Trunk Neural Crest Cells Research Articles

Antisense knockdown of the β1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing β1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of β1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with β1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after β1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.

Alterations in Ca2+-dependent and cAMP-dependent signaling pathways affect neurogenesis and melanogenesis of quail neural crest cells in vitro.

Trunk neural crest cells primarily form neurons, nerve supportive cells of the peripheral nervous system and melanocytes. We are interested in signal transduction pathways that affect the production of peripheral neurons or melanocytes. Quail neural crest cell cultures were treated with a variety of drugs that affect components of protein kinase A- (PKA-), protein kinase C- (PKC-) and inositol-3-phosphate- (I3P-) dependent pathways. Forskolin, a drug that increases cAMP levels, augmented melanocyte populations and reduced neuronal populations in our cultures. H8 and H89, two drugs that inhibit PKA, reduced melanocyte populations well below control levels. Down regulation of PKC with a phorbol ester, PMA, or with calphostin C inhibited neurogenesis. PMA also enhanced melanogenesis. Increasing intracellular calcium levels (with A23187 or thapsigargin) resulted in cultures with few melanocytes but many neurons, compared to untreated controls. An antagonist of the I3P pathway, wortmannin, prevented the appearance of neurons but did not affect melanocyte populations. In summary, molecules that altered the PKA-dependent pathway affected melanogenesis. Manipulations of the I3P and/or PKC-dependent pathways influenced neurogenesis. Stimulation of one pathway often inhibited appearance of cells associated with the alternative pathway.

Regulated gene expression of hyaluronan synthases during Xenopus laevis development

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.

Long-distance cue from emerging dermis stimulates neural crest melanoblast migration.

Neural crest melanoblasts display unique navigational abilities enabling them to colonize the dorsal path between ectoderm and somite. One signal shown here to elicit melanoblast migration is a chemotactic cue supplied by the emerging dermis. Until dermis emerges, melanoblasts fail to enter the dorsal path. The dermis emerges from a site that is too distant to stimulate migration by cell contact. Instead, surgeries show that dermis elicits migration from a distance. When dermis is grafted distally, neural crest cells enter the path precociously. Moreover, large grafts recruit melanoblasts from the control sides (without increasing crest cell numbers) as well as a few crest cells from ventral somite. Because other grafted tissues fail to stimulate migration, the dermis stimulus is specific. This report is the first documentation that trunk neural crest cells can be guided chemotactically. It also extends evidence that migration is exquisitely sensitive to temporal-spatial patterns of somite morphogenesis. Developmental Dynamics 229:99-108, 2004.

Significance of the cranial neural crest.

The cranial neural crest has long been viewed as being of particular significance. First, it has been held that the cranial neural crest has a morphogenetic role, acting to coordinate the development of the pharyngeal arches. By contrast, the trunk crest seems to play a more subservient role in terms of embryonic patterning. Second, the cranial crest not only generates neurons, glia, and melanocytes, but additionally forms skeletal derivatives (bones, cartilage, and teeth, as well as smooth muscle and connective tissue), and this potential was thought to be a unique feature of the cranial crest. Recently, however, several studies have suggested that the cranial neural crest may not be so influential in terms of patterning, nor so exceptional in the derivatives that it makes. It is now becoming clear that the morphogenesis of the pharyngeal arches is largely driven by the pharyngeal endoderm. Furthermore, it is now apparent that trunk neural crest cells have skeletal potential. However, it has now been demonstrated that a key role for the cranial neural crest streams is to organise the innervation of the hindbrain by the cranial sensory ganglia. Thus, in the past few years, our views of the significance of the cranial neural crest for head development have been altered. Developmental Dynamics 229:5-13, 2004.

Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro.

During development neural crest cells give rise to a wide variety of specialized cell types in response to cytokines from surrounding tissues. Depending on the cranial-caudal level of their origin, different populations of neural crest cells exhibit differential competence to respond to these signals as exemplified by the unique ability of cranial neural crest to form skeletal cell types. We show that in addition to differences in whether they respond to particular signals, cranial neural crest cells differ dramatically from the trunk neural crest cells in how they respond to specific extracellular signals, such that under identical conditions the same signal induces dissimilar cell fate decisions in the two populations in vitro. Conversely, the same differentiated cell types are induced by different signals in the two populations. These in vitro differences in neural crest response are consistent with in vivo manipulations. We also provide evidence that these differences in responsiveness are modulated, at least in part, by differential expression of Hox genes within the neural crest.

Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells.

Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells.

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration.

Integrin α5β1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and “synergy” sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin α5β1.

Sox10 is required for the early development of the prospective neural crest in Xenopus embryos

The Sox family of transcription factors has been implicated in the development of different tissues during embryogenesis. Several mutations in humans, mice, and zebrafish have shown that depletion of Sox10 activity produces defects in the development of neural crest derivatives, such as melanocytes, ganglia of the peripheral nervous system, and some specific cell types as glia. We have isolated the Xenopus homologue of the Sox10 gene. It is expressed in prospective neural crest and otic placode regions from the earliest stages of neural crest specification and in migrating cranial and trunk neural crest cells. Loss-of-function experiments using morpholino antisense oligos against Sox10 produce a loss of neural crest precursors and an enlargement of the surrounding neural plate and epidermis. This effect of Sox10 depletion is produced during some of the earliest steps of neural crest specification, as is shown by the inhibition in the expression of Slug and FoxD3, which are early markers of neural crest specification. In addition, we show that Sox10 depletion leads to an increase in apoptosis and a decrease in cell proliferation in the neural folds, suggesting that Sox10 could work as a survival as well as a specification factor in neural crest precursors during premigratory stages. Although some of the deficiencies found in the Waardenburg syndrome and in the Hirschprung disease could be associated with a failure of the development of crest derivatives during the late phase of its development, or even during adulthood, our results suggest that inhibition of Sox10 activity produces an earlier failure of neural crest precursors. In experiments where melanocytes and ganglia were induced in vivo and in vitro, we were able to block their development by inhibiting Sox10 activity. These results are compatible with an additional late role of Sox10 on development of neural crest derivatives, as it has been previously proposed. We show that Sox10 expression is dependent on FGF and Wnt activity, both in the neural crest and in the otic placode territories. Finally, in order to establish the position of Sox10 in the hierarchical cascade of gene activation required for neural crest specification, we used inducible forms of the wild type and dominant negatives for the Snail and Slug genes. Our results show that Snail is able to control Sox10 expression. However, the overexpression of Slug was not able to upregulate Sox10 expression. Taken together, these results indicate that Sox10 may lie between Snail and Slug in the genetic cascade that controls neural crest development.

Neural crest motility and integrin regulation are distinct in cranial and trunk populations

The neural crest is a transient cell population that travels long distances through the embryo to form a wide range of derivatives. The extensive migration of the neural crest is highly unusual and incompletely understood. We examined the ability of neural crest cells (NCCs) to migrate under different conditions in vitro. Unlike most motile cell types, avian NCCs migrate efficiently on a wide range of fibronectin concentrations. Strikingly, the migration of NCCs on laminin depends on the axial level from which the crest is derived. On high concentrations of laminin, cranial NCCs migrate at approximately twice the rate of trunk NCCs and show greater persistence, a higher percentage of migratory cells, and a less organized cytoskeleton. The difference in migration between cranial and trunk neural crest is not due to transcriptional differences in integrin mRNA, but rather to differences in posttranslational regulation. Overexpression of a single integrin is sufficient to significantly slow the migration velocity of cranial neural crest cultured on high laminin densities. These results demonstrate that neural crest cells accommodate a wide range of ECM concentrations in vitro and suggest that differences in integrin regulation along the anterior–posterior axis may contribute to differences in neural crest migration and cell fate.

The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo

The ontogeny of neural crest cells (NCC) involves a number of orchestrated variety of derivatives, including components of the peripheral nervous system and melanocytes. Thus, it represents an excellent model system to investigate mechanisms controlling epithelial–mesenchymal transitions, cell migration and differentiation, as well as cell proliferation and death. We have established a new transgenic line expressing the Cre recombinase under the control of the human tissue plasminogen activator promoter (Ht-PA). The activity of the reporter in the Ht-PA-Cre/R26R embryos is observed as early as Theiler stage 12 in the cephalic mesenchyme. Later, the targeted cells include all the known derivatives of cranial, vagal, and trunk NCC, including craniofacial structures and cranial ganglia, cardiac and endocrine derivatives, melanocytes, peripheral, and enteric nervous system. At the vagal level, the location of presumptive enteric NCC differs from their avian counterparts in their ability to invade the mesenchyme lateral to the neural tube. In contrast to the Wnt1-Cre line, the Ht-PA-Cre line does not target the central nervous system and therefore renders it more specific for NCC. Our Ht-PA-Cre mice represent a novel model to specifically target conditional mutations in migratory NCC.

Ephrin-as cooperate with EphA4 to promote trunk neural crest migration.

Trunk neural crest cells delaminate from the dorsal neural tube and migrate on two distinct pathways: a dorsolateral route, between the ectoderm and somites,and a ventromedial route, through the somitic mesoderm. Neural crest cells that migrate ventromedially travel in a segmental manner through rostral half-somites, avoiding caudal halves. Recent studies demonstrate that various molecular cues guide the migration of neural crest cells, primarily by serving as inhibitors to premature pathway entry orby preventing neural crest from entering inappropriate territories. Trajectories of migrating trunk neural crest are well organized and generally linear in nature, suggesting that positive, migration-promoting factors may be responsible for this organized cell behavior. However, the identity of these factors and their function are not well understood. Here we examine the expression of members of the EphA subclass of receptor tyrosine kinases and ephrins using RT-PCR and immunocytochemistry. Neural crest cells express ephrins and EphA4 at distinct stages during their migration. In functional analyses, addition of ephrin-A2-, ephrin-A5-, and EphA4-Fc disrupted the segmental organization of trunk neural crest migration in explants: neural crest cells entered rostral and caudal halves of somites. Finally, to test the specific effects of these factors on cell behavior, neural crest cells were exposed in vitro to substrate-bound EphA and ephrin-As. Surprisingly, neural crest cells avoided ephrin-A2 or ephrin-A5 substrates; this avoidance was abolished by the addition of EphA4. Together, these data suggest that ephrin-As and EphA4 cooperate to positively promote the migration of neural crest cells through rostral half somites in vivo.

Trunk Neural Crest Has Skeletogenic Potential

During early vertebrate development, neural crest cells emerge from the dorsal neural tube, migrate into the periphery, and form a wide range of derivatives. There is, however, a significant difference between the cranial and trunk neural crest with respect to the diversity of cell types that each normally produces. Thus, while crest cells from all axial levels form neurons, glia, and melanocytes, the cranial crest additionally generates skeletal derivatives such as bone and cartilage; trunk crest cells are generally thought to lack skeletogenic potential. Here, we show, however, that if avian trunk neural crest cells are cultured in appropriate media, they form both bone and cartilage cells, and if placed into the developing head, they contribute to cranial skeletal components. Thus, the neural crest from all axial levels can generate the full repertoire of crest derivatives. The skeletogenic potential of the trunk neural crest is significant, as it was likely realized in early vertebrates, which had extensive postcranial exoskeletal coverings.

Forskolin effect on the lineage specification of trunk neural crest cellsin vitro

Recent evidence has suggested that trunk neural crest cell generally assumed to have equivalent differentiation potentials, demonstrate differentiation bias along the anterior/posterior axis. In amphibian and fish, neural crest cells give rise to three chromatophore types, melanophores, xanthophores, and iridophores. Each pigment cell type has distinct characteristics but there is speculation about the cellular plasticity that exists among them. Neural crest cells migrate along specific routes, ventromedially and dorsolaterally. Neural crest cells that travel dorsolaterally are the first cells to begin migration in the axolotl and are the major contributors to the visible pigment pattern. Many factors and mechanisms that are responsible for guiding migratory neural crest cells along potential pathways or determining their fate remain unknown. A single lineage of the crest, which becomes restricted to one of the three pigment cell types, gives us the opportunity to examine the existence of neural crest ste...

