Endogenous galectins and effect of galectin hapten inhibitors on the differentiation of the chick mesonephros.

Galectins are galactoside-binding lectins. In the mesonephros of the chick embryo, the 16-kDa galectin is abundant in the glomerular and tubular basement membranes where it colocalizes with fibronectin and laminin. To test whether galectin-glycoprotein interactions could play a role in mesonephric development, the effects of the galectin hapten inhibitors thiodigalactoside (TDG) and lactose on the differentiation of the cultured mesonephros were investigated. When compared to control saccharide-free or maltose-treated cultures, mesonephroi cultured in the presence of TDG and lactose exhibited defects in tissue organization. These included a distorted tubule shape, pseudo-stratification of the tubular epithelium, and detachment of glomerular podocytes from the basement membrane. The presence of molecular differentiation markers in the developing mesonephros was investigated. In vivo, expression of the epithelial-specific cell adhesion molecule E-cadherin is restricted to differentiated tubular epithelial cells, whereas the intermediate filament protein vimentin is present in mesonephrogenic mesenchyme and is undetectable in tubular epithelial cells. In mesonephroi cultured in the absence of sugars or in the presence of maltose, the expression pattern of these two marker molecules resembles that found in the mesonephros in vivo. In contrast, in the mesonephroi cultured in the presence of TDG and lactose, the epithelial tubular cells expressing E-cadherin also express vimentin. Re-expression of vimentin in the tubular epithelial cells could indicate a partial reversal to a mesenchymal phenotype. Results suggest that galectin-glycoprotein interactions in the basement membrane are important in the maintenance of the renal epithelial phenotype. Dev Dyn 1999;215:248-263.

Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly.

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell-cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.

Hapten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo.

The early chick blastoderm expresses two endogenous galactose-binding lectins of 14 kDa and 16 kDa. We have studied the effect the lectin hapten inhibitors thiodigalactoside and the synthetic neoglycoprotein lactosyl-bovine serum albumin as well as polyclonal anti-lectin antibodies on the development of early chick embryos cultured in a defined medium. Controls consisted of maltose, maltosyl bovine serum albumin and rabbit IgG. Embryos treated at the onset of cell migration during early gastrulation underwent blastoderm retraction with decrease in surface area. In addition, they exhibited a lack of demarcation between the presumptive embryonic area (area pellucida) and the presumptive extraembryonic area (area opaca). These blastoderms also lacked a primitive streak, that is, the structure that forms in the area pellucida during gastrulation as cell migrate to form the endodermal and mesodermal layers of the embryo. Embryos treated at later stages of gastrulation showed development similar to that of controls in that they were able to undergo early organogenesis. The results suggest that lectin mediated mechanisms are essential for the migratory movements of early gastrulation and that, at late gastrulation, other mechanisms exist in the embryo to compensate for lectin function.

The cells of the dorsal iris involved in lens regeneration are myoepithelial cells whose cytoskeleton changes during cell type conversion.

During newt lens regeneration, the pigmented epithelial cells (PECs) of the dorsal iris dedifferentiate and give rise to a new lens. We have studied the cytoskeleton of the PECs using iris flat mounts and sections. In flat-mount iris preparations stained by labelled phalloidin three main regions can be recognized: the pupillary (P) ring, the middle (M) ring, and the more external junctional (J) ring. The cells of the P ring that give rise to the lens have an elongated spindle shape and exhibit an elaborate cytoskeleton of actin filament bundles oriented along the long axis of the cells, reminiscent of myoepithelial or smooth muscle cells. These cells express smooth muscle-specific alpha actin, muscle gamma actin and cytokeratin II, and adhere to each other through the cell adhesion molecule A-CAM. During dedifferentiation, actin staining increases considerably as the actin filament bundles thicken and shorten and then accumulate preferentially in the apical and basel regions of the elongating lens fibres. Cytokeratin II, which is also organized as fibrils along the long axis of the normal iris PECs, increases progressively during dedifferentiation, when it is organized as a thick band surrounding the nucleus. The expression of this protein is repressed during lens fibre differentiation, but is retained in mitotic cells. The data suggest that during cell type conversion some cytoskeletal proteins increase and reorganize, while others disappear during lens fibre differentiation.

