Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan.

It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r )of 360-530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to 'attract' moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve 'haptotactic-like' motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells.

Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio).

The hypochord of the zebrafish embryo (Danio rerio) emerges at the 9-somite stage as a single row of cells in the dorsomedial endoderm immediately ventral to the notochord. It is recognizable from the 2(nd) or 3(rd) somite and extends along the trunk to the same extent as the somites. A basal lamina surrounds the hypochord and its cells are slightly larger than the nearby endoderm cells. TEM studies have shown that the hypochord cells contain, in addition to mitochondria, well-developed rough endoplasmic reticula and Golgi networks, indicating synthetic activity. Once formed, the hypochord will stay in close association with the notochord, and this axial complex gradually moves dorsally, separating the hypochord from the endoderm as a one-cell-wide, rod-like structure that is bean-shaped in transverse section. This is the situation in the 15-somite embryo, at the level of the 4-5(th) somites. In the gap between the hypochord and the endoderm, angioblast cells aggregate and start to form the dorsal aorta, which becomes intimately associated with the hypochord. In the 17-somite embryo the aortic rudiment is established just ventral to the hypochord as a tube with a lumen. As development proceeds, the size of the hypochord decreases. In the pec fin embryo the hypochord is still recognizable in the posterior trunk, but has apparently vanished in anterior regions. The temporal correlation between the appearance of the hypochord and the formation of the dorsal aorta, coupled with the intimate relationship between these structures, suggest that the hypochord may play a role in the positioning of the dorsal aorta.

Reduced epidermal expression of a PG-M/versican-like proteoglycan in embryos of the white mutant axolotl.

Axolotl embryos have previously been used to study neural crest cell migration. In embryos of the normal wild type, neural crest cells migrate subepidermally to form pigment cells. In the trunk of the white mutant embryo, these cells are unable to migrate, possibly due to an inherited delay in the maturation of the local extracellular matrix. The present investigation reveals a reduced incorporation of [35S]sulfate into PG-M/versican-like proteoglycans synthesized in epidermal explants from the dorsal trunk of white mutant embryos during stages pertinent to migration. This is the major form of proteoglycans in the subepidermal matrix, where they are assembled in large disulfide-stabilized supramolecular complexes. The reduction in [35S]sulfate incorporation is not due to qualitative differences between wild-type and white mutant proteoglycans but is paralleled by a reduced expression of mRNA for the core protein of the PG-M/versican-like proteoglycan. We conclude that a reduced amount of these proteoglycans is produced by the white mutant embryo during the period critical for migration.

Hypochord, an enigmatic embryonic structure: study of the axolotl embryo.

The hypochord of the axolotl embryo is first visible at an early tailbud stage, forming a rod-like structure, situated immediately under the notochord. A profusion of extracellular matrix fibrils is attached to the dorsolateral regions of the hypochord, linking it with the somites. A basal lamina develops around the hypochord, indicating an epithelial type of cell differentiation. Abundant rough endoplasmic reticula in the hypochord cells suggest lively synthetic activity. Prospective endoderm cells were vitally labeled with the lipophilic dye 1,1-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) at the gastrula stage. Cells labeled with the dye were later found in the hypochord as well as in the gut endoderm. This shows that the hypochord is of endodermal origin, contrary to recent suggestions that the hypochord is of mesodermal origin, but consistent with histological data. After about 8 days of existence, the hypochord disappears. Experimental results, using an apoptosis detection kit, indicate that the hypochord cells may disintegrate by a type of apoptotic cell death. The close association between the hypochord and developing dorsal aorta suggests that the hypochord could be involved in the positioning of the dorsal aorta, which forms under it.

PG-M/versican-like proteoglycans are components of large disulfide-stabilized complexes in the axolotl embryo.

