Population Of Neural Crest Cells Research Articles

Speed from recycling

On page 545, Strachan and Condic show that neural crest cells (NCCs) can be speedy if they remember to recycle receptors. The speed gained from their conservation efforts may expose them to differentiation pathways that other NCCs miss out on. Figure Integrin α6 (green) is returned to the cell surface from recycling endosomes (red). Populations of NCCs differ in their speed on various matrices. Cranial NCCs, for instance, pick up speed on higher concentrations on laminin, whereas trunk NCCs keep a slow pace. The new results show that the cranial population is able to move quickly because they both recycle and reduce the numbers of their laminin-binding integrin receptors. More laminin might actually slow cells by promoting strong adhesions. But cranial NCCs avoided this slowdown by removing laminin receptors (integrin α6) from the cell surface. The removed α6 receptors were internalized into recycling endosomes and sent back to the surface. Through this recycling pathway, receptors that are freed from adhesions at the trailing edge may be quickly moved to the leading edge, where new adhesions are needed. Indeed, inhibition of α6 receptor recycling slowed NCC migration on high concentrations of laminin, but had no affect on low laminin levels. On fibronectin, which binds to integrin α5, the surface levels of integrins α5 and α6 were unchanged, and α5 recycling was not increased. The lack of α5 recycling may explain why the speed of cranial NCCs does not vary based on fibronectin concentrations. Integrin recycling may explain in part how some sets of NCCs acquire their fate. Upon emerging from the neural tube, NCCs migrate throughout the body and differentiate into cell types as varied as bone, nerves, and pigment cells. NCCs that move quickly on laminin may receive one set of differentiation instructions at their destination. By the time latecomers arrive, the environment may have changed such that these NCCs are told to adopt a different fate.

Neural crest cell origin for intrinsic ganglia of the developing chicken lung

The development of intrinsic ganglia, comprised of neurons and glia cells that innervate airway smooth muscle, is a recognized component of the growing lung. However, the embryological origin of these neurons and glia is unclear. The lung buds develop as an outgrowth of the foregut, which contains migrating neural crest cells (NCC) that ultimately give rise to the enteric nervous system (ENS) along the entire length of the gut. It has therefore been proposed that the intrinsic ganglia of the lung arise from a subset of NCC that leave the gut and migrate into the lung buds during early development. We have tested this hypothesis using quail-chick interspecies grafting to selectively label the hindbrain-derived neural crest cell population that colonizes the gut. In conjunction with antibody labeling and in situ hybridization, we demonstrate that: (i) lung ganglia arise from vagal NCC that migrate from the foregut into the lung buds; (ii) like ENS precursors, these NCC express the transcription factor Sox10, and the receptors EDNRB and RET; (iii) the co-receptor for RET, GFRα1, is expressed in the lung mesenchyme and in ganglia; (iv) ganglia persist within the lung throughout development and contain cells immunopositive for the pan-neuronal markers ANNA-1 and PGP9.5, the inhibitory neurotransmitter NO, as shown by NADPH-diaphorase staining, and the glial marker GFAP.

The neural crest: basic biology and clinical relationships in the craniofacial and enteric nervous systems.

The highly migratory, mesenchymal neural crest cell population was discovered over 100 years ago. Proposals of these cells' origin within the neuroepithelium, and of the tissues they gave rise to, initiated decades-long heated debates, since these proposals challenged the powerful germ-layer theory. Having survived this storm, the neural crest is now regarded as a pluripotent stem cell population that makes vital contributions to an astounding array of both neural and non-neural organ systems. The earliest model systems for studying the neural crest were amphibian, and these pioneering contributions have been ably refined and extended by studies in the chick, mouse, and more recently the fish to provide detailed understanding of the cellular and molecular mechanisms regulating and regulated by the neural crest. The key questions regarding control of craniofacial morphogenesis and innervation of the gut illustrate the wide range of developmental contexts in which the neural crest plays an important role. These questions also focus attention on common issues such as the role of growth factor signaling in neural crest cell development and highlight the central role of the neural crest in human congenital disease.