Expression of the galactose-binding lectins during the formation of organ primordia in the chick embryo.

Early chick embryos contain two beta-galactoside-binding lectins of 16 kDa and 14 kDa. using several antisera to these proteins, we have studied lectin expression at embryonic stages when the segregation and early differentiation of organ primordia are taking place. With antisera to the 16 kDa lectin that display similar immunoreactivity in immunoblot analysis, we show that these antisera exhibit varying immunoreactivity in embryo sections. One antiserum reacts preferentially with a matrix form of lectin while another detects mainly a cellular form of this protein. During early development, galactoside-binding lectins of the matrix type are expressed in the vitelline membrane, the outer and inner limiting membranes of the neural tube, the surface of the notochord and the coelomic surface of the cardiac rudiments. The cellular form of the lectin occurs in the intracellular yolk of early embryos, in the primordial germ cells, the myocardium, in the early myotome, and in a cohort of cells which are presumed to belong to the neural crest. Our results indicate that, although all of the antisera recognize the intracellular lectin of the extraembryonic endoderm, some antisera to the 16 kDa lectin exhibit preferential reactivity with different lectin isoforms. The extracellular matrix form of lectin is transiently expressed during early development at the stages when the segregation of organ primordia is occurring. It's expression could be related to the acquisition of polarity in developing epithelia. Results also suggest that various versions of the same protein may perform distinct developmental roles in the embryo.

Different immunoreactivities of anti-soluble lactose lectin antisera to tissues from early chick embryos: a histochemical study.

The location of soluble lactose-binding proteins (S-lac lectins) has been studied by immunohistochemical methods during morphogenesis of the chick embryo, when segregation and early differentiation of organ primordia was occurring. Using a panel of polyclonal antisera raised to various purified lectin preparations, we observed striking differences in the antigenic properties of these antisera, indicating that diverse versions of the lectins may be expressed during development. The antisera referred to as anti-L-16, anti-M-16, anti-S-14 and anti-I-14 were respectively raised to native or denatured 16 kDa lectins from adult liver and embryonic muscle and to 14 kDa lectins from embryonic skin and adult intestine. Having determined the optimal immunohistochemical conditions in the preparation of embryo sections (fixation, embedding, sectioning) we show that anti-L-16, anti-S-14 and anti-I-14 mostly bind the lectins expressed at the cell surface, in the extracellular matrix and in some released secretion. As previously shown, anti-L-16 and anti-S-14 are also able to recognize the cytoplasmic form of some migrative lectin-rich cells (primitive streak, neural crest cells, germ cells). Anti-M-16 was bound exclusively to the cytoplasmic form of the 16 kDa lectin in the same cell lines as above and also in some others, such as in the notochord, the myotomal part of the somites, the pharyngeal endoderm and the cardiac muscle. These different antigenic properties may be applied to the accurate mapping of various lectin isoforms and evaluation of the respective contribution of their intra- and extracellular variants during development and differentiation.

On the possible role of endogenous lectins in early animal development.

In this review I have tried to summarize the information available on the lectins of developing embryos. The emerging evidence indicates that during fertilization carbohydrate-binding proteins play a role in sperm adhesion and in the reorganization of the extracellular matrix of the fertilized egg. Results also indicate that in adult tissues lectins participate in cell recognition and adhesion, and that several galactose-binding lectins function as receptors for laminin and, in principle could also interact with polylactosamine groups of other extracellular matrix glycoproteins. Since in developing embryos lectins are located at the cell surface, and colocalize with extracellular matrix glycoproteins, they could play a role in transitory adhesive interactions and in the segregation of organ primordia. On the basis of experiments in cultured cell lines, it has been suggested that lectins are involved in lysosomal and nuclear glycoprotein transport. These carbohydrate-binding proteins could also regulate development by modulating these processes in the embryo. Since galactose-binding lectins are mitogenic, and are present in high concentration in the chick yolk sac, these proteins could be released into the embryonic circulation, bind to cells expressing appropriate receptors, and act as growth regulators, by modulating cell division of specific cell lineages.

