Pathways and mechanisms of avian trunk neural crest cell migration and localization

Crest cells individualized at the dorsal border of the neural tube, while they became surrounded by a fibronectin-rich matrix. Crest cells initiated their migration between the basement membranes of the neural tube and the ectoderm. In the vagal region, crest cells migrated in a fibronectin-rich environment between the ectoderm and the dermomyotome, very rapidly reaching the apex of the pharynx. In the trunk region, crest cells opposite the bulk of the somite accumulated at the junction between the somite, the neural tube, and the ectoderm; they resumed their migration at the onset of the dissociation of the somite into dermomyotome and sclerotome. Migration occurred more ventrally along the neural tube; nevertheless, the formation of the rapidly expanding sclerotome prevented crest cells from reaching the paranotochordal region. Thereafter, crest cells accumulated between the neural tube, the dermomyotome, and the sclerotome, where ultimately they formed the dorsal root ganglia. In contrast, cells opposite the intersomitic space did not encounter these obstacles and utilized a narrow pathway formed between the basement membranes of the two adjacent somites. This pathway allowed crest cells to reach the most ventral regions of the embryo very rapidly; they accumulated along the aorta to form the aortic plexuses, the adrenal medulla, and the sympathetic ganglia. The basic features of the migration pathways are (1) a strict delimitation by the fibronectin-rich basement membranes of the surrounding tissues, (2) a formation of space concomitant with the migration of crest cells, (3) a transient existence: continued migration is correlated with the presence of fibronectin, whereas cessation is correlated with its focal disappearance. The crest cells are characterized by their inability to traverse basement membranes and penetrate within tissues. We propose that the combination of active proliferation, unique motility properties, and the presence of narrow pathways are the major mechanisms ensuring correct directionality. Morphologically defined transient routes of migration along with developmentally regulated changes in the extracellular matrix and in the adhesive properties of crest cells are most probably involved in their stabilization in defined territories and their aggregation into ganglia.     

Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells

Summary Immunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm, but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1–2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.     

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.     

The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo

In recent studies of chick embryos, the cranial paraxial mesoblast was found to be patterned into segmental units termed somitomeres. Anterior to the first segmental cleft, seven contiguous segments are aligned, with somitomeric interfaces forming grooves at right angles to the midline. In this study, the morphological relationship between the migratory pathways of cranial neural crest cells and patterned primary mesenchyme was analyzed with the scanning electron microscope, utilizing stereo imaging. In addition, the development of neuromeres in the adjacent neural tube was monitored. It was found that cranial neural crest first appears along the dorsal midline as a ridge of cells which loosens from the wall of the neural tube and migrates laterally as discrete populations. The mesencephalic crest appears first, immediately following neural tube fusion at that level, and migrates over the dorsal surface of the adjacent third somitomere and into the grooves formed by its juncture with the second and fourth somitomeres. Later, the addition of prosencephalic and rostral rhombencephalic crest extends the mesencephalic population to form a shelf of crest which spreads over the dorsal surface of the first four somitomeres. Component cells of this most cranial crest shelf become oriented and mimic the metameric pattern of the subjacent somitomeres. Crest cells adjacent to the fifth somitomeres appear along the midline, but do not migrate, creating a gap anterior to the otic crest. By stage 9, a narrow finger-like segment of the otic crest migrates from a specific neuromere into the grooved interface between the fifth and sixth somitomeres, delimiting the rostral border of the otic placode in the ectoderm above. By the end of stage 9, crest cells delimiting the caudal border of the placode have migrated along the interface of the seventh and eighth somitomeres. The crest cells adjacent to the sixth and seventh somitomeres, between the rostral and caudal otic populations, appear but do not migrate, remaining condensed along the midline. Thus, otic crest cells form a ring which circumscribes the invaginating otic placode. This study suggests that the precise distribution of cranial neural crest cells may result from their introduction at specific times, as specific populations from specific brain regions (neuromeres), onto a patterned mesodermal layer.     

