Concentration dependence of inductive activity in the mixture of lens epithelium proteins

As shown elsewhere, the mixture of proteins secreted by lens epithelium cells in the process of microcultivation can selectively induce eye and forebrain tissues in the early gastrula ectoderm (Zemchikhina et al., 2000, 2003). In the present work, the dependence of inductive activity of this protein mixture on its concentration in culture solution has been studied. The test-system was the early gastrula ectoderm of Xenopus laevis frogs. The results of the experiments revealed no direct dependence of the spectrum of induced tissues on the concentration of the protein mixture. At a concentration of 0.5 mg/ml, brain appeared being accompanied by retina, pigmented epithelium, and lentoids, while at 0.031 mg/ml a perfect lens developed along with brain, retina and pigmented epithelium. At 0.125 mg/ml not only brain with accompanying structures but also muscle fibers were equally differentiated. These data suggest a new approach to the problem of dependence of the character of induction on the concentration of inducing factors, and they enable us to suppose that this dependence is not realized as a simple concentration dependence but may de determined by some adaptive, yet not elucidation processes.     

Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.     

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.     

Isolation and effects of inducing factors secreted by the lens epithelium cells

Our previous studies showed that, unlike tissue extracts, the cells of living organs secrete substances capable of inducing the same organ rudiments in the early gastrula ectoderm (EGE). In this work, the molecular nature of these substances was studied. The porcine lens epithelium was chosen for the initial analysis. When cultivated, this epithelium secreted a mixture of proteins, which were separated by gel-filtration. Both the total protein mixture and its individual fractions were tested for their inducing capacity using the early gastrula ectoderm of Rana temporaria. Unexpected results were obtained, which indicated that (a) the mixture of native proteins secreted by lens epithelium has a selective inducing capacity differing from those of individual fractions isolated from this mixture and (b) each fraction has a specific effect, but all of them cause the induction of neural tissue or sensory organs. These results (obtained for the first time) suggest that the inducing capacity of individual protein fractions is wider than that of the total protein mixture secreted by lens epithelium. This fact raises a question concerning the relationships between the mechanisms underlying the corresponding inducing effects.     

Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis

Previously, the only anuran amphibian known to regenerate the lens of the eye was Xenopus laevis. This occurs during larval stages through transdifferentiation of the outer cornea epithelium under control of factors presumably secreted by the neural retina. This study demonstrates that a distantly related species, X. tropicalis, is also able to regenerate lenses through this process. A transgenic line of X. tropicalis was used to examine the process of cornea-lens transdifferentiation in which green fluorescent protein (GFP) is expressed in differentiated lens cells under the control of the Xenopus gamma1-crystallin promoter element. Unlike X. laevis, the process of cornea-lens transdifferentiation typically occurs at a very low frequency in X. tropicalis due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening. The inner cornea endothelium serves as a key physical barrier that normally prevents retinal signals from reaching the outer cornea epithelium. If this barrier is circumvented by implanting outer cornea epithelium of transgenic tadpoles directly into the vitreous chamber of non-transgenic X. tropicalis larval eyes, a higher percentage of cases formed lenses expressing GFP. Lenses were also formed if these tissues were implanted into X. laevis larval eyes, suggesting the same or similar inducing factors are present in both species. When pericorneal ectoderm and posteriolateral flank ectoderm were implanted into the vitreous chamber, only in rare cases did pericorneal ectoderm form lens cells. Thus, unlike the case in X. laevis, competence to respond to the inducing factors is tightly restricted to the cornea epithelium in X. tropicalis. As controls, all these tissues were implanted into the space located between the inner and outer corneas. None of these implants, including outer cornea epithelium, exhibited GFP expression. Thus, the essential inductive factors are normally contained within the vitreous chamber. One explanation why this type of lens regeneration is not seen in some other anurans could be due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening once the original lens is removed. These findings are discussed in terms of the evolution of this developmental process within the anurans.     