Large disulfide-stabilized proteoglycan complexes were previously shown to be synthesized by the epidermis of axolotl embryos during stages crucial to subepidermal migration of neural crest cells. We now show that the complexes contain PG-M/versican-like monomers in addition to some other component with low buoyant density. Metabolically 35S-labeled proteoglycans were extracted from epidermal explants and separated by size exclusion chromatography and density equilibrium gradient centrifugation. The complexes, which elute in the void volume on Sepharose CL-2B, were recovered at buoyant density 1.42 g/ml in CsCl gradients, whereas the monomer proteoglycans, which could only be liberated from the complexes by reduction, had a higher buoyant density (1.48 g/ml). The native complexes did not aggregate with hyaluronan. The purified complexes reacted with antibodies against a portion of a cloned PG-M/versican-like axolotl proteoglycan. These antibodies were found to stain the subepidermal matrix of axolotl embryos, suggesting that the proteoglycan complexes are encountered by neural crest cells during subepidermal migration. From Western blot analysis, the core protein of the PG-M/versican-like monomers was found to be of similar size ( approximately 500 kDa) as those of PG-M/versican variants of other species. Another chondroitin sulfate proteoglycan that was present in small amounts in the epidermal extracts was found to be distinctly different from the similarly sized PG-M/versican-like monomers.

Improved preservation of the subepidermal extracellular matrix in axolotl embryos using electron microscopical techniques based on cryoimmobilization.

The purpose of this metholdological survey was to find optimal methods for the fixation and demonstration of glycosaminoglycans, mainly hyaluronan, and proteoglycans, in subepidermal extracellular matrix (ECM) regions of axolotl embryos. We compared living ECM in the laser-scanning microscope (LSM) with chemically fixed or cryoimmobilized extracellular matrix in the transmission (TEM) and scanning electron microscope (SEM). The gel-like structure of living extracellular matrix in the LSM undoubtedly provides the most natural state, whereas shrinkage of the extracellular matrix occurs during conventional fixation and dehydration for TEM or SEM. Among the methods used for fixation and processing of subepidermal extracellular matrices for SEM, plunge-freezing/freeze-drying is to be preferred. Still more satisfying, however, are results obtained with high-pressure frozen/freeze-substituted ECM material in the TEM, for which 10% polyvinyl pyrrolidon +7% methanol was used as a cryoprotectant before high-pressure freezing. In these specimens, no freeze-damage could be observed and they could be regarded as adequately frozen. Conversely, the yield in adequately frozen specimens without cryoprotection was insufficient. In these specimens, the ECM contained honeycomb-like structures which, in the current literature, are regarded as hyaluronan.

What insights into the phenomena of cell fate determination and cell migration has the study of the urodele neural crest provided?

In this review we ask whether studies on the development of the urodele neural crest (NC) have provided special insights into the fate and migration of these cells when compared to other amphibian embryos or those of higher vertebrates. We recognize that during the first half of this century and even before, urodele embryos were the favorite objects of experimental embryology for studying the development of mesenchymal derivatives and their participation, together with mesodermal mesenchyme, in the development of the neuro- and viscerocranium. Furthermore, the NC was discovered to be the source of cranial sensory and spinal ganglia, and the influence of the somites on the localization of the latter was clearly pointed out. In addition, pioneering studies were devoted to the NC-derived pigment cells. Investigations in this field concentrated on their migration in the embryo and in vitro, and on the mechanisms underlying larval pigment pattern formation. It is mainly in these three areas that the urodele embryo has served as a tool for gaining major results and defining the concepts of classical embryology. Even today, when the interest has shifted towards the molecular biology in Xenopus, chicks and mice, the urodele embryo with its large cells, convenient for injections, is a potential model for future lineage studies and knockout experiments. And furthermore, as important concepts of vertebrate development are defined in the urodele, future studies in these embryos may link the disciplines of development and evolution.

Neural crest cell migration and pigment pattern formation in urodele amphibians.