The expression pattern of a novel Deltex homologue during chicken embryogenesis

We isolated a partial cDNA encoding a novel chicken homologue of human Deltex (DTX1), a member of the Notch signaling pathway. The cDtx2 sequence showed higher homology to KIAA0937 protein (92% identical) than to DTX1 (68% identical). cDtx2 is expressed widely in the epiblast at stage 4. Later in development it is expressed in many neural and sensory structures, such as neural tube, migrating neural crest cells, epidermal placodes, cranial ganglia, and the optic and otic vesicles.     

Control of neural crest cell dispersion in the trunk of the avian embryo

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Do vertebrate neural crest and cranial placodes have a common evolutionary origin?

Two embryonic tissues-the neural crest and the cranial placodes-give rise to most evolutionary novelties of the vertebrate head. These two tissues develop similarly in several respects: they originate from ectoderm at the neural plate border, give rise to migratory cells and develop into multiple cell fates including sensory neurons. These similarities, and the joint appearance of both tissues in the vertebrate lineage, may point to a common evolutionary origin of neural crest and placodes from a specialized population of neural plate border cells. However, a review of the developmental mechanisms underlying the induction, specification, migration and cytodifferentiation of neural crest and placodes reveals fundamental differences between the tissues. Taken together with insights from recent studies in tunicates and amphioxus, this suggests that neural crest and placodes have an independent evolutionary origin and that they evolved from the neural and non-neural side of the neural plate border, respectively.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

Cranial neural crest emergence and migration in the Mexican axolotl (Ambystoma mexicanum)

The timing and pattern of cranial neural crest cell emergence and migration in the Mexican axolotl, Ambystoma mexicanum, are assessed using scanning electron microscopy (SEM). Cranial neural crest cells emerge and begin to migrate at the time of neural fold closure and soon form three distinct streams. The most anterior (mandibular) stream emerges first, at the level of the mesencephalon. Cells in this stream migrate rostroventrally around the optic vesicle. The second (hyoid) and third (branchial) streams emerge in close succession at the level of the rhombencephalon and extend ventrolaterally. Cells forming the hyoid stream migrate rostral to the otic vesicle, whereas the branchial stream divides into two parallel streams, which migrate caudal to the otic vesicle. At later stages (stage 26 onwards) the cranial neural crest cells disperse into the adjacent mesoderm and can no longer be followed by dissection and SEM. The pattern of cranial neural crest emergence and migration, and division into migratory streams is similar to that in other amphibians and in the Australian lungfish (Neoceratodus forsteri). Emergence of crest cells from the neural tube, relative to the time of neural tube closure, occurs relatively late in comparison to anurans, but much earlier than in the Australian lungfish. These results establish a morphological foundation for studies in progress on the further development and fate of cranial neural crest cells in the Mexican axolotl, as well as for studies of the role of cranial neural crest in cranial patterning.     

A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.     

Integrin-α5 coordinates assembly of posterior cranial placodes in zebrafish and enhances Fgf-dependent regulation of otic/epibranchial cells

Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

Neural crest and placode interaction during the development of the cranial sensory system

In the vertebrate head, the peripheral components of the sensory nervous system are derived from two embryonic cell populations, the neural crest and cranial sensory placodes. Both arise in close proximity to each other at the border of the neural plate: neural crest precursors abut the future central nervous system, while placodes originate in a common preplacodal region slightly more lateral. During head morphogenesis, complex events organise these precursors into functional sensory structures, raising the question of how their development is coordinated. Here we review the evidence that neural crest and placode cells remain in close proximity throughout their development and interact repeatedly in a reciprocal manner. We also review recent controversies about the relative contribution of the neural crest and placodes to the otic and olfactory systems. We propose that a sequence of mutual interactions between the neural crest and placodes drives the coordinated morphogenesis that generates functional sensory systems within the head.     

Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hybridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and 1-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex.