The appearance of alpha-crystallin in relation to cell cycle phase in the embryonic mouse lens

Specific protein synthesis in the embryonic mouse lens was studied by immunofluorescence with antisera to adult mouse lens or crystallin fractions. Positive reactions were first detected in a few cells of the lens cup 18-24 hr after contact between optic vesicle and presumptive lens ectoderm had been established. During formation of the lens vesicle a rapidly increasing fraction of cells produced crystallins. At the time of detachment of the vesicle from the surface all cells of its posterior wall showed immunofluorescence. After fiber elongation became distinct cells of the anterior epithelium began to fluoresce and shortly afterwards the entire rudiment produced crystallins. The early reactions were due entirely to the presence of alpha-crystallin. Reactions were restricted to the lens. Thus, in the mouse as in other species crystallins were detectable by immunofluorescence in vivo only after lens morphogenesis was well underway and only in the lens rudiment itself. Cells first synthesizing crystallins always had an elongated shape and their nuclei were in a basal position. A few hours later mitotic cells displayed fluorescence. Taking into account earlier found relations between cell morphology and cell cycle phase, this indicates that alpha-crystallin is first demonstrable in the S-or early G-2 phase of the cell cycle, and that the start of its synthesis does not preclude continued cell replication. It is interesting that the cellular location, cell cycle phase, and developmental stage, in which crystallins first appear, are comparable in mouse and chick embryo. Yet, entirely different proteins are involved: alpha-crystallin in the first, delta-crystallin in the latter. Implications of this for our understanding of lens induction are discussed.     

Induction of the Eye Lens

In general terms embryonic induction involves the association of embryonic tissues and leads to tissue differentiation. It is one of the known essential processes leading to the normal development of embryos. However, despite its importance, very little is known about the mechanisms of inductive interactions. For example, what is the nature of communication between tissues, how does this communication effect the synthetic activity of the cells, and once a new pattern of synthesis has been established how is the sequence of events leading to tissue differentiation co-ordinated? The answers to these questions will come only from the intensive study of inductive interactions and tissue differentiation at all levels from the morphological to the molecular.
One of the best known examples of induction, at least superficially, is the differentiation of lens from head ectoderm after its interaction with optic vesicle. The popularity of this tissue with embryologists may be attributed to its accessibility of manipulation because of its position on the outside of the embryo. In addition, its distinct morphology and specific biochemical composition make it relatively easy to determine whether the lens differentiates after experimental treatment. About the turn of this century lens differentiation was thought to depend on the specific interaction of just two embryonic tissues, head ectoderm and neuro-ectoderm (optic vesicle). However, experimental analysis since then has revealed that this oversimplified view of lens induction is incorrect. In fact there is evidence that a large number of other tissues besides embryonic head ectoderm can differentiate into lens and that other conditions besides the presence of optic vesicle can induce lens differentiation. The purpose of this work is to review the evidence on lens induction and based on this, to determine what we know about the mechanism(s) controlling this process.     

Changes in the Glycoprotein Concentration of the Extracellular Matrix between Lens and Optic Vesicle Associated with Early Lens Differentiation

The glycoproteins of the extracellular interface between the lens rudiment and the optic vesicle of the chicken embryo were demonstrated histochemically and their concentration was measured by microspectrophotometry through-out the period of lens induction. The concentration reaches a peak immediately preceding the maximum elongation of the lens cells and the onset of the synthesis of lens-specific proteins, suggesting the involvement of matrix density increase in lens induction.     

FGF signals induce Caprin2 expression in the vertebrate lens

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.     

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro

We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/polyacryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies, within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either alpha- or beta-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.     

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro

Abstract. We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/poly-acryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies. within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either α or β-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.     

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.     

Reinvestigation of the role of the optic vesicle in embryonic lens induction

The induction of the lens by the optic vesicle in amphibians is often cited as support for the view that a single inductive event can lead to determination in a multipotent tissue. This conclusion is based on transplantation experiments whose results indicate that many regions of embryonic ectoderm which would normally form epidermis can form a lens if brought into contact with the optic vesicle. Although additional evidence argues that during normal development other tissues, acting before the optic vesicle, also contribute to lens induction, it is still widely held, on the basis of these transplantation experiments, that the optic vesicle alone can elicit lens formation in ectoderm. While testing this conclusion by transplanting optic vesicles beneath ventral ectoderm in Xenopus laevis embryos, it became apparent that contamination of optic vesicles by presumptive lens ectoderm cells can generate lenses in these experiments, illustrating the need for adequate host and donor marking procedures. Since previous studies rarely used host and donor marking, it was not clear whether they actually demonstrated that the optic vesicle can induce lenses. Using careful host and donor marking procedures with horseradish peroxidase as a lineage tracer, we show that the optic vesicle cannot stimulate lens formation in neurula- or gastrula-stage ectoderm of Xenopus laevis. Since the general conclusion that the optic vesicle is sufficient for lens induction rests on studies in many organisms, we felt it was important to begin to test this conclusion in other amphibians as well. Similar experiments were therefore performed with Rana Palustris embryos, since it was in this organism that optic vesicle transplant studies had originally argued that this tissue alone can cause lens induction. Under conditions similar to those used in the original report, but with careful controls to assess the origin of lenses in transplants, we found that the optic vesicle alone cannot elicit lens formation. Our data lead us to propose that the optic vesicle in amphibians is not generally sufficient for lens induction. Instead, we argue that lens induction occurs by a multistep process in which an essential phase in lens determination occurs as a result of inductive interactions preceding contact of ectoderm with the optic vesicle.     