The gastrulating chick blastoderm contains 16-kDa and 14-kDa galactose-binding lectins possibly associated with an apolipoprotein.

Extracts from chick blastoderms were subjected to affinity chromatography on lactoside-Sepharose. Lactose-eluted fractions were examined by gradient SDS-PAGE with silver staining, as well as by immunoblot analysis using antibodies to the chicken galactose-binding lectins of 14 kDa and 16 kDa and to an apolipoprotein of chicken very low density lipoprotein (Apo-VLDL-II). Fractions containing the highest lectin activity contained four main bands. One, unidentified, comigrated with albumin; two bands were identified by immunoblotting as the 14-kDa and 16-kDa lectins. The fourth band comigrated with Apo-VLDL-II and in immunoblot analysis reacted with antibodies to this apolipoprotein. In our electrophoretic system this protein migrates close to bovine trypsin inhibitor and has an apparent molecular weight of 6500 +/- 500. The present studies establish the identity of this previously described 6.5 kDa protein (Zalik et al. J. Cell. Sci. 88, 483, 1987) as Apo-VLDL-II. While the 16-kDa lectin was present consistently in all the affinity-purified preparations, the relative frequencies of the 14-kDa lectin and Apo-VLDL-II varied. In sections of primitive streak blastoderms, lectin immunofluorescence was present in the lowest, most ventral area of the primitive groove and in the cells emerging laterally from the groove to form the endoderm. Cells of the extraembryonic endoderm also displayed high lectin immunoreactivity. The localization of the lectins is similar to the one described previously for Apo-VLDL-II. Double immunofluorescence staining indicates that Apo-VLDL-II and the lectin(s) colocalize. The copurification and colocalization of Apo-VLDL-II and the lectins in the chick blastoderm suggest that this apolipoprotein may associate with the galactose-binding lectins or may display lectin activity.

Fibronectin distribution during cell type conversion in newt lens regeneration.

The distribution of fibronectin during the cell type conversion from iris into lens that occurs in newt lens regeneration was studied by immunofluorescence. Newts were lentectomized and irises at different stages of dedifferentiation and redifferentiation were examined using as a probe a rabbit antiserum prepared to Xenopus plasma fibronectin. In the normal iris, fibronectin is predominantly located at the basal surface of the pigmented iris epithelial cells. During activation and early dedifferentiation fibronectin staining is progressively displayed at the basolateral and apical surface of the depigmenting cell, to eventually surround the surface of the dedifferentiated cells. As cells redifferentiate into lens fibers, staining for cell surface fibronectin decreases and is displayed mainly in the nascent lens capsule. Fibronectin deposition may be associated with the formation of intercellular spaces during dedifferentiation. The fibronectin-rich extracellular matrix could be important in cell reprogramming.

Expression of an endogenous galactose-binding lectin in the early chick embryo.

The gastrulating chick blastoderm contains lectin activity specific for beta-D-galactoside groups. The galactose-binding lectin isolated by affinity chromatography on rho-aminophenyl-beta-D-lactoside separates into two bands when studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One of these LII has a relative molecular mass of 70 (+/- 2) X 10(3) while the other LI is a polypeptide that migrates with the dye front in 10% gels. We have prepared an antiserum against this lectin preparation and have affinity-purified antibodies against LI. When embryos at stages 3-7 were examined by immunofluorescence using the affinity-purified antibodies, lectin was expressed in cells at the lowest portions of the primitive streak as well as in cells migrating laterally from this region to form the endoderm. Lectin was also expressed by the cells of the extra-embryonic endoderm and the primordial germ cells of the proximal area opaca. In transfers of gradient gels stained with affinity-purified antibodies against LI, this lectin had an approximate molecular weight of 6.5 X 10(3). Our results indicate that this lectin is expressed in areas that are undergoing cell spreading.

Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration.

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (non-lens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 3.5-4.0. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.

Release of beta-D-galactoside-binding lectins into the cavities of aggregates of chick extraembryonic endoderm cells.

Dissociated cells from the extraembryonic endoderm of gastrulating chick embryos form aggregates when cultured in rotating flasks. The large cellular aggregates are initially solid but subsequently cavitate to form hollow, thin-walled vesicles. These cells also contain an endogenous beta-D-galactoside-binding lectin. Previous work has shown that high extracellular concentrations of this lectin are associated with decreased cell-cell adhesion [Milos, N. and S.E. Zalik: Differentiation 21, 175-182 (1982)]. We have removed the fluid contents from aggregates cultured for 24 and 48 h and tested them for the presence of lectin activity. The results demonstrate that lectin activity is detectable in a higher number of aggregates cultured for 24 as opposed to 48 h (75% vs. 28%, respectively). The lectin activity per aggregate is also higher in aggregates cultured for 24 h (180 vs. 67 hemagglutinating units, respectively, for 24- and 48-h aggregates). Thus, at the time when cells are moving apart from one another during aggregate cavitation, detectable lectin activity is released into the vesicular contents of the aggregate.

Studies on the endogenous galactose-binding lectin during early development of the embryo of Xenopus laevis.

Embryos of the frog Xenopus laevis at cleavage, blastula, gastrula and neurula stages contain a galactose-specific lectin. Extracts of gastrula embryos show the highest specific activity for this lectin compared to the other stages. Haemagglutinating activity of crude extracts is inhibited by lactose, alpha-galactose, beta-galactose, alpha Gal(1----4) beta Gal, beta Gal(1----3) alpha GalNAc, beta Gal(1----3) beta GlcNAc, beta Gal (1----4) beta GlcNAc, and most effectively by the disaccharide alpha Gal(1----3) beta Gal. Lectin from all stages was purified by absorption to galactose-linked immunoadsorbent or by affinity chromatography on a column of p-aminophenyl-beta-D-lactoside coupled to Sepharose 4B. In order to identify a single lectin band under reducing conditions in sodium dodecyl sulphate/polyacrylamide electrophoresis SDS/PAGE, it was necessary to treat aqueous suspensions of the purified lectin with chloroform/methanol (2:1, v/v). The lectin remained in the aqueous layer and gave rise on SDS/PAGE to a distinct band of 65 500 +/- 2780 molecular weight. Aqueous suspensions of the purified lectin that were not subjected to extraction with chloroform/methanol gave rise to several bands. Isoelectric focussing of the purified lectin resulted in two bands that separated at pI 4.3 and 4.5. In aqueous solution in the presence of lactose the chloroform/methanol-treated lectin appears to be an aggregate of apparent molecular weight of 375 000; the non-treated lectin under the same conditions has an apparent molecular weight of 490 000.

Comparison of newt lens regeneration stimulating activity in preparations of mammalian thyrotropin and fibroblast growth factor purified by various methods.

