Crystallins during Xenopus laevis free lens formation

Summary The ontogeny and localization of crystallins during free lens development (i.e. lens development without the optic vesicle) were investigated in Xenopus laevis using the indirect immunofluorescence staining method with an antiserum raised against homologous total lens soluble proteins. Since the developing free lenses pass through stages similar to those of the lenses regenerated from the inner cell layer of the outer cornea following lentectomy in the same species Freeman's classification was used to identify the stages of free lens development. The first appearance of a positive reaction occurred at early stage IV in a number of cells in an area where future lens fibre cells would develop. With further differentiation of the free lens more and more cells in the fibre area started to show a positive reaction and the first positive reaction in the epithelium was observed late in stage V. Histological examination revealed that a fully differentiated free lens and a normally developed lens are similar but that the free lens is smaller.     

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.     

Regulation of cell-cell adhesion during Dictyostelium development

During development of Dictyostelium, four adhesion systems have been identified and adherens junction-like structures have been discovered in the fruiting body. The temporal and spatial expression of cell adhesion molecules (CAMs) is under stringent developmental control, corresponding to major shifts in morphological complexity. Genetic manipulations, including over-expression and knockout mutations, of the adhesion genes, cadA (encoding DdCAD-1), csaA (gp80) and lagC (gp150), have shed light on new roles for cell adhesion molecules in aggregate size regulation, cell-type proportioning, cell differentiation and cell sorting. As cell-cell interactions remain highly dynamic within cell streams and aggregates, mechanisms must exist to facilitate the rapid assembly and disassembly of adhesion complexes. Studies on gp80 have led to a model for the rapid assembly of adhesion complexes via lipid rafts.     

PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis

We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin β1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated α5β1 integrin and cytoskeleton strength during cell movement.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

The distribution of E-cadherin during Xenopus laevis development

A vast amount of experimental evidence suggests that cell surface molecules involved in cell-to-cell and/or cell-to-substrate interactions participate in the control of basic events in morphogenesis. E-cadherin is a cell adhesion molecule directly implicated in the control of Ca2(+)-dependent interactions between epithelial cells. We report here the patterns of expression of E-cadherin in developmental stages of Xenopus laevis ranging from early embryo to adult using immunofluorescence microscopy. Although its distribution shares some similarities with those of L-CAM in the chicken and E-cadherin/Uvomorulin in the mouse, the distribution of E-cadherin in Xenopus presents several peculiar and unique features. In early stages of Xenopus development, E-cadherin is not expressed. The molecule is first detectable in the ectoderm of late gastrulas (stage 13-13.5 NF). At this time both the external and the sensory layer of the nonneural ectoderm accumulate high levels of E-cadherin while the ectoderm overlying the neural plate and regions of the involuting marginal zone (IMZ) not yet internalized by the movements of gastrulation are E-cadherin-negative. Unlike most other species, endodermal cells express no or very low levels of E-cadherin up to stage 20 NF. Endodermal cells become strongly E-cadherin-positive only when a well-differentiated epithelium forms in the gut. No mesodermal structures are stained during early development. In the placodes, in contrast to other species, E-cadherin disappears very rapidly after placode thickening. During further embryonic development E-cadherin is present in the skin, the gut epithelium, the pancreas, many monostratified epithelia and most glands. Hepatocytes are stained weakly while most other tissues, including the pronephros, are negative. In the mesonephros, the Wolffian duct and some tubules are positive. During metamorphosis a profound restructuring of the body plan takes place under the control of thyroid hormones, which involves the degeneration and subsequent regeneration of several tissues such as the skin and the gut. All newly formed epithelia express high levels of E-cadherin. Surprisingly, degenerating epithelia of both skin and intestine maintain high levels of the protein even after starting to become disorganized and to degenerate. In the adult, staining is strong in the skin, the glands, the lungs, the gut epithelium and the pancreas, weak in the liver and absent from most other tissues. Our results show that the expression of E-cadherin in Xenopus is strongly correlated with the appearance of differentiated epithelia.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Lens-forming competence in the epidermis of Xenopus laevis during development

