Preservation of the morphological and molecular stability of embryonic tissues.

There is presently great interest in using early embryonic tissues, particularly human tissue, for studies of protein and gene expression. Embryonic human tissue is very fragile, and delays often occur before it can be properly prepared for scientific study. Using chick embryos, we have studied the effects of delaying fixation or biochemical isolation on the preservation of cytological characteristics and biochemical molecules. Our study shows that by 60 min post-harvest, tissue morphology and immunofluorescence staining degrades, but the total mRNA profile remains stable. This study suggests that the time between removal of the tissue and fixation is critical to the results and that the critical time is much shorter for embryonic tissues than for more developed tissues. Our results have implications for all research where embryonic tissues are harvested but not processed immediately.

Expression of the endogenous galactoside-binding lectin of Xenopus laevis during cranial neural crest development: lectin localization is similar to that of members of the N-CAM and cadherin families of cell adhesion molecules.

Cranial neural crest of Xenopus laevis at different stages of development from neurulation to metamorphosis was studied for the expression of the endogenous galactoside-binding lectin of Xenopus using immunocytochemistry. The presence and localization of members of the N-CAM and cadherin cell adhesion families were also investigated. Lectin and the other known cell adhesion molecules are expressed throughout development and their localization patterns change coordinately depending on the development stage. All the molecules colocalize. The results suggest that all of these molecules, including the lectin, may be involved in cranial development, possibly in cellular adhesion.

A monoclonal antibody against neural crest-stage Xenopus laevis lectin perturbs craniofacial development of Xenopus.

A monoclonal antibody that we have produced against the neural crest-stage galactoside-binding lectin of Xenopus laevis has been used to confront neural crest cells in Xenopus embryos in vivo. Confronted tadpoles have been raised to stage 47 (premetamorphosis) and examined for jaw defects in whole mounts and tissue sections. Antibody treatment correlates with no grossly defective lower jaw development in 19% of tadpoles. An increase in protrusion of the lower jaw and a decrease in the number of chondrocytes in the head is also noted. Further biochemical studies of the lectin reveal that it is an aggregate. Agglutinating activity resides in doublet protein that binds the monoclonal antibody. The results therefore suggest that interfering with the portion of the lectin complex that has the agglutinating activity can have significant effects on craniofacial development.

Confrontation of developing melanophore bars of dark and white axolotls with endogenous dark-embryo mannose-binding lectin correlates with melanophore bar disruption.

Lectins are carbohydrate-binding proteins suggested to be important in embryonic cell adhesion/differentiation. Dark and white axolotls contain an endogenous mannose-binding lectin that is especially prevalent during larval melanophore pattern formation (Martha et al., 1990). To determine if this lectin can alter melanophore patterning, lectin extracts have been isolated from Dark embryos by affinity chromatography. The main protein band is 44K on SDS-PAGE. Dark and white embryos at the early chromatophore migration stage have been confronted with Dark lectin or its nonmetabolized inhibitor, 2-deoxyglucose (2-DG). The barred melanophore pattern of both genotypes is disrupted by lectin or 2-DG treatment suggesting that endogenous mannose-binding lectin and its receptor participate in bar formation.

Defective development of the craniofacial/digestive complex of Xenopus laevis after treatment with endogenous galactoside-binding lectin or its hapten inhibitor thiodigalactoside.

The regions of the developing craniofacial skeleton and gut of Xenopus laevis have been confronted in vivo with purified embryonic galactoside-binding lectin or its hapten inhibitor thiodigalactoside (TDG). Confrontation was carried out at stage 24-26 (cranial neural crest migrating). Further development of the head skeleton and gut has been monitored in living animals and in histological cross-sections of selected head regions. Lectin treatment correlates with the development of larger heads than controls. TDG treatment correlates with the development of narrower heads than controls. After both treatments, head cartilages are composed of fewer total chondrocytes. Both neural crest and non-neural crest cartilages are affected. The gut forms larger, irregular coils after lectin or TDG confrontation. The results suggest that galactoside-binding lectin/galactoside-bearing receptor adhesive interactions are important in development of the craniofacial/visceral skeleton and gut.

