xsnail, and Xtwist all are expressed in the presumptive neural folds and are thought to delineate the presumptive neural crest. However, their interrelationship and relative spatiotemporal distributions are not well understood. Here, we present a detailed in situ hybridization analysis of the relative patterns of expression of these transcription factors from gastrulation through neurulation and post-neural crest migration. The three genes mark the prospective neural crest and roof plate, coming on sequentially, with Xsnail preceding Xslug preceding Xtwist. By combining gene expression analysis with a fate map of the same region using DiI labeling, we determined the correspondence between early and late domains of gene expression. At the beginning of gastrulation, Xsnail is present in a unique domain of expression in a lateral region of the embryo in both superficial and deep layers of the ectoderm, as are Xslug and Xtwist. During gastrulation and neurulation, the superficial layer moves faster toward the dorsal midline than the deep layer, producing a relative shift in these cell populations. By early neurula stage, the Xsnail domain is split into a medial domain in the superficial ectoderm (fated to become the roof plate) and a lateral domain in the deep layer of the ectoderm (fated to become neural crest). Xsnail is down-regulated in the most anterior neural plate and up-regulated in the posterior neural plate. Our results show that changes in the expression of Xsnail, Xslug, and Xtwist are a consequence of active cell movement in some regions coupled with dynamic changes in gene expression in other regions.
Asunto(s)
Biomarcadores , Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Animales , Hibridación in Situ , Cresta Neural/metabolismo , Xenopus/embriologíaRESUMEN
The inner ear is induced from cranial ectoderm adjacent to the hindbrain. Despite almost a century of study, the molecular mechanisms of inner ear induction remain obscure. We have identified four genes expressed very early in the anlage of the inner ear, the otic placode. Pax-2, Sox-3, BMP-7 and Notch are all expressed in placodal ectoderm from the 4-5 somite stage (ss) onwards, well before the otic placode becomes morphologically visible at the 12-14ss. We have used these four molecular markers to show that cranial ectoderm becomes specified to form the otic placode at the 4-6ss, and that this ectoderm is committed to a placodal fate by the 10ss. We also demonstrate that much of the embryonic ectoderm is competent to generate an otic placode if taken at a sufficiently early age. We have mapped the location of otic placode-inducing activity along the rostrocaudal axis of the embryo, and have determined that this activity persists at least until the 10ss. Use of the four molecular otic placode markers suggests that induction of the otic placode in birds occurs earlier than previously thought, and proceeds in a series of steps that are independently regulated.
Asunto(s)
Oído Interno/embriología , Inducción Embrionaria/fisiología , Factor de Crecimiento Transformador beta , Animales , Biomarcadores , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular , Embrión de Pollo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oído Interno/metabolismo , Ectodermo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Homeodominio/genética , Proteínas de la Membrana/genética , Factor de Transcripción PAX2 , Codorniz , Conejos , Receptores Notch , Factores de Transcripción SOXB1 , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Neural crest cells can be induced by an interaction between neural plate and ectoderm. To clarify the timing and nature of these inductive interactions, we have examined the time of competence of the neural plate to become neural crest as well as the time of neural fold specification. The neural plate is competent to respond to inductive interactions with the nonneural ectoderm for a limited period, rapidly losing its responsive ability after stage 10. In contrast, nonneural ectoderm from numerous stages retains the ability to induce neural crest cells from competent neural plate. When neural folds are explanted to test their ability to produce neural crest without further tissue interactions, we find that folds derived from all rostrocaudal levels of the open neural plate are already specified to express the neural crest marker Slug. However, additional signals may be required for maintenance of Slug expression, since the transcript is later down-regulated in vitro in the absence of tissue interactions. Taken together, these results suggest that there are multiple stages of neural crest induction. The earliest induction must have occurred by the end of gastrulation, since the newly formed neural fold population is already specified to form neural crest. However, isolated neural folds eventually down-regulate Slug, suggesting a second phase that maintains neural crest formation. Thus, induction of the neural crest may involve multiple and sustained tissue interactions.
