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.     

Biochemical investigation of lens induction in vitro. I. Induction properties of the eye cup and ectodermal response

1. Optic cups of 48, 72 and 96 hours old chick embryos were prepared, cultured and recombined with ectoderm. With the optic cups of 48 hours old embryos, lens formation occurred in 16% of the cases. With the optic cups of 72 hours old embryos, lens formation occurred in 28% of the cases. Optic cups of 96 hours old embryos were not able to induce a lens. 2. The optic cup proved to be able to induce a lens more than once. 3. Ectoderm of the head of 72 hours old embryos was still able to form a lens. 4. Using homogenized eye cups of 72 hours old embryos, lens induction occurred only in a few cases. When the optic cups were cut into small pieces, lens induction occurred in 30% of the cases. This suggests that intact cells are necessary to obtain lens induction.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

Positional heterogeneity of interaction between fragments of avian limb bud in organ culture

We have previously succeeded in culturing whole leg bud from stage 21-23 chick embryos and observed a leg structure with typical cartilage pattern in vitro. In the present study, we have attempted the organ culture of the fragmented leg bud and investigated its capacity to form cartilage. Leg buds from stages 17-21 chick embryos were dissected into four pieces in the anteroposterior sequence (named 1, 2, 3, and 4, respectively) and cultured on a membrane filter in a medium consisting of Ham's F-12, chick serum, and chick embryo extract. After 6 days in culture, two central fragments (2 and 3) developed into large cartilaginous masses, while anterior (1) and posterior (4) fragments formed few or small cartilaginous masses. In addition, when these less chondrogenic fragments were combined, pinned together, and cultured, large cartilaginous masses were formed from 1 + 4 combinations but not from 1 + 1 or 4 + 4 combinations. These observations were analyzed quantitatively by measurement of 35SO4 incorporation into the sulfated glycosaminoglycan (S-GAG) and of final DNA content per explant, and by histological reconstruction of the chick-quail chimera explant. The results showed that (a) the 1 + 4 combination resulted in higher S-GAG synthesis and final DNA content than the 1 + 1 or 4 + 4 combinations in stage 18 and 21 leg buds (P less than 5%); (b) removal of ectoderm from the leg bud inhibited the increase observed for the 1 + 4 combination; c) in chick-quail chimera explants the cartilage formed from the 1 + 4 combination was largely of fragment 1 origin. These results demonstrate, first, the presence of a difference in chondrogenic capacity along the anteroposterior axis in the leg bud and, second, the occurrence of an interaction between anterior and posterior fragments which mimics the effects of grafting a zone of polarizing activity (ZPA). The mechanism of ZPA function is still unknown but the ectoderm may play some role. Some roles for ectoderm in ZPA function and differences in mesodermal responsiveness to ZPA factor(s) are suggested.     

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.     

Differentiation of presumptive lens ectoderm of the chick in vitro studied with immunofluorescence techniques

The differentiation capacity of presumptive lens ectoderm was studied in the chick by an in vitro technique using the appearance of central nervous system or lens-specific antigens as indicators of differentiation. Handling the explants resulted in 'autodifferentiation' of both antigens, but co-culture with alcohol-killed primitive node or optic cup material could induce much stronger differentiation. Little specificity exists in the reaction and a hypothesis is presented whereby selection between the two differentiation pathways is thought to be due mainly to maturation within the ectoderm and the inducing tissue plays a minor qualitative role.     

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.     

Experimental manipulation leading to induction of dorsal ectodermal ridges on normal limb buds results in a phenocopy of the eudiplopodia chick mutant

Elongation of chick limb buds depends on the presence of the apical ectodermal ridge which is induced by subjacent limb bud mesoderm. Recombination experiments have shown that the limb bud mesoderm loses the capacity to induce ridges by late stage 17. Moreover, in normal limb development only one ridge forms. However, in the eudiplopodia chick mutant accessory ectodermal ridges form on the dorsal surface of limb buds as late as stage 22. Tissue recombinant experiments show that the mutation affects the ectoderm, extending the time it responds to ridge induction (Fraser and Abbott, 1971a, Fraser and Abbott, 1971b while the mesoderm is normal. The result is polydactyly, with extra digits dorsal to the normal digits. Because eudiplopodia limb bud dorsal mesoderm can induce ridges at stage 22 but is unaffected by the gene, genetically normal dorsal limb bud mesoderm may also be able to induce ridges after stage 17. To test this possibility we grafted stages 14–18 flank ectoderm to normal limb bud dorsal mesoderm and found that mesoderm from stages 17 through 20 was able to induce a ridge and subsequently dorsal digits developed. Limbs with duplicate digits were similar to eudiplopodia limbs. In other experiments, stage 18, 19, and 20 leg bud dorsal ectoderm did not form ridges when grafted to leg bud dorsal mesoderm of the same stage, indicating a lack of response to the mesoderm. Finally, the inductive capacity of limb bud mesoderm appeared to be reduced compared to mesoderm at pre-limb bud stages. These experiments demonstrate a spatially generalized potential in limb bud dorsal mesoderm to induce ridges during the stages when the apical ridge is induced. The determination of where the ridge will form and the acquired inability of limb bud dorsal ectoderm to respond to induction by underlying mesoderm are necessary early pattern forming events which assure that a single proximodistal limb axis will form.     