The therapeutic potential of stem cells in the treatment of craniofacial abnormalities

Anomalies associated with the vertebrate head and face account for a third of all reported major birth defects. Of the principle cell populations that participate in formation of the craniofacial complex, the neural crest is central, generating much of the peripheral nervous system and constituting the predominant connective tissue-forming mesenchyme of the facial skeleton. Many craniofacial anomalies are, therefore, largely attributed to defects in neural crest cell development. Neural crest cells exhibit many of the features of stem cells; they are multipotent, remarkably plastic and have a limited capacity for self-renewal. This article will review recent studies that demonstrate the ability of stem cells to generate neural crest cell populations that form appropriate neural crest derivatives in the developing craniofacial complex, and will discuss the potential application for stem cells in the treatment of craniofacial disorders.

AP-2 and HNK-1 define distinct populations of cranial neural crest cells.

To determine if distinct populations of cranial neural crest cells (CNCC) exist by characterization of their divergent gene expression patterns. Identification of unique populations of CNCC was determined by a combination of lineage and immunohistochemical analyses. Department of Pathology, Children's Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, PA 19104. We found antibodies of two proteins previously described as identifying all CNCC, label three populations of CNCC at specific time-points. Furthermore, the activating protein 2 (AP-2) expressing CNCC become neural or mesenchymal NCC derivatives whereas the HNK-1 labeled cells do not participate in the mesenchymal lineage. These data provide molecular markers for unique CNCC fates and thus will be invaluable in the characterizing of craniofacial anomalies related to defects in NCCS. In addition, our data suggest AP-2 may function in determining the unique mesenchymal fate of CNCCs.

Multipotentiality of the neural crest

Multiple neural and non-neural cell types arise from the neural crest (NC) in vertebrate embryos. Recent work has provided evidence for multipotent stem cells and intermediate precursors in the early NC cell population as well as in various NC derivatives in embryos and even in adult. Advances have been made towards understanding how cytokines, regulatory genes and cell–cell interactions cooperate to control commitment and differentiation to pigment cells, glia and neurone subtypes. In addition, NC cell fates appeared to be unstable, as differentiated NC cells can reverse to multipotent precursors and transdifferentiate in vitro.

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.

Development of teeth in chick embryos after mouse neural crest transplantations.

Teeth were lost in birds 70-80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crest-derived mesenchymal cells.

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.

Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells.

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.

Book reviews: 1

Book reviews: 1

Chimeric Analysis of Retinoic Acid Receptor Function during Cardiac Looping

Retinoids (vitamin A and its derivatives) play essential roles during vertebrate development. Vitamin A deprivation leads to severe congenital malformations affecting many tissues, including diverse neural crest cell populations and the heart. The vitamin A signal is transduced by the retinoic acid receptors (RARα, RARβ, and RARγ). However, these receptors exhibit considerable functional redundancy, as judged by the mild phenotype of RAR single null mutants relative to the defects evoked by loss of multiple RARs. To circumvent this redundancy, the endogenous RARγ2 allele was replaced with a ligand-binding RARγ mutant (RARγE305) by gene targeting in mouse embryonic stem (ES) cells. Chimeric embryos derived from hemizygous RARγE305 ES cells displayed several defects similar to those observed in certain RAR double null mutants, including hypoplasia or absence of the caudal pharyngeal arches and myocardial deficiencies. The latter defects were not due to abnormal cardiac specification as affected hearts still expressed chamber-specific markers in an appropriate manner. Chimeras also displayed cardiac looping anomalies, which were associated with a reduction of Pitx2. This work suggests a role for RAR signaling in late looping morphogenesis and illustrates the utility of using a dominant-negative gene substitution approach to circumvent the functional redundancy inherent to the RAR family.