In larval X. laevis the capacity to regenerate a lens under the influence of inductive factors present in the vitreous chamber is restricted to the outer cornea and pericorneal epidermis (Lentogenic Area, LA). However, in early embryos, the whole ectoderm is capable of responding to inductive factors of the larval eye forming lens cells. In a previous paper, Cannata et al. (2003) demonstrated that the persistence of lens-forming competence in the LA is the result of early signals causing lens-forming bias in the presumptive LA and of late signals from the eye causing cornea development. This paper analyzes 1) the decrease of the lens-forming capacity in ectodermal regions both near LA (head epidermis) and far from LA (flank epidermis) during development, 2) the capacity of the head epidermis and flank epidermis to respond to lens-competence promoting factors released by an eye transplanted below these epidermal regions, and 3) the eye components responsible for the promoting effect of the transplanted eye. Results were obtained by implanting fragments of ectoderm or epidermis into the vitreous chamber of host tadpoles and by evaluating the percentage of implants positive to a monoclonal antibody anti-lens. These results demonstrated that the lens-forming competence in the flank region is lost at the embryonic stage 30/31 and is weakly restored by eye transplantation; however, lens-forming competence in the head region is lost at the larval stage 48 and is strongly restored by eye transplantation. The authors hypothesize that during development the head ectoderm outside the LA is attained by low levels of the same signals that attain the LA and that these signals are responsible for the maintenance of lens-forming competence in the cornea and pericorneal epidermis of the larva. In this hypothesis, low levels of these signals slacken the decrease of the lens-forming competence in the head ectoderm and make the head epidermis much more responsive than the flank epidermis to the effect of promoting factors released by a transplanted eye. Results obtained after transplantation of eyes deprived of some components indicate that the lens and the retina are the main source of these promoting factors. The immunohistochemical detection of the FGFR-2 (bek variant) protein in the epidermis of stage 53 larvae submitted to eye transplantation at stage 46 showed that the eye transplantation increased the level of FGFR-2 protein in the head epidermis but not in the flank epidermis, indicating that the lens-forming competence in X. laevis epidermis could be related to the presence of an activated FGF receptor system in the responding tissue.     

Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta- catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549- 557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E- cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Expression of muscle LIM protein during early development in Xenopus laevis

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.     

Regenerative capability in the hindlimb of Xenopus laevis during ontogenetic development

The regenerative capacity of hindlimb of Xenopus laevis was investigated by amputating the limbs at four levels in various developmental stages including younger postmetamorphosed froglets. Amputations of limbs were performed at the base of limb in stages 50, 51, 52, 53, 54, 55, 58, and 60 (Nieuwkoop and Faber's table), at the middle of limb bud in stages 50, 51, 52 and 54, and at mid-thigh and mid-shank in stages 58 and 60, and the froglets in 2 and 3 cm in snout-vent length. In the present experiments the regenerative capacity of limbs was expressed by the rate of regeneration and morphogenesis. Tadpoles in the stages after 55 failed to regenerate when the limbs were amputated at base level, but individuals in all the other experimental series exhibited regeneration in various rates irrespective of the level of amputation and the stage. The regenerative capacity increased distally along the proximo-distal axis of the limb when amputated at the same stage, while regeneration was better in younger stages than that in older stages when amputations were made at the same levels. The regenerates obtained by amputation of limbs in stages between 50 and 54, were mainly digitated in that they had 5 toes with 3 claws which is the same pattern with the normal limb, 4 toes with 2 claws, 3 toes with 2 claws or one, and 2 toes with one claw etc. Tadpoles at stage 50 could regenerate toes and claws without defect, but in the later the regenerative capacity gradually declined by reducing the number of toes and claws and accompanied by malformation of skeleton as the stage proceeded. The tadpoles in stages after 58, and the froglets of 2 and 3 cm, produced various types of heteromorphic regenerates of shapes such as cone, spike or rod of which the centra were occupied with cartilage rods. However these regenerates showed no morphological differences according to the developmental stages. These heteromorphic regenerates continued their growth even after one year without any sign of development of digitated feet.