The distribution of competence for adenohypophysis development in the ectoderm of chick embryos

The ability of various zones of the cephalic and trunk ectoderm to differentiate into adenohypophysis after the contact with the bottom of the prosencephalon was studied in tissue culture of chick embryos as the stage of 10-13 somites. Stomodeal presumptive lens ectoderm and lateral cephalic ectoderms were shown to be competent for development into adenohypophysis. In all cases adenohypophyseal cords were formed in the zones of ectoderm contact with the brain. The cords contained antigens A-2, A-3 specific for chicken adenohypophysis as well as ACTH and beta-lopotropin. Trunk ectoderm proved to be incapable to differentiate into adenohypophysis.     

Axonal pathfinding in organ-cultured embryonic avian retinae

Eye cups from stage 14-28 (E2 to E5) chick and quail embryos consisting of neural retina, lens, and vitreous body were cultured for 1 or 2 days. These eyes expanded by proliferation of the retinal cells and the surface areas of the retinae increased several-fold. The area covered by ganglion cells and axons also expanded in vitro. [3H]Thymidine labeling showed extensive proliferation of the neuroepithelial cells including the formation of new ganglion cells. Culturing eyes from embryos before stage 17 results, as in vivo, in the generation of the first ganglion cells of the retina, but unlike in the in vivo situation, the outgrowing axons always formed a random fiber net in the central portion of the retina. A defined axonal pattern identical to the in vivo developed only in specimens from embryos of stage 17 and older. Some aberrant axons, however, were also observed at the retinal periphery in specimens from embryos of more advanced stages (20-24), but only during the second day of culturing. Axons in retinae from embryos of stages 23 to 26 heading toward the optic fissure often crossed the fissure and, in contrast to the situation in vivo, invaded the opposite retinal side. These axons of wrong polarity followed the pathways of axons growing centripetally but in reverse direction. This suggests that the polarity of growing nerve fibers and their course are determined by different factors. Culturing the eyes of embryos from stages 20 to 25 in the presence of antibodies showed that the antibodies penetrated the entire retina with 6 hr. Neither anti-N-CAM nor the T-61 antibody--both recognizing membrane proteins of retinal cells--affected the growth of the eyes in vitro. The development of the axonal pattern in vitro was not affected by incubation with N-CAM-antibodies at concentrations up to 500 micron/ml, whereas the T-61 antibody which is known to block neurite extention in vitro (S. Henke-Fahle, W. Reckhaus, and R. Babiel (l984). "Developmental Neuroscience: Physiological, Pharmacological, and Clinical Aspects," pp. 393-398. Elsevier, Amsterdam/New York) showed inhibition of axonal growth in retina cultures at 50 micron/ml. These results indicate that the eye cultures can be used as a test system for antibodies against antigens which could be involved in axon extension and neurite pathfinding in situ.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Water-soluble proteins in early amphibian embryos. III. Immunoelectrophoretic analysis of the antigenic changes in the ectoderm of the early gastrula and neural plate during development

20 water-soluble antigen have been identified with the help of rabbit antisera to extracts of the early gastrula ectoderm and neural plate in Rana temporaria. All of them were also found in the early blastula embryos and unfertilized eggs. The identified antigens are characterized by a definite embryospecificity. As the development proceeds, the concentration of these antigens in the embryonic tissues decreases until the complete disappearance of corresponding immunoelectrophoretic reactions. By this characteristic all antigens under study are subdivided into four groups: I--five antigens identified at the early developmental stages only (until hatching, stage 29); II--nine antigens present up to stages 33--35; III--three antigens followed up to stages 39--40 (well formed tadpole); IV--three antigens were found at all developmental stages under study up to stages 45--47. 11 out of 20 identified antigens have antigenic similarity with the proteins of blood serum of adult amphibians. Besides, the early gastrula ectoderm contains antigens similar with those of the brain of adult amphibians.     

Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti-XHE (Xenopus hatching enzyme) as a marker. The anterio-dorsal ectoderm excised from stage 12-13 (mid-late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio-, but not the anterio-dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA-1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.     

SCANNING ELECTRON MICROSCOPICAL APPEARANCE OF CELLS FROM XENOPUS LAEVIS EMBRYOS OF DIFFERENT STAGES

The scanning electron microscopical appearances of cells isolated from different regions of Xenopus laevis embryos of different stages, and cultured in vitro have been compared. Blastula inner ectoderm cells initially show filopodia, then become flattened onto the substrate and then form pseudopodia. Blastula outer ectoderm cells are initially similar, but do not form pseudopodia. Most of the ectoderm cells from gastrulae and neurulae are featureless. Endoderm cells from blastulae do not initially form filopodia, but later form pseudopodia. Most of the endoderm cells from gastrulae and neurulae show neither filopodia nor pseudopodia, but in the gastrula some elongated, cylindrical cells are observed. Thus cells change their appearance after the three hour culture period; cells from different regions of embryos of the same stage show different appearances in vitro ; and cells from equivalent regions of embryos of different stages show different behaviours in vitro.     

The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo

Summary Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating³H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of³H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.     

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.     

Defining progressive stages in the commitment process leading to embryonic lens formation

The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well‐studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ～ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment. genesis 50:728–740, 2012. © 2012 Wiley Periodicals, Inc.