Immunofluorescent studies on aphakia,a mutation of a gene involved in the control of lens differentiation in the mouse embryo

Aphakia, an autosomal recessive single gene mutation in the mouse, seriously affects the development of the ocular lens. Up to advanced stages of lens invagination morphogenesis proceeds normally. In the late lens cup and early lens vesicle stage, however, the epithelium of the lens rudiment becomes disorganized and the lumen of the vesicle fills up with rounded cells, apparently released from the epithelium. The lens stalk persists frequently. Probably as a consequence of the aphakic state other parts of the eye secondarily become abnormal.Immunofluorescent studies were done on embryonic normal and aphakia eyes with antisera against adult mouse crystallins. In the normal embryo the first positive reactions were found in the late lens cup stage ($\text{10}\text{3}\text{4}\text{–11}$ days of gestation). By Day 12 all cells of the lens vesicle were brightly fluorescent. A day later the cells of the posterior wall, now lens fibers, had elongated sufficiently to obliterate the lumen of the vesicle. The entire organ was highly fluorescent, indicating that all of its cells contained large amounts of crystallins. The mutant lens, studied over the same time span, showed no reaction at all. The most likely explanation is, that the multiple structural genes, which normally must be involved in the production of the crystallins, are not expressed up to this time in the mutant.The combination of morphological and biochemical defects suggests that the gene involved in the mutation somehow functions in the control of lens differentiation.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. I. Composition and properties of water-soluble lens antigens]

Antisera were obtained to the total extract and individual electrophoretic fractions of lens proteins: alpha-, beta-, gamma1- and gamma2-crystallins. The crystallins under study are immunochemically heterogenous: each class of lens proteins contains 2--4 antigens. Using the indirect method of fluorescent antibodies, it was established that the appearance of crystallins during development coincided with the onset of formation of the presumptive lens fibers. No crystallins were found in the lens placode and early lens vesicle. gamma-Crystallins appear later than the other lens proteins and are characteristic, mainly, for the lens fibers; at the advanced stages of organogenesis gamma-crystallins are regularly found in the epithelial cells of the developing lens as well.     

Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye

Chimaeric mice were made by aggregating Pax6(-/-) and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell-autonomous removal of Pax6(-/-) cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6(-/-) cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6(-/-) cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6(-/-) optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.     

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.     

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.     

The lens: a classical model of embryonic induction providing new insights into cell determination in early development

The lens was the first tissue in which the concept of embryonic induction was demonstrated. For many years lens induction was thought to occur at the time the optic vesicle and lens placode came in contact. Since then, studies have revealed that lens placodal progenitor cells are specified already at gastrula stages, much earlier than previously believed, and independent of optic vesicle interactions. In this review, I will focus on how individual signalling molecules, in particular BMP, FGF, Wnt and Shh, regulate the initial specification of lens placodal cells and the progressive development of lens cells. I will discuss recent work that has shed light on the combination of signalling molecules and the molecular interactions that affect lens specification and proper lens formation. I will also discuss proposed tissue interactions important for lens development. A greater knowledge of the molecular interactions during lens induction is likely to have practical benefits in understanding the causes and consequences of lens diseases. Moreover, knowledge regarding lens induction is providing fundamental important insights into inductive processes in development in general.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

Functions of crystallins in and out of lens: Roles in elongated and post-mitotic cells

The vertebrate lens evolved to collect light and focus it onto the retina. In development, the lens grows through massive elongation of epithelial cells possibly recapitulating the evolutionary origins of the lens. The refractive index of the lens is largely dependent on high concentrations of soluble proteins called crystallins. All vertebrate lenses share a common set of crystallins from two superfamilies (although other lineage specific crystallins exist). The α-crystallins are small heat shock proteins while the β- and γ-crystallins belong to a superfamily that contains structural proteins of uncertain function. The crystallins are expressed at very high levels in lens but are also found at lower levels in other cells, particularly in retina and brain. All these proteins have plausible connections to maintenance of cytoplasmic order and chaperoning of the complex molecular machines involved in the architecture and function of cells, particularly elongated and post-mitotic cells. They may represent a suite of proteins that help maintain homeostasis in such cells that are at risk from stress or from the accumulated insults of aging.     

Ontogeny and localization of the α, β, and γ crystallins in newt eye lens development

The α-, β-, and γ-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the α-, β-, and γ-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. β-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. γ-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and α-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for β-crystallins until late in lens morphogenesis, and α- and γ-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.     

Chick lens differentiation in vitro

If that portion of a chick embryo destined to form the eye, the lens, part of the brain and the surrounding head region be removed from the chick before the lens has begun to develop, and placed in a liquid culture medium for four days, a structure resembling the lens will form in appropriate proximity to the presumptive retina, and the cells of the lens will synthesize proteins unique to and characteristic of the lens. The time course of morphological development of such explants is here described. In vitro the lens placode dose not invaginate to form a lens vesicle, as it does in ovo. Instead placode cells elongate directly to form fibre cells. The shape of the lens formed in this aberrant manner is remarkably similar to that of normal lens. At least one, and probably all three, of the characteristic crystallins of the lens form in these aberrant lenses, the cytological and biochemical differentiation of which proceeds normally, despite failure of the normal morphogenetic activities of the organ.