This review deals with research on the development and differentiation of the neural crest (NC) in amphibians carried out during the past twenty years. First, earlier studies on the migration and differentiation of NC cells in vitro are summarized. These studies include the modes of NC cell migration and their differentiation into chondroblasts, perichondral cells, neurons, Schwann cells and pigment cells (melanophores and xanthophores). Then a summary is given on the development of cranial sensory ganglia and enteric ganglia in Xenopus laevis. In the subsequent sections, mechanisms of NC cell migration are investigated in Ambystoma mexicanum, the Mexican axolotl (wild-type and white mutant) using ultrastructural, immunohistochemical and biochemical methods. In wild-type or dark axolotl embryos, pigment cells leave the NC and migrate out under the epidermis, whereas in the white mutant, pigment cells remain closely confined to the original position of the NC. This system provides an excellent model for analyzing NC cell migration in vertebrate embryos. Further sections deal with the development of larval pigment patterns in Triturus alpestris, (horizontal melanophore stripes) and Ambystoma mexicanum (vertical melanophore bars). Comparing the formation of these patterns shows that two different principles exist in the distribution of pigment derivatives of the NC: patterns following environmental cues (Triturus) and those ignoring these cues, relying solely on cell-cell interactions (Ambystoma). Other studies relate to evolutionary perspectives in pigment pattern formation. They are based on phylogenetic analyses of North American ambystomatids, combined with data on pigment patterns and their formation where such data are available. These studies have shown that vertical bars which develop from aggregates in the NC string are an evolutionary innovation, compared to the more primitive horizontal stripes lacking aggregates in the NC. Thus, in this review we show that the NC of amphibians (T. alpestris, Xenopus laevis, dark and white axolotls and other ambystomatids) may be used for various analyses concerning the migration and differentiation of its derivatives, as well as for studies on the formation and evolution of pigment patterns.

Distribution of keratan sulphate and chondroitin sulphate in wild type and white mutant axolotl embryos during neural crest cell migration.

In embryos of the white mutant axolotl, prospective pigment cells are unable to migrate from the neural crest (NC) due to a deficiency in the subepidermal extracellular matrix (ECM). This raises the question of the molecular nature of this functional defect. Some PGs can inhibit cell migration on ECM molecules in vitro, and an excess of this class of molecules in the migratory pathways of neural crest cells might cause the restricted migration of prospective pigment cells seen in the white mutant embryo. In the present study, we use several monoclonal antibodies against epitopes on keratan sulphate (KS) and chondroitin sulphate (CS) and LM immunofluorescence to examine the distribution of these glycosaminoglycans at initial (stage 30) and advanced (stage 35) stages of neural crest cell migration. Most KS epitopes are more widely distributed in the white mutant than in the wild type embryo, whereas CS epitopes show very similar distributions in mutant and wild type embryos. This is confirmed quantitatively by immunoblotting: certain KS epitopes are more abundant in the white mutant. TEM immunogold staining reveals that KS as well as CS are present both in the basal lamina and in the interstitial ECM in both types of embryos. It remains to be investigated whether the abundance of certain KS epitopes in the white mutant embryo might contribute to the deficiency in supporting pigment cell migration shown by its ECM.

The development of the neural crest in amphibians.

Our review deals with the development of the neural crest (NC) in amphibians. We will consider relevant aspects of evolution, ontogeny, migration and differentiation, and investigate principal problems such as the regulation of NC cell determination, pathway selection and destination recognition. Earlier data and more recent findings will be presented. The NC probably evolved about 440 million years ago from the anlagen of epidermal nerve plexuses in protochordates. In urodele amphibians, the prospective NC is already present in the early gastrula as a narrow band of ectodermal cells between the prospective epidermis and the prospective neural plate. The NC proper develops later from the apices of the neural folds and forms, after neural fold fusion, a transient cellular ridge on the dorsal surface of the neural tube. NC cells migrate extensively into various body regions and give rise to a wide variety of derivatives including the mesenchymal elements of the skull, the neural and glial precursors of the peripheral nervous system and the pigment cells. NC cell migration is stimulated by components of the extracellular matrix (ECM) and may conveniently be analyzed in the system of wild-type (dark) and white mutant axolotl embryos. Skeletal elements of the head derive from cranial NC cells following an interaction with pharyngeal endoderm. Other derivatives of the NC are the ganglia of the peripheral nervous system in the head (sensory ganglia; mostly mixed origin with placodal material) and trunk (spinal, sympathetic and enteric ganglia). Pigment cells also derive from the NC and become arranged into uniform or banded pigment patterns.