Transient expression of the bHLH factor neurogenin-2 marks a subpopulation of neural crest cells biased for a sensory but not a neuronal fate.

Lineage-tracing experiments have indicated that some premigratory neural crest cells (NCCs) are pleuripotent, generating sensory and sympathetic neurons and their associated glia. Using an inducible Cre recombinase-based fate mapping system, we have permanently marked a subpopulation of NCCs that expresses Ngn2, a bHLH transcription factor required for sensory neurogenesis, and compared its fate to the bulk NCC population marked by expression of Wnt1. Ngn2(+) progenitors were four times more likely than Wnt1(+) NCCs to contribute to sensory rather than sympathetic ganglia. Within dorsal root ganglia, however, both Ngn2- and Wnt1-expressing cells were equally likely to generate neurons or glia. These data suggest that Ngn2 marks an NCC subpopulation with a predictable fate bias, early in migration. Taken together with previous work, these data suggest that NCCs become restricted to sensory or autonomic sublineages before becoming committed to neuronal or glial fates.

Cranial neural crest-cell migration in the direct-developing frog, Eleutherodactylus coqui: molecular heterogeneity within and among migratory streams

Direct development is a specialized reproductive mode that has evolved repeatedly in many different lineages of amphibians, especially anurans. A fully formed, albeit miniature adult hatches directly from the egg; there is no free-living larva. In many groups, the evolution of direct development has had profound consequences for cranial development and morphology, including many components that are derived from the embryonic neural crest. Yet, the developmental bases of these effects remain poorly known. In order to more fully characterize these changes, we used three molecular markers to analyze cranial neural crest-cell emergence and migration in the direct-developing frog, Eleutherodactylus coqui: HNK-1 immunoreactivity, Dlx protein expression, and cholinesterase activity. Our study validates and extends earlier results showing that the comprehensive changes in embryonic cranial patterning, differentiation, and developmental timing that are associated with direct development in Eleutherodactylus have not affected gross features of cranial neural crest biology: the relative timing of crest emergence and the number, configuration and identity of the principal migratory streams closely resemble those seen in metamorphic anurans. The three markers are variably expressed within and among neural crest-cell populations. This variation suggests that determination of cranial neural crest-cells may already have begun at or soon after the onset of migration, when the cells emerge from the neural tube. It is not known how or even if this variation correlates with differential cell lineage or fate. Finally, although HNK-1 expression is widely used to study neural crest migration in teleost fishes and amniotes, E. coqui is the only amphibian known in which it effectively labels migrating neural crest-cells. There are not enough comparative data to determine whether this feature is functionally associated with direct development or is instead unrelated to reproductive mode.

The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.

How the considerable diversity of neural crest (NC)-derived cell types arises in the vertebrate embryo has long been a key question in developmental biology. The pluripotency and plasticity of differentiation of the NC cell population has been fully documented and it is well-established that environmental cues play an important role in patterning the NC derivatives throughout the body. Over the past decade, in vivo and in vitro cellular approaches have unravelled the differentiation potentialities of single NC cells and led to the discovery of NC stem cells. Although it is clear that the final fate of individual cells is in agreement with their final position within the embryo, it has to be stressed that the NC cells that reach target sites are pluripotent and further restrictions occur only late in development. It is therefore a heterogenous collection of cells that is submitted to local environmental signals in the various NC-derived structures. Several factors were thus identified which favor the development of subsets of NC-derived cells in vitro. Moreover, the strategy of gene targeting in mouse has led at identifying new molecules able to control one or several aspects of NC cell differentiation in vivo. Endothelin peptides (and endothelin receptors) are among those. The conjunction of recent data obtained in mouse and avian embryos and reviewed here contributes to a better understanding of the action of the endothelin signaling pathway in the emergence and stability of NC-derived cell phenotypes.

Zebrafish deadly seven Functions in Neurogenesis

In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon–Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon–Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch–Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch–Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5).

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.