Probing the functions of endogenous lectins: effects of a monoclonal antibody against the neural crest-stage lectin of Xenopus laevis on trunk development.

Trunk neural crest of Xenopus laevis has been confronted prior to migration with whole or fragments of a monoclonal antibody raised against the carbohydrate-binding site of the endogenous neural crest-stage galactoside binding lectin (Milos et al.: Anat. Embryol., 182:319-327 '90). External fin formation was inhibited in confronted regions but extensive internal matrix develops suggesting interiorization of fin tissue. In the regions of missing fin, the myotomes overgrew the neural tube and dorsal root ganglion neuronal numbers became more variable. Melanophore numbers in regions of missing fin did not change but a significantly greater proportion of the pigment cells localized on the muscle surface that had overgrown the neural tube suggesting that the pigment cell population redistributed itself to occupy a greater myotomal area.

Alterations of heart development in Xenopus laevis by galactoside-binding lectin or its sugar hapten inhibitor.

The early heart anlagen of Xenopus laevis embryos were exposed to purified embryonic galactoside-binding lectin or its potent hapten inhibitor thiodigalactoside (TDG). Heart development was then studied using a variety of microscopical techniques. Conotruncal morphology and positioning with respect to the ventricle are altered in treated animals. In 34% of animals treated with lectin and 35% treated with TDG, the conotruncus leaves the ventricle from an abnormal location. Lectin or TDG treatments are also correlated with altered conotruncal shape, with the conotruncal regions showing greater radii of curvature compared to controls. Conotruncal myocyte differentiation is altered by the test treatments, with lack of development of organized myofibrillar arrays. Conotruncal cushion development is also affected. Changes occur in the shape and size of the primary conotruncal cushion, and alterations of outflow tract septation develop. Less maturation of ventricular myocytes is also observed in test animals. The results suggest that galactose-lectin interactions are important in heart development.

Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis.

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24-26, 32, 41 and 47. The main lectin activity at st. 24-26 is ß-galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.

Mesoderm and jaw development in vertebrates: the role of growth factors.

The head and neck arise during development as the result of a complex series of cellular and molecular interactions that begin in the fertilized egg. In this article, the role of an important class of molecules, growth factors, is examined in two main steps of the developmental sequence: the initial induction of mesoderm and the later induction of jaw cartilage and bone. The article focuses particularly on the roles of members of the transforming growth factor-beta (TGF-beta), fibroblast growth factor (FGF), and epithelial growth factor (EGF) families in these processes and current models of growth factor involvement. Possible experiments for the future are discussed.

Studies on cellular adhesion of Xenopus laevis melanophores: pigment pattern formation and alteration in vivo by endogenous galactoside-binding lectin or its sugar hapten inhibitor.

We have studied the development of Xenopus laevis tail melanophores and the effects on these cells on confrontation with endogenous X. laevis galactoside-binding lectin or its sugar hapten inhibitor thiodigalactoside (TDG). An initial population of unpigmented cells differentiates into melanophores on the dorsal surface of the neural tube, and on the dorsal and ventral apices of the myotomes, forming the larval pattern. Melanophores secondarily populate the flank, forming a spaced arrangement which is later transformed into a dorsal and ventral strip. A technique has been developed for confrontation of premigratory neural crest with purified lectin or TDG. These molecules impact on tail melanophores. With lectin treatment melanophore numbers decrease, and cell morphologies and arrangements change. TDG treatment, however, primarily affects pigment cell morphology. These results suggest that both galactoside-bearing receptors for this lectin and the lectin itself can affect melanophores in this species of frog.

Localization of endogenous galactoside-binding lectin during morphogenesis of Xenopus laevis.

A monoclonal antibody has been produced against Xenopus laevis galactoside-binding neural-crest-stage lectin. This antibody inhibits lectin-mediated hemagglutination. Using this antibody in conjunction with immunohistochemical techniques, lectin deposition has been studied in embryos and tadpoles at different stages of morphogenesis, from initial neural crest migration, up to the formation of a swimming tadpole. Lectin levels change during development in different regions of the embryo and tadpole, decreasing in migratory cells, and increasing in sites where cells become more adhesive to one another. The results suggest that galactoside-binding lectins may be an important class of cellular adhesion molecules during these stages of development.