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

The development of the larval pigment patterns in Triturus alpestris and Ambystoma mexicanum.

1. Melanophores and xanthophores are pigment cell derivatives of the NC. In amphibian embryos they migrate from their original position on the neural tube dorsally (into the dorsal fin) as well as laterally (between somites and epidermis) and arrange themselves into typical pigment patterns of the skin. We investigated pigment pattern formation in two species of tailed amphibians, Triturus alpestris (alpine newt) and Ambystoma mexicanum (Mexican axolotl). In larvae of T. alpestris alternating longitudinal stripes or bands of melanophores and xanthophores develop, whereas in larvae of A. mexicanum a barred pattern with alternating transverse bands of melanophores and xanthophores is formed. Iridophores, a third type of pigment cell, are present later in both species and therefore play no role during early larval pigment pattern development. Visibly differentiated melanophores and xanthophores can be distinguished from each other under the light microscope by their contents of black melanins and yellow pterins respectively. With the dopa reaction (indicates tyrosinase in melanophores), and ammonia treatment (stimulates pterin fluorescence in xanthophores), the pigment cell phenotypes can be visualized even before their normal visible differentiation. In the TEM, melanophores and xanthophores can be distinguished from each other by their morphologically distinct pigment organelles and in the SEM by their different surface structure. 2. Because of the NC origin of melanophores and xanthophores and the ease with which these cells can be demonstrated even before they are visible from outside, their different arrangements in Triturus and axolotl embryos offer suitable model systems for studying the migration, interaction and localization of NC derivatives in relation to specific environmental influences. The environment of NC cells are the neural tube, epidermis, somites and lateral plate mesoderm, and the subepidermal ECM, a network of collagen fibrils associated with glycosaminoglycans, proteoglycans and glycoproteins. 3. Development of the pigment pattern in T. alpestris: Melanophores and xanthophores start to leave the NC at stage 28, melanophores slightly earlier than xanthophores. Both cell types become scattered in the dorsolateral trunk. In contrast to melanophores in the axolotl, melanophores in T. alpestris cannot be demonstrated with the dopa reaction before they become visibly black. From stage 29+ onwards, melanophores start to accumulate in zones alongside the dorsal and lateral somite edges, where they form compact stripes later. Xanthophores can be demonstrated from stage 28+ onwards only with the SEM (by means of their specific surface structures) or with the fluorescence microscope (by means of their fluorescing pterins). At state 34, xanthophores become visible externally as yellow cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Timing in the regulation of neural crest cell migration: retarded "maturation" of regional extracellular matrix inhibits pigment cell migration in embryos of the white axolotl mutant.

In larvae of the white axolotl mutant (Ambystoma mexicanum), contrary to normal dark ones, trunk pigmentation is restricted because the epidermis is unable to support subepidermal migration of pigment cells from the neural crest (NC). This study examines whether the subepidermal extracellular matrix (ECM) is the defective component which prevents pigment cell migration in the white embryo. We transplanted subepidermal ECM, adsorbed in vivo on membrane microcarriers, from and to white and dark embryos in various combinations. White embryos have demonstrated normal NC cell migration along the medioventral pathway, and in order to test the effects of medial ECM on subepidermal migration, this ECM was similarly transplanted. Carriers with ECM attached were inserted subepidermally in host embryos at a premigratory NC stage. Control carriers without ECM and carriers with subepidermal ECM from white donors did not affect NC cell migration in white or dark embryos. In contrast, subepidermal ECM from dark donors triggered NC cell migration in the subepidermal space of both white and dark hosts. Remarkably, subepidermal ECM from white donors which were older than those normally used also stimulated migration in embryos of both strains. Likewise, medial ECM from white donors elicited migration in white as well as dark hosts. Pigment cells occurred among those NC cells that were stimulated to migrate in response to contact with ECM on carriers. These results indicate that the subepidermal ECM of the white embryo is transiently defective as a substrate for pigment cell migration, implying that "maturation" of the ECM is retarded beyond the times during which pigment cells are able to respond. In contrast, the medial ECM of the white embryo appears to mature normally. These findings suggest that the effect of the d gene is expressed regionally through the subepidermal ECM during a limited period of development. Hence, the action of the d gene seems to retard ECM maturation, bringing it out of phase with the migratory capability of the pigment cells. We propose that such a shift in relative timing of the developmental phenomena involved inhibits pigment cell migration in embryos of the white axolotl mutant and, accordingly, that the restricted pigmentation of the mutant larva is generated through heterochrony.