Segmental organization of neural crest migration.

Avian neural crest cells migrate on precise pathways to their target areas where they form a wide variety of cellular derivatives, including neurons, glia, pigment cells and skeletal components. In one portion of their pathway, trunk neural crest cells navigate in the somitic mesoderm in a segmental fashion, invading the rostral, while avoiding the caudal, half-sclerotome. This pattern of cell migration, imposed by the somitic mesoderm, contributes to the metameric organization of the peripheral nervous system, including the sensory and sympathetic ganglia. At hindbrain levels, neural crest cells also travel from the neural tube in a segmental manner via three migratory streams of cells that lie adjacent to even-numbered rhombomeres. In this case, the adjacent mesoderm does not possess an obvious segmental organization, compared to the somitic mesoderm at trunk levels. Thus, the mechanisms by which the embryo controls segmentally-organized cell migrations have been a fascinating topic over the past several years. Here, I discuss findings from classical and recent studies that have delineated several of the tissue, cellular and molecular elements that contribute to the segmental organization of neural crest migration, primarily in the avian embryo. One common theme is that neural crest cells are prohibited from entering particular territories in the embryo due to the expression of inhibitory factors. However, permissive, migration-promoting factors may also play a key role in coordinating neural crest migration.

Xenopus ADAM 13 is a metalloprotease required for cranial neural crest-cell migration

Background: Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context.Results: Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate.Conclusions: Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.

Multiple roles of mouse Numb in tuning developmental cell fates

Background: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive.Results: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest–derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons.Conclusions: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Specific pan-neural crest expression of zebrafish Crestin throughout embryonic development.

Zebrafish crestin was identified in a screen for genes dependent on cyclops function and is a member of a family of retroelements (Rubinstein et al. [2000] Genesis 26:86-97). We report here a detailed description of crestin mRNA expression during zebrafish embryogenesis. Crestin expression was first observed during the onset of somitogenesis in cells of the neural crest domain of the ectoderm. Crestin expression was subsequently observed in premigratory cranial and trunk neural crest cells and then in actively migrating crest cells. Cell counts of crestin-expressing premigratory trunk neural crest cells strongly suggest that crestin is expressed by all neural crest cells at this stage. Crestin expression co-localized with a battery of markers for premigratory neural crest cells, developmentally distinct neural crest-derived precursor sublineages, and overtly differentiated neural crest-derived cell types. Expression of crestin is gradually downregulated in overtly differentiated cells. Our results indicate that crestin is a specific pan-neural crest marker throughout zebrafish embryogenesis.

Patterns of migration and regulation of trunk neural crest cells in zebrafish (Danio rerio).

Regulation is the replacement of lost, undifferentiated embryonic cells by neighboring cells in response to environmental signals. Neural crest cells, embryonic cells unique to craniates, are good candidates for studies of regulation because they are pluripotent, and thus might be able to alter their behavior in response to environmental signals. This study investigated regulation for the loss of trunk neural crest (TNC) cells, specifically pigment derivatives, in the zebrafish, Danio rerio. The first part of the study clarifies and extends what has previously been described on normal patterns of TNC migration and differentiation. These data were then used to address the hypothesis that there is regulation for loss of TNC, and that regulation would vary with the amount removed, the position or stage of removal. Zebrafish TNC cells are large and numerous. SEM and Dil labeling revealed that TNC cells undergo several successive waves of 'sheet' and 'segmental' migration, beginning as early as the 12 somite stage. Dil-labeled TNC cells often migrated several somite lengths anteriorly and posteriorly along the trunk axis to form glial cells, ganglia, pigment, ectomesenchyme and tail reticular cells. Regulation occurred on a sliding scale, ranging from complete to incomplete. Defects in development and/or pigmentation occurred if large regions of TNC cells were removed, or if cells were removed from anterior (cardiac) and posterior (tail) extremities of the trunk. Melanophores were the cell type most visibly affected by TNC extirpations. Otherwise, pigmentation was remarkably normal. We propose that the completeness of regulation largely depends upon healing of the overlying epidermis.