Developmentally regulated lectin in dark versus white axolotl embryos.

The white mutant of the Mexican axolotl, A. mexicanum, involves an ectodermal defect which prevents melanophore colonization. Endogenous lectins have been suggested to function in neural crest-derived melanophore adhesion in other animals. To determine if differences in endogenous lectins exist in dark and white axolotls during melanophore colonization, white and dark ectoderm and carcass tissues have been assayed for lectin activity at premigratory, early migratory, and late migratory neural crest stages. Lectin content (specific for D-glucosamine, N-acetyl-D-glucosamine and D-mannose) increases significantly during early migration only in dark ectoderm and white carcass tissues, whereas white ectoderm and dark carcass lectin activities remain close to premigration levels. Neural crest cells in these embryos are associated with regions of high lectin activity suggesting that the differences in endogenous lectins may be involved in establishment of the dark/white phenotype.

Involvement of endogenous galactoside-binding lectin of Xenopus laevis in pattern formation of Xenopus neurites in vitro.

Galactoside-binding lectin has been purified from Xenopus laevis embryos at the stage of neural crest migration. Addition of this lectin to neurite cultures correlates with the appearance of fascicles of greater diameter and shorter length compared with controls. Lectin-treated neurites are also more spread out on the substratum than their controls. The potent hapten inhibitor of the endogenous lectin, thiodigalactoside (TDG), was also added to these cultures. TDG-treated neurites are less well spread out than the controls; fascicle diameters and lengths are not altered. These results suggest that galactoside-bearing receptors and endogenous galactoside-binding lectin are present in these neurites and can participate in controlling neuronal morphogenesis in vitro, although to differing extents.

The effects of various nutritional supplements on the growth, migration and differentiation of Xenopus laevis neural crest cells in vitro.

This study investigates the nutritional requirements of Xenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, alpha-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conducive environment tested for cell migration and adhesion.

Studies on cellular adhesion of Xenopus laevis melanophores: modulation of cell-cell and cell-substratum adhesion in vitro by endogenous Xenopus galactoside-binding lectin.

We have investigated cell-cell and cell-substratum adhesion of Xenopus laevis neural crest cells at various stages of melanophore differentiation. Single-cell suspensions were obtained by trypsinization and aggregated in a cell-cell adhesion assay. Unpigmented cells did not adhere while the rate of adhesion of melanophores correlated with the degree of melanization. Melanophore cell-cell adhesion decreased significantly in the presence of beta-galactosidase, which suggests that cell-surface galactose is involved. Beta-galactoside-binding lectin has been isolated and purified from embryos at the stage of neural crest migration. When added to aggregating cells smaller, looser clusters formed compared to controls. When lectin was added to cells in stationary culture to test cell-substratum adhesion, melanophores spread more smoothly and formed more regular spacing patterns. These results suggest that this lectin can modulate receptors used in cell-cell and cell-substratum adhesion of melanophores.

Cell surface carbohydrate involvement in controlling the adhesion and morphology of neural crest cells and melanophores of Xenopus laevis.

Pieces of dorsal neural tube (stages 22-23) or late neural crest tissue (stages 24-26) of Xenopus laevis were cultured. Migratory cells moved out of explants to form an outgrowth of multipolar melanophores on the substratum. Treatment with beta-galactosidase (0.1-0.4 U/ml) to remove cell surface galactose was correlated with detachment of melanophores. In the presence of lower concentrations of this enzyme the shapes of these cells were converted to arborized, spidery morphologies and cell movement was inhibited. Unpigmented cells were affected more slowly. Neuraminidase treatment, to remove cell surface sialic acid and expose more galactose, only affected melanophores. These became increasingly spread on the substratum and cell overlap was observed. These results suggest that the relative amounts of galactose and sialic acid at the cell surface become increasingly important in controlling cell adhesion as X. laevis neural crest cells migrate and differentiate into melanophores.