Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.     

Ephrin-B ligands play a dual role in the control of neural crest cell migration

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. Our aim was to determine how neural crest cells that are specified as neurons and glial cells only migrate ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, we asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, we detected the expression of ephrin-B ligands in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, we show that ephrins block dorsolateral migration of early-migrating neural crest cells because when we disrupt the Eph-ephrin interactions by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, we discovered the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridisation, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signalling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signalling to the actin cytoskeleton, potentially involving the activation of Cdc42/Rac family of GTPases. In support of this hypothesis, we show that adhesion of early migratory cells to an ephrin-B-derivatized substratum results in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin.     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects

Background  

Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood.     

Factors controlling cardiac neural crest cell migration

Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continues migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration and condensation of these cells. This Review elucidates what is currently known about these factors.Key words: cardiac neural crest, migration, signaling, matrix, pharyngeal arches, rhombencephalic streams     

Excitatory Eph receptors and adhesive ephrin ligands

Ephrins are cell surface associated ligands for Eph receptor tyrosine kinases and are implicated in repulsive axon guidance, cell migration, topographic mapping and angiogenesis. During the past year, Eph receptors have been shown to associate with glutamate receptors in excitatory neurons, suggesting a role in synapse formation or function. Moreover, ephrin/Eph signaling appears to regulate neural stem cell proliferation and migration in adult mouse brains. The mode of action of ephrin/Ephs has been expanded from repulsion to adhesion and from cell surface attachment to regulated cleavage.     

Disruption of segmental neural crest migration and ephrin expression in delta-1 null mice

Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, we examined neural crest migration and ephrin expression in Delta-1 mutant mice. Using Sox-10 as a marker, we noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice.     

Factors controlling cardiac neural crest cell migration

Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continue migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration, and condensation of these cells. This review elucidates what is currently known about these factors.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.     

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.     

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.     

The effect of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in vitro

We have investigated the morphology and migratory behavior of quail neural crest cells on isolated embryonic basal laminae or substrata coated with fibronectin or tenascin. Each of these substrata have been implicated in directing neural crest cell migration in situ. We also observed the altered behavior of cells in response to the addition of tenascin to the culture medium independent of its effect as a migratory substratum. On tenascin-coated substrata, the rate of neural crest cell migration from neural tube explants was significantly greater than on uncoated tissue culture plastic, on fibronectin-coated plastic, or on basal lamina isolated from embryonic chick retinae. Neural crest cells on tenascin were rounded and lacked lamellipodia, in contrast to the flattened cells seen on basal lamina and fibronectin-coated plastic. In contrast, when tenascin was added to the culture medium of neural crest cells migrating on isolated basal lamina, a significant reduction in the rate of cell migration was observed. To study the nature of this effect, we used human melanoma cells, which have a number of characteristics in common with quail neural crest cells though they would be expected to have a distinct family of integrin receptors. A dose-dependent reduction in the rate of translocation was observed when tenascin was added to the culture medium of the human melanoma cell line plated on isolated basal laminae, indicating that the inhibitory effect of tenascin bound to the quail neural crest surface is probably not solely the result of competitive inhibition by tenascin for the integrin receptor. Our results show that tenascin can be used as a migratory substratum by avian neural crest cells and that tenascin as a substratum can stimulate neural crest cell migration, probably by permitting rapid detachment. Tenascin in the medium, on the other hand, inhibits both the migration rates and spreading of motile cells on basal lamina because it binds only the cell surface and not the underlying basal lamina. Cell surface-bound tenascin may decrease cell-substratum interactions and thus weaken the tractional forces generated by migrating cells. This is in contrast to the action of fibronectin, which when added to the medium stimulates cell migration by binding both to neural crest cells and the basal lamina, thus providing a bridge between the motile cells and the substratum.     

A paraxial exclusion zone creates patterned cranial neural crest cell outgrowth adjacent to rhombomeres 3 and 5.

Cranial neural crest cell migration is patterned, with neural crest cell-free zones adjacent to rhombomere (R) 3 and R5. These zones have been suggested to result from death of premigratory neural crest cells via upregulation of BMP-4 and Msx-2 in R3 and R5, consequent to R2-, R4-, and R6-derived signals. We reinvestigated this model and found that cell death detected by acridine orange staining in avian embryos varied widely numerically and in pattern, but with a tendency for an elevated zone centered at the R2/3 boundary. In situ hybridization of BMP-4 mRNA resolved to centers at R3 and R5 but Msx-2 resolved to the R2/3 border with only a faint smear from R5 to R6. Outgrowth of neural crest cells was less in isolated R3 cultures than in R1+2, R2, and R4 cultures, but R3 showed neither a decrease in outgrowth of neural crest cells nor an increase in cell death when cocultured with R1+2, R2, or R4. In addition, in serum-free culture, exogenous BMP-4 strikingly reduced neural crest cell outgrowth from R1+2 and R4 as well as R3. Thus we cannot confirm the role of intraneural cell death in patterning rhombomeric neural crest outgrowth. However, grafting quail R2 or R4 adjacent to the chick hindbrain demonstrated a neural crest cell exclusion zone next to R3 and R5. We suggest that one important pattern determinant for rhombomeric neural crest cell migration involves the microenvironment next to the neural tube.     

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.