Effect of heterogeneous inductors on the ectoderm of the early gastrula in Rana temporaria in vitro. 5. Biochemical analysis of induction-active extracts from chick embryo retina and brain

The water extracts from the retina and brain of 7-8-day old chick embryos were centrifuged at 20,000 g; sediments were discarded and supernatants were additionally centrifuged at 110,000 g. The inductive activity of supernatants (20,000 and 110,000 g) and sediments (110,000 g) was estimated in vitro on the Rana temporaria early gastrula ectoderm. The neutralizing activity was related exclusively to the soluble fractions of the extracts from the chick embryo retina and brain. The lens-inducing activity appeared to be characteristic of both the supernatants and the microsome fractions of these extracts. A comparative biochemical analysis of the extracts (isoelectrofocusing, electrophoresis in the presence of sodium dodecylsulfate, electroblotting) has shown that the chick embryo retina and brain are similar by the spectrum and properties of peptides. It is suggested that the similarity of the extracts inducing effect on the early gastrula ectoderm is due to the presence of the same proteins (peptides) in the retina and brain. Peptides with a positive immunohistochemical reaction to vimentin and peptides of neurofilaments were found in trace quantities in the retina and brain extracts by means of immunoelectroblotting.     

Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikha?lov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.     

Compatibility of chick embryo eye anlagen with the ectoderm of the early amphibian gastrula in vitro

Eye vesicles were isolated from the early chick embryos (stage 9+ after Hamburger and Hamilton, 1951) and combined with the Rana temporaria early gastrula ectoderm (EGE) in vitro. The tissues were jointly incubated in medium 199 diluted twice with deionized water at 22 +/- 1 degree for 7-8 days or the eye vesicles were removed from the EGE ectoderm within 16-18 h. At the joint long-term incubation of these tissues, a toxic effect of the chick embryonic tissues on the EGE cells was noted. In none of the experiments, the inducing effect of the eye vesicle on the EGE was found. Similar data were obtained when the EGE was jointly cultivated with the brain (stage 9-10) and retina (stage 15) of chick embryos. The brain of the chick embryos at stage 15 exerted a weak neuralizing effect on the EGE. In the control experiments, the eye vesicles explanted with the chick embryonic ectoderm remained viable till the end of cultivation but no lentoids formed in the ectoderm. The absence of lens-inducing effect at the joint cultivation of the chick embryonic eye vesicles with the EGE is considered as a result of disturbance of the synthesis or secretion of the corresponding agents rather than a sequence of the species "incompatibility" of the inductor and reacting tissue. Hence, the use of "xenogenic" tissue recombinants is not justified when analyzing the lens-inducing activity of the eye vesicles.     

Distribution of differentiation potentials and the conditions for their realization in the amphibian neuroectoderm

The X. laevis neuroectoderm (NE) at the mid and late gastrula stages is capable to form mesoderm in vitro after its separation from mesoderm. This capacity is inherent in posterior 2/3 of NE underlied by axial mesoderm in the embryo and forming deuterencephalic and trunk regions of the brain in the normal development. The archencephalic 1/3 of NE of the late gastrula, underlied in the embryo by prechordal plate, is capable of differentiation into archencephalic regions of the brain, rather than into mesoderm. For the typical differentiation of archencephalic NE to be realized, it should be surrounded by the outer ectoderm layer. In the absence of the latter, the whole explant develops into retina and brain only. Inside the closed explants, ectomesenchyme and melanophores arise and the eye material is subdivided into retina and pigmented epithelium. The archencephalic NE, dissociated to individual cells and wrapped into epidermis, forms much more ectomesenchyme and melanophores than the usual NE explants.     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

Cellular heredity, its alteration and organ restoration

Patterns of cell heredity in vertebrates and possibility of its alteration, i. e. artificial tissue metaplasia, are considered. These problems are compared with the well studied phenomenon of metaplasia in eyes of the newt in which the removal of some parts of the eye leads to natural metaplasia, an initial step for restoration of eye parts. A brief analysis of sequence of inductive processes in development shows that by the end of the period of induction and the onset of terminal differentiation the maximum concentration of specific inducing agents in induced rudiments can be expected. This suggestion was confirmed by the experiments of specific assimilatory induction in gastrula ectoderm and artificial conversion of pigmented epithelium in retina and lens tissues. On the basis of the data reported and in comparison with the theories of intragenomic regulation, a new hypothesis of cell heredity is put forward. The basic idea of this hypothesis is that the regulatory genes can switch on the genes responsible for the synthesis of terminal proteins via inducing proteins; the latter can simultaneously programm the function of regulatory genes, initiating their own synthesis. Due to such a feedback mechanism forming during development, stable cell types arise. Their inheritance can be altered by the introduction of new inducing agents in parallel with the elimination of conditions stabilizing cell differentiation. Possible ways for application of artificial metaplasia for restoration of eye defects in medical practice are considered.     

Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.

The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.     

DIFFERENCE IN INDUCTIVE EFFECT OF LIVER TISSUES WITH AND WITHOUT PERISINUSOIDAL BASEMENT MEMBRANE

Differential inductive capacities among liver tissues of several animals were examined by anticipating the correlation between the capacity and the completness of perisinusoidal basement membrane.
The reacting tissue was competent ectoderm of gastrula of Triturus pyrrhogaster , and the inductive effects of livers on the ectoderm were tested by explantation method. The inductive effect of livers being devoid of the membrane (chick and guinea pig) was neural and the tissues having the dense well-developed membrane (reptiles) produced an assembly of neural and meso-dermal tissues, such as notochord and somite or muscle. The livers with the membrane being of intermediate grade of development ( calf, Triturus and mouse) induce mesodermal tissues, but not frequently, together with neural tissue or alone. The liver tissue was more active in mesodermal induction in proportion to the completeness of the perisinusoidal basement membrane.
On the basis of these data the difference in inductive capacity among liver tissues from different kinds of animals were discussed.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

Regeneration of the amphibian retina: role of tissue interaction and related signaling molecules on RPE transdifferentiation

Regeneration of eye tissue is one of the classic subjects in developmental biology and it is now being vigorously studied to reveal the cellular and molecular mechanisms involved. Although many experimental animal models have been studied, there may be a common basic mechanism that governs retinal regeneration. This can also control ocular development, suggesting the existence of a common principle between the development and regeneration of eye tissues. This notion is now becoming more widely accepted by recent studies on the genetic regulation of ocular development. Retinal regeneration can take place in a variety of vertebrates including fish, amphibians and birds. The newt, however, has been considered to be the sole animal that can regenerate the whole retina after the complete removal of the retina. We recently discovered that the anuran amphibian also retains a similar ability in the mature stage, suggesting the possibility that such a potential could be found in other animal species. In the present review article, retinal regeneration of amphibians (the newt and Xenopus laevis) and avian embryos are described, with a particular focus on transdifferentiation of retinal pigmented epithelium. One of the recent progresses in this field is the availability of tissue culture methods to analyze the initial process of transdifferentiation, and this enables us to compare the proliferation and neural differentiation of retinal pigmented epithelial cells from various animal species under the same conditions. It was revealed that tissue interactions between the retinal pigmented epithelium and underlying connective tissues (the choroid) play a substantial role in transdifferentiation and that this is mediated by a diffusible signal such as fibroblast growth factor 2. We propose that tissue interaction, particularly mesenchyme-neuroepithelial interaction, is considered to play a fundamental role both in retinal development and regeneration.     

Xenopus frizzled-2 is expressed highly in the developing eye, otic vesicle and somites.

Wnts are secreted signaling molecules implicated in a large number of developmental processes. Frizzled proteins have been identified as likely receptors for Wnt ligands in vertebrates and invertebrates. To assess the endogenous role of frizzled proteins during the development of Xenopus laevis, we have identified several frizzled homologs. Here we report the cloning and expression of Xenopus frizzled-2 (xfz2). Xfz2 shows high sequence homology to rat and human frizzleds-2. It is expressed in the developing embryo from late gastrula stages onward. Xfz2 has a wide domain of expression but is concentrated in the eye anlage, otic vesicle, and developing somites.     

Formation of mesodermal pattern by secondary inducing interactions

Summary A highly purified vegetalizing factor induces endoderm preferentially in amphibian gastrula ectoderm. After combination of this factor with less pure fractions, a high percentage of trunks and tails with notochord and somites are induced. The induction of these mesodermal tissues depends on secondary factors which may act on plasma membrane receptors of the target cells. The secondary factors are probably proteins as they are inactivated by trypsin or cellulose-bound proteinase K. They are not inactivated by thioglycolic acid.The implication of these findings for tissue determination and differentiation in normal development in relation to the anlageplan for endoderm and mesodermal tissues is discussed.     

Dissection,Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation^1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates ^12,14-18.While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells ^7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues ^{8,19-22,24-33}. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level ^{5,8,21,24,27-30,33-39}. Xenopus laevis, a classic model system for the study of early neural development ^{19,27,29,31-32,40-42}, serves as a particularly suitable system for retinal primary cell culture ^10,38,43-45.Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction ^25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products ^10,24,44-45.However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).     

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.