The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

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.     

Correlation between immunodifferentiation and histodifferentiation in presumptive lens ectoderm of the chick in vitro

The ability of stage-4-9 chick presumptive lens ectoderm to undergo nervous tissue or lens differentiation was studied in vitro. The tissue was cultured alone or co-cultured with alcohol-killed primitive node or optic cup as inducer. Immunofluorescence was studied on paraffin-wax preparations, which were then studied histologically. An attempt was made to correlate immunological and histological differentiation. The presumptive lens ectoderm differentiated both nervous tissue and lens structures in all stages, regardless of the presence or absence of an inducer. The outcome, however, was improved when an inducer was included. The inducers were not qualitatively specific. The stage-4 ectoderm proved to be more apt than older stages to differentiate nervous tissue and form neural tube-like structures. In the former stage, lens differentiation occurred with less readiness. Older stages differentiated lens structures readily and also showed immunological signs of nervous tissue differentiation. No indication of histological differentiation, however, was apparent and no neural tube-like structures formed.     

Neural differentiation of ectodermal explants of the early gastrula from Rana temporaria under the action of various mitogens and kainic acid

The following mitogens: concanavalin A (con A), phytohemagglutinin (PHA), hydra growth factor (HGF) as well as neurotoxic agent kainic acid, caused neural differentiation (N) effects differed in value and also in character of dependence on concentration of the agent. The lowest effective concentration of con A was 75 micrograms/ml (15% neural differentiation, treatment during 3 h), and the effect reached maximum of 50-60% at 100-200 micrograms/ml. Con A concentration 50 micrograms/ml showed no effect but after 1% rabbit gamma-globulin was added, 17% neural differentiation was detected. N-effects observed after treatment of explants with con A (200 micrograms/ml, 3h) at 2 degrees and 21 degrees were similar (58 and 42% respectively). Minimum PHA concentration used (6 micrograms/ml, 18h) led to neural differentiation in 5% of explants. N-effect of PHA increased along with the concentration of the lectin and was most pronounced at 25 micrograms/ml. However, further increase in concentration (up to 200 micrograms/ml) resulted in decrease of its N-effect to 13%. At 12 micrograms/ml PHA exerted not only neural differentiation, but also lens-inducing (32%) action on the ectoderm. N-effect of HGF (2.5, 25 and 250 micrograms/ml) was lower as compared with the maximum effects of con A and PHA (30-35%). No correlation of HGF inducing action with its concentration was observed. Kainic acid showed weak N-effect (20-30%) at 1 and 10 micrograms/ml. Higher concentration (100 micrograms/ml) had no N-effect, but in 27% of explants "free" lentoids were found. Oubain (10(-3) and 10(-4) M) and HEPES (20 mM) did not affect the differentiation of explants.     

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.     

Reliability of delta-crystallin as a marker for studies of chick lens induction

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the non-lens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.     

Concanavalin A induces neural tissue and cartilage in amphibian early gastrula ectoderm

We have studied in vitro differentiation of explants of the amphibian (Rana temporaria) early gastrula ectoderm after treatment with various concentrations (50–300 μg/ml) of ‘free’ and Sepharose-bound concanavalin A (Con A). The explants were incubated with Con A for 3 h at 20°C; the rolling up of the explants was prevented by using special weights. We have demonstrated that: (1) free Con A has an inducing action on the explants in the concentration range 100–300 μg/ml medium; (2) when treated with Con A the explants produce neural tissue (50–70%), cartilage (20–40%) and, rarely, lentoids (5–10%); (3) the frequency of neural and cartilage inductions was similar at various Con A concentrations; (4) α-methyl-d-mannoside pyranoside inhibited the Con A effects; (5) Sepharose-bound Con A had no effect on the explants, although it was bound to the cell surface of the ectoderm inner layer. Possible mechanisms of the neuralizing and chondrogenic effects of Con A on ectodermal explants are discussed.     

Transferrin enhances lentoid differentiation in rat egg cylinders cultivated in a chemically defined medium

Rat egg cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically-defined medium. The purpose of the experiment was to determine whether the terminal tissue differentiation is modified by human transferrin. The control sets were grown in medium with or without rat serum. In explants treated with transferrin, groups of atypical cells of the ocular lens (lentoids) appeared more frequently than in both control sets; however neuroblasts were observed as often as in the serum-supplemented medium. Bovine serum albumin (BSA) stimulated the differentiation of neuroblasts but did not promote lentoid formation. We conclude that human transferrin does stimulate the differentiation of lentoids in rat embryonic explants, but the mechanism of its action remains unknown.     

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.     

A novel fork head gene mediates early steps during Xenopus lens formation.

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.     

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.     

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.     

BRAIN INDUCTION IN VARIOUSLY AGED PRESUMPTIVE ECTODERMS BY HEAD ORGANIZER IN CYNOPS PYRRHOGASTER

Brain formation in variously aged presumptive ectoderms of Cynops pyrrhogaster under the influence of the head organizer was examined by the sandwich method. The head organizer was obtained from the middle portion of the archenteron roof at the slit-blastopore stage. The presumptive ectoderm was taken from 0- to 36-hr exogastrulae. Exogastrulae were prepared from the earliest gastrulae just before invagination (0-hr embryos). The presumptive neural plate overlying the archenteron roof used as organizer was cultivated in an envelope of belly ectoderm from an early neurula.
The following results were obtained: 1) Brain induction was almost entirely restricted to explants covered with 6-hr ectoderm and its frequency was low. 2) The presumptive neural plate above the head organizer was almost completely determined as neural tissues. 3) The head organizer showed a tendency to differentiate into more endodermal and less mesodermal tissues than those expected from its prospective fate.
Brain induction in normal development and the relationship between neural tissue formation in variously aged presumptive ectoderms and the time necessary for neural induction are discussed.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

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.     

Critical period in the work of the form-inducing apparatus of the lens in chick embryos, detected after chloramphenicol application

The inhibitor of protein synthesis chloramphenicol (2 mg/egg) was applied to elucidate the critical period in the chicken lens development. Chloramphenicol injected before incubation and at 24, 30 and 48 h of incubation did not prevent the formation of parts in the lens-inducing apparatus (the optic vesicle, and the presumptive lens ectoderm), but the injection at the stage of 24--30 h of incubation resulted, in many survived for 5--7 days of incubation, in lack of the lens. Therefore, it is possible to speak about a disturbance in the activity of the inducing apparatus during the period of determination, or about a critical period of the lens development.     

Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.