Asunto(s)
Inducción Embrionaria/genética , Cresta Neural/citología , Cresta Neural/embriología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Trasplante de Tejido Encefálico , Células Cultivadas , Embrión de Pollo , Colágeno , Regulación hacia Abajo , Ectodermo/citología , Ectodermo/metabolismo , Gástrula/citología , Gástrula/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas In Vitro , Mesodermo/citología , Mesodermo/metabolismo , Cresta Neural/metabolismo , Codorniz , Factores de Transcripción de la Familia Snail , Factores de Tiempo , Factores de Transcripción/biosíntesisRESUMEN
The trigeminal and epibranchial placodes of vertebrate embryos form different types of sensory neurons. The trigeminal placodes form cutaneous sensory neurons that innervate the face and jaws, while the epibranchial placodes (geniculate, petrosal and nodose) form visceral sensory neurons that innervate taste buds and visceral organs. In the chick embryo, the ophthalmic trigeminal (opV) placode expresses the paired homeodomain transcription factor Pax3 from very early stages, while the epibranchial placodes express Pax2. Here, we show that Pax3 expression in explanted opV placode ectoderm correlates at the single cell level with neuronal specification and with commitment to an opV fate. When opV (trigeminal) ectoderm is grafted in place of the nodose (epibranchial) placode, Pax3-expressing cells form Pax3-positive neurons on the same schedule as in the opV placode. In contrast, Pax3-negative cells in the grafted ectoderm are induced to express the epibranchial placode marker Pax2 and form neurons in the nodose ganglion that express the epibranchial neuron marker Phox2a on the same schedule as host nodose neurons. They also project neurites along central and peripheral nodose neurite pathways and survive until well after the main period of cell death in the nodose ganglion. The older the opV ectoderm is at the time of grafting, the more Pax3-positive cells it contains and the more committed it is to an opV fate. Our results suggest that, within the neurogenic placodes, there does not appear to be a two-step induction of 'generic' neurons followed by specification of the neuron to a particular fate. Instead, there seems to be a one-step induction in which neuronal subtype identity is coupled to neuronal differentiation.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Ectodermo/fisiología , Neuronas/citología , Ganglio Nudoso/embriología , Animales , Embrión de Pollo , Técnicas de Cultivo , Proteínas de Unión al ADN/metabolismo , Ectodermo/citología , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso , Neuronas/fisiología , Ganglio Nudoso/citología , Ganglio Nudoso/fisiología , Factor de Transcripción PAX2 , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Codorniz/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r )of 360-530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to 'attract' moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve 'haptotactic-like' motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/fisiología , Proteínas de la Matriz Extracelular , Ácido Hialurónico/metabolismo , Cresta Neural/citología , Proteoglicanos/fisiología , Agrecanos , Animales , Anticuerpos/química , Western Blotting , Bovinos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos , Fibronectinas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Membranas Intracelulares , Lectinas Tipo C , Microscopía Electrónica , Cresta Neural/embriología , Isoformas de Proteínas , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular , Células Tumorales Cultivadas , VersicanosRESUMEN
Previous analyses of single neural crest cell trajectories have suggested important roles for interactions between neural crest cells and the environment, and amongst neural crest cells. To test the relative contribution of intrinsic versus extrinsic information in guiding cells to their appropriate sites, we ablated subpopulations of premigratory chick hindbrain neural crest and followed the remaining neural crest cells over time using a new in ovo imaging technique. Neural crest cell migratory behaviors are dramatically different in ablated compared with unoperated embryos. Deviations from normal migration appear either shortly after cells emerge from the neural tube or en route to the branchial arches, areas where cell-cell interactions typically occur between neural crest cells in normal embryos. Unlike the persistent, directed trajectories in normal embryos, neural crest cells frequently change direction and move somewhat chaotically after ablation. In addition, the migration of neural crest cells in collective chains, commonly observed in normal embryos, was severely disrupted. Hindbrain neural crest cells have the capacity to reroute their migratory pathways and thus compensate for missing neural crest cells after ablation of neighboring populations. Because the alterations in neural crest cell migration are most dramatic in regions that would normally foster cell-cell interactions, the trajectories reported here argue that cell-cell interactions have a key role in the shaping of the neural crest migration.