Irises of the newt Notophthalmus viridescens will regenerate a new lens in organ culture in the presence of the bovine thyrotropin preparation NIH-TSH-B8. It is not certain, however, whether thyrotropin itself is responsible for this stimulatory effect. To elucidate this problem further we compared the lens regeneration stimulating activity of thyrotropin preparations from several species, prepared by various methods. The lowest effective concentrations were approximately 3.0 micrograms ml-1 for the bovine NIH-TSH-B8 and 1.4 micrograms ml-1 for the ovine NIAMDD-oTSH-9 preparations. At those lowest concentrations, lenses with elongating lens fiber cells (stage 6) and enlarged lens fiber core (stage 8) were obtained, respectively, and the lens-fiber-specific protein gamma-crystallin was present in both cases. The crude bovine thyrotropin fraction, Sigma-TS-10, stimulated lens regeneration only at the highest concentration, 1400 micrograms ml-1. Bovine Pierce-bTSH, the purest thyrotropin preparation, stimulated lens regeneration sporadically at the lower concentration of 0.04 micrograms ml-1 up to the advanced stage 9 with large lens fiber core and flattened lens epithelium in one of nineteen irises. The pituitary fibroblast growth factor is a known contaminant of thyrotropin preparations. The preparation CR-FGF-40002 at concentrations between 0.001 and 0.1 microgram ml-1 did not promote lens regeneration. Therefore, the lens regeneration stimulating activity in thyrotropin preparations is not attributable to the fibroblast growth factor, and may also be independent of thyrotropin because the lens regeneration stimulating activity is not proportional to the thyrotropic activity in the preparations examined.

Intercellular relationships during cavitation of aggregates of extraembryonic endoderm cells from gastrulating chick embryos.

Extraembryonic endoderm cells from gastrulating chick embryos undergo epiboly and change from a multilayered cell group to a single cell layer surrounding the yolk. Single cell suspensions from this cell layer can aggregate in vitro to form aggregates that cavitate. To study the stages of cavitation aggregates were harvested after different times in culture, and fixed and processed for light and electron microscopy. In aggregates harvested at 75 min of culture cell contact consisted of areas of parallel and close membrane apposition and interdigitation. Desmosomes were occasionally observed. Aggregates in the early stages of cavitation (24 h) contained numerous intercellular spaces bordered by irregularly shaped cells which appeared to be digesting their yolk and releasing material extracellularly. Long cytoplasmic projections were extended into these spaces. In addition to regions of parallel membrane apposition and interdigitation, desmosomes and adherens junctions were observed. Cells closer to the periphery of the aggregates displayed fewer cell projections and also showed signs of release of material extracellularly. After 48 h of culture, a single smooth-walled central cavity was present and cells still exhibited signs of extracellular release of material. These same cell shapes and intercellular junctions were also observed when area opaca tissue dissected from gastrulating embryos was examined. Aggregates of different sizes were created and cultured. The results suggest that a critical tissue mass may be important for cavitation.

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm beta-D-galactoside-binding lectin and by beta-galactosidase.

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.

Mechanisms of adhesion among cells of the early chick blastoderm: role of the beta-D-galactoside-binding lectin in the adhesion of extraembryonic endoderm cells.

Cells from the extraembryonic endoderm of the gastrulating chick embryo contain a beta-D-galactoside-binding lectin inhibited by thiodigalactoside (TDG). TDG inhibits the aggregation of freshly prepared cells. In these fresh cell suspensions, adhesion is also inhibited when purified lectin is added to the aggregation assay. If these cells are incubated at 22 degrees C their adhesion decreases. Associated with this is an increase in lectin activity in the cell supernatants. In these incubated cells aggregation is stimulated by TDG and desialyzed fetuin. These data suggest that the lectin may have a role to play in cellular adhesion. Under some experimental conditions extraembryonic endoderm cells from rosettes with trypsinized glutaraldehyde-fixed rabbit erythrocytes. This phenomenon is inhibited, to a certain extent, by TDG.

Distribution of a galactose-specific lectin in endoderm cells from early chick embryos.

Cells from the endoderm of the area opaca of gastrulating chick embryos were maintained in stationary cultures, stained with antibodies against the endogenous beta-D-galactoside-binding lectin and examined by immunofluorescence. In the majority of cells fluorescence was present as an irregular circular web in the central cytoplasm. In cells that appeared to be migrating increased fluorescence was observed in the peripheral cytoplasm and retraction fibers. In regions where a portion of the cell was detaching from the substratum high fluorescence was observed in the extracellular "footprints" deposited by the cell.