Cell interactions within nascent neural crest cell populations transiently promote death of neurogenic precursors.

We have previously shown that cultured trunk neural crest cell populations irreversibly lose neurogenic ability when dispersal is prevented or delayed, while the ability to produce other crest derivatives is retained (Vogel, K. S. and Weston, J. A. (1988) Neuron 1, 569-577). Here, we show that when crest cells are prevented from dispersing, cell death is increased and neurogenesis is decreased in the population, as a result of high cell density. Control experiments to characterize the effects of high cell density on environmental conditions in culture suggest that reduced neurogenesis is the result of cell-cell interactions and not changes (conditioning or depletion) of the culture medium. Additionally, we show that the caspase inhibitor zVAD-fmk, which blocks developmentally regulated cell death, rescues the neurogenic ability of high density cultures, without any apparent effect on normal, low-density cultures. We conclude, therefore, that increased cell interaction at high cell densities results in the selective death of neurogenic precursors in the nascent crest population. Furthermore, we show that neurogenic cells in cultured crest cell populations that have dispersed immediately are not susceptible to contact-mediated death, even if they are subsequently cultured at high cell density. Since most early migrating avian crest cells express Notch1, and a subset expresses Delta1 (Wakamatsu, Y., Maynard, T. M. and Weston, J. A. (2000) Development 127, 2811-2821), we tested the possibility that the effects of cell contact were mediated by components of a Notch signaling pathway. We found that neurogenic precursors are eliminated when crest cells are co-cultured with exogenous Delta1-expressing cells immediately after they segregate from the neural tube, although not after they have previously dispersed. We conclude that early and prolonged cell interactions, mediated at least in part by Notch signaling, can regulate the survival of neurogenic cells within the nascent crest population. We suggest that a transient episode of cell contact-mediated death of neurogenic cells may serve to eliminate fate-restricted neurogenic cells that fail to disperse promptly in vivo.

Stem cell factor and/or endothelin-3 dependent immortal melanoblast and melanocyte populations derived from mouse neural crest cells.

Stem cell factor (SCF) and endothelin-3 (ET3) are both necessary for melanocyte development. In order to obtain immortal cell populations of melanoblasts that can survive without feeder cells, we first obtained an immortal cell population of neural crest cells (NCCs) from Sl/+ and +/+ mice of strain WB by incubating with a culture medium supplemented with SCF and ET3, and then we designated them as NCC-SE3 cells. NCC-SE3 cells were bipolar, polygonal, or round in shape and possessed melanosomes of stages I-III (mainly stage I). They were positive to dihydroxyphenylalanine (DOPA) reaction and expressed KIT (a receptor tyrosine kinase), tyrosinase, tyrosinase-related protein-1 (TRP1), tyrosinase-related protein-2 (TRP2), and endothelin-B receptor (ETRB) as determined by immunostaining. We next cultured NCC-SE3 cells by changing culture medium from the one supplemented with SCF + ET3 to the one supplemented with SCF or ET3. NCC-SE3 cells cultured with ET3 alone, designated as NCC-E3 cells, were bipolar in shape and had mainly stage II melanosomes and expressed the same proteins as did NCC-SE3 cells. However, NCC-SE3 cells cultured with SCF alone, designated as NCC-S4.1 cells, were polygonal in shape and had mainly stage I melanosomes. They are thought to be more immature because they were positive to KIT, TRP1, and TRP2, but not to ETR(B), tyrosinase, and DOPA reaction. When 12-O-tetradecanoylphorbol 13-acetate and cholera toxin were added to the culture medium, NCC-S4.1 cells changed shape from polygonal to bipolar and became DOPA-positive. This suggests that NCC-S4.1 cells are melanoblasts that have the potential to differentiate into melanocytes. These cell populations will be extremely useful to study factors that affect melanocyte development and melanogenesis.

Fluorescence-activated cell sorting of EGFP-labeled neural crest cells from murine embryonic craniofacial tissue.

During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.