Local embryonic matrices determine region-specific phenotypes in neural crest cells.

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.

Promotion of chromatophore differentiation in isolated premigratory neural crest cells by extracellular matrix material explanted on microcarriers.

This study was undertaken to determine whether premigratory neural crest cells of the axolotl embryo differentiate autonomously into chromatophores, or whether stimuli from the environment, particularly from the extracellular matrix, are required for this process. Neural crest cells were excised from the dorsal part of the premigratory crest cord and cultured alone, either in a serum-free salt solution or in the presence of fetal calf serum (FCS), and together with explants of the neural tube or dorsal epidermis. A "microcarrier" technique was developed to assay the possible effects of subepidermal extracellular matrix (ECM) on chromatophore differentiation. ECM was adsorbed in vivo onto microcarriers prepared from Nuclepore filters, by inserting such carriers under the dorsolateral epidermis in the embryonic trunk. Neural crest cells were then cultured on the substrate of ECM deposited on the carriers. Melanophores were detected by DOPA incubation, revealing phenol oxidase activity, or by externally visible accumulation of melanin. Prospective xanthophores were visualized before they became overtly differentiated by alkali-induced pteridine fluorescence. Isolated premigratory neural crest cells did not transform autonomously into any of these phenotypes. Conversely, coculture with the neural tube or the dorsal epidermis, and also the initial presence or later addition of FCS during incubation, resulted in differentiation of neural crest cells into chromatophores. Both chromatophore phenotypes were also expressed on the ECM substrate deposited on the microcarriers. The results indicate that neural crest cells do not differentiate autonomously into melanophores and xanthophores, but that interactions with components of, or factors associated with the extra cellular matrix surrounding the premigratory neural crest and present along the dorsolateral migratory pathway are crucial for the expression of these chromatophore phenotypes in the embryo.

Stimulation of initial neural crest cell migration in the axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers.

The present experiments were designed to test whether the onset of neural crest cell migration in the embryonic axolotl trunk is stimulated by surrounding tissues and their associated extracellular matrix (ECM). Tissue grafts, or embryonic ECM adsorbed in vivo onto inert "microcarriers" prepared from Nuclepore filters, were placed close to the premigratory neural crest cells, and the embryos were then incubated to a specific stage. The experiments were evaluated with light microscopy, SEM, and TEM. It was found that grafts from the dorsal epidermis were especially effective in locally stimulating initial neural crest cell migration in the region under the graft. The microcarrier experiments showed that the subepidermal ECM alone could initiate neural crest cell migration, implying that the ECM of the epidermal grafts was the stimulating factor. These results indicate that the premigratory neural crest cells along the trunk have migratory capability but that they need to be triggered from the environment, probably from the surrounding ECM, to start migration. It is proposed that ECM, as substrate for cell locomotion, initiates and regulates the onset of neural crest cell migration.

Tetracycline influence on leukocyte functions.

Two tetracyclines, lymecycline and doxycycline, were investigated with reference to modification of granulocyte function. Both preparations influenced granulocyte adherence, migration, quantitative phagocytosis and induced surface membrane morphological changes. Doxycycline caused more pronounced effects than lymecycline.