Asunto(s)
Tipificación del Cuerpo , Encéfalo/embriología , Cresta Neural/embriología , Cresta Neural/fisiología , Óvulo/fisiología , Animales , Comunicación Celular , Movimiento Celular , Embrión de Pollo , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Microscopía por Video , Factores de TiempoRESUMEN
Within the bilaterally symmetric vertebrate body plan, many organs develop asymmetrically. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is one of the earliest proteins to be asymmetrically expressed in the chicken embryo and that its activity is required during gastrulation for proper establishment of the left-right axis. Blocking N-cadherin function randomizes heart looping and alters the expression of Snail and Pitx2, later components of the molecular cascade that regulate left-right asymmetry. However, the expression of other components of this cascade (Nodal and Lefty) was unchanged after blocking N-cadherin function, suggesting the existence of parallel pathways in the establishment of left-right morphogenesis. Here, the results suggest that N-cadherin-mediated cell adhesion events are required for establishment of left-right asymmetry.
Asunto(s)
Tipificación del Cuerpo , Cadherinas/fisiología , Desarrollo Embrionario , Gástrula/fisiología , Corazón/embriología , Proteínas Nucleares , Transactivadores , Receptores de Activinas Tipo II , Activinas , Animales , Anticuerpos Monoclonales/inmunología , Cadherinas/genética , Cadherinas/inmunología , Adhesión Celular , Embrión de Pollo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Hibridación in Situ , Inhibinas/fisiología , Factores de Determinación Derecha-Izquierda , Mesodermo/fisiología , Morfogénesis , Proteína Nodal , Factores de Transcripción Paired Box , Proteínas/genética , Proteínas/fisiología , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
We have examined the ability of normal and heterotopically transplanted neural crest cells to migrate along cranial neural crest pathways in the axolotl using focal DiI injections and in situ hybridization with the neural crest marker, AP-2. DiI labeling demonstrates that cranial neural crest cells migrate as distinct streams along prescribed pathways to populate the maxillary and mandibular processes of the first branchial arch, the hyoid arch and gill arches 1-4, following migratory pathways similar to those observed in other vertebrates. Another neural crest marker, the transcription factor AP-2, is expressed by premigratory neural crest cells within the neural folds and migrating neural crest cells en route to and within the branchial arches. Rotations of the cranial neural folds suggest that premigratory neural crest cells are not committed to a specific branchial arch fate, but can compensate when displaced short distances from their targets by migrating to a new target arch. In contrast, when cells are displaced far from their original location, they appear unable to respond appropriately to their new milieu such that they fail to migrate or appear to migrate randomly. When trunk neural folds are grafted heterotopically into the head, trunk neural crest cells migrate in a highly disorganized fashion and fail to follow normal cranial neural crest pathways. Importantly, we find incorporation of some trunk cells into branchial arch cartilage despite the random nature of their migration. This is the first demonstration that trunk neural crest cells can form cartilage when transplanted to the head. Our results indicate that, although cranial and trunk neural crest cells have inherent differences in ability to recognize migratory pathways, trunk neural crest can differentiate into cranial cartilage when given proper instructive cues.
Asunto(s)
Ambystoma mexicanum/embriología , Encéfalo/embriología , Cresta Neural/fisiología , Animales , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/fisiología , Morfogénesis , Mutación , Cresta Neural/citología , Cresta Neural/trasplante , Proteínas Recombinantes/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterotópicoRESUMEN
The neural crest is a transient population of precursor cells that arises at the border between the neural plate and prospective epidermis in vertebrate embryos. The earliest known response to neural-crest-inducing signals is the expression of the zinc-finger transcription factors slug and snail. Although it is widely believed that these transcription factors play an essential role in neural crest development, relatively little is understood about their mechanism of action during this process. We have previously shown that overexpression of XSlug leads to expanded expression of neural crest markers and an excess of at least one neural crest derivative, melanocytes. In order to further investigate XSlug function, we overexpressed mutant constructs in which the DNA-binding domain was fused to either the activation domain from Gal4 or the repressor domain from Drosophila Engrailed. The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. In contrast, overexpression of either the activation domain fusion or the DNA-binding domain alone was found to inhibit XSlug function. Using a hormone-inducible inhibitory mutant, we show that inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration, demonstrating for the first time that this transcriptional repressor is required during multiple stages of neural crest development.