Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.     

c-myc mRNA is elevated as differentiating lens cells withdraw from the cell cycle

Explants of the central region of lens epithelia from early chicken embryos differentiate in vitro to form lens fiber cells when cultured in the presence of chicken vitreous humor. Hybridization of a 32P-labeled v-myc viral oncogene DNA probe to RNA extracted from differentiating explants and immobilized on nitrocellulose filters indicates that levels of 2.5 kb c-myc mRNA are transiently elevated 5-10-fold in the differentiating cells. Increased levels of c-myc mRNA are observed within 30 min of the initiation of differentiation in vitro and persist for 8-9 h. Thymidine labeling of nuclei in differentiating explants indicates that entry of cells into S phase is inhibited during this period, as differentiating cells complete a final round of mitosis and withdraw from the cell cycle. Levels of c-myc mRNA are also elevated in the peripheral region of the lens epithelium, which contains cells undergoing differentiation in vivo, suggesting that the regulation of c-myc mRNA which occurs in vitro may also occur in vivo. c-myc mRNA, c-fos mRNA, and c-src mRNA showed distinct patterns of regulation associated with lens fiber formation in vivo, thus providing evidence that the regulation of c-myc mRNA is specific to this proto-oncogene. The finding that c-myc mRNA undergoes a specific, transient elevation in differentiating lens cells as they withdraw from the cell cycle contrasts with a large body of evidence linking enhanced c-myc expression with increased cell proliferation.     

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.     

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.     

The effect of growth rate on differentiation of chick embryo limb bud mesenchyme in organ culture

Small explants of limb bud mesenchyme of day chick embryos which form muscle in organ culture synthesize proportionally less protein than DNA than do large explants which form cartilage. Chondrogenesis occurred in the central area of greatest population density in reaggregating limb bud cells, myotubes in areas of lesser density and fibroblasts in the sparsely populated periphery. Small explants grown in microdrops in plastic dishes undergo less cell division and form cartilage, but not muscle. Small explants on lens paper undergo more cell division and form muscle, but not cartilage.     

Phosphatidylcholine and phosphatidylethanolamine metabolism during lens fiber cell formation

Phosphatidylinositol is metabolized with a half-life of about 5 h in lens epithelial cells of 6-day-old embryonic chickens. When these cells differentiate to form lens fiber cells, however, phosphatidylinositol turnover virtually ceases. The present study was undertaken to determine whether there is a similar change in the metabolism of phosphatidylcholine and phosphatidylethanolamine. [32P]Orthophosphate was injected into 6-day-old chicken embryos, and the incorporation of label into phosphatidylcholine and phosphatidylethanolamine was followed for 48 h. The specific activities of the precursors phosphorylcholine and phosphorylethanolamine were also measured during this time. The data were then analysed by means of a simple kinetic model to determine the rate of synthesis and the half-life of each phospholipid. The results showed that phosphatidylcholine is synthesized at a rate of about 1.2 X 10(-20) mol/s per cell in the lens epithelial cells, and 6.4 X 10(-20) mol/s per cell in the fiber cells. Phosphatidylethanolamine is synthesized at approximately 0.9 X 10(-2)) mol/s per cell in the epithelial cells, and 4.0 X 10(-20) mol/s per cell in the fiber cells. Both phospholipids are stable in both the epithelial cells and in the fiber cells, with half-lives of 48 h or greater. Thus, although phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol all experience an increase in synthesis following lens fiber formation, the previously observed decrease in phosphatidylinositol turnover accompanying differentiation is a specific effect.     

Demonstration of Transdifferentiation of Neural Retina from Pigmented Retina in Culture1

The formation of neural retina (NR) from retinal pigmented epithelium (RPE) of chick embryos in culture was investigated. In cultures of explants of PRE, depigmented, preretinal foci, consisting of 50 to 100 cells appeared in the pigmented central portion of the explant within three days. Then these depigmented cells increased rapidly in number and by about day 14 they formed characteristic spherical bodies, which were identified as a neural retinal-like structure (NR structure) by electron microscopic observations. Culture of explants of RPE from embryos of different stages showed that the capacity of embryonic RPE to form an NR structure decreased steadily with embryonic age from st. 24 to 27. At and after stage 27, no foci leading to the neural retinal differentiation were formed in the explants. Medium conditioned by cell cultures of chicken embryonic NR, RPE or chondrocytes had no effect on the formation of NR structures by explants of RPE.     

The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.     

The high turnover rate of phosphatidylinositol in chicken heart ventricular cells during the earlier stages of development

The number of the chicken ventricular cells develops exponentially up to Day 12 in the developmental stages of embryos, after which the number gradualy decreases. The phosphatidylinositol metabolism at various stages in development (Days 5 to 21) has been studied. Ventricular cells were incubated in a physiological solution containing ³²P_i, or [1,3-³H]glycerol. Radioactivities incorporated into the phosphatidylinositol were estimated. The specific activity of [1,3-³H]glycerol, taken into phosphatidylinositol, at Day 12, was shown to be approximately equal to that of the other classes of phospholipids. However, the rate of labeling of ³²P_i into the phosphatidylinositol was extremely high in comparison with the other classes of phospholipids in the same ventricles. These results show that there is a rapid turnover of the phosphorylinositol moiety in the phosphatidylinositol in the earlier stages of development. This high turnover rate of the phosphatidylinositol was observed up to Day 12, after which it began to decrease. This turning point of the phosphatidylinositol metabolism coincided well with the decrease of the rate of cell proliferation. Therefore, this rapid turnover of phosphatidylinositol could have a specific functional role related to cell division. This rapid turnover of the phosphorylinositol moiety of the phosphatidylinositol associated with ventricular cell proliferation at different embryonic stages is reported for the first time.     

NCAM in the differentiation of embryonic lens tissue

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.     

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.     

Early Determination of Developmental Fate in Presumptive Intestinal Endoderm of the Chicken Embryo

The endodermal epithelia of esophagus, proventriculus and gizzard of 6-day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract.
The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6-day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland-like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization.
These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into intestinal epithelium.     

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.     

Primary Cultures of Embryonic Chick Lens Cells as a Model System to Study Lens Gap Junctions and Fiber Cell Differentiation

A major limitation in lens gap junction research has been the lack of experimentally tractable ex vivo systems to study the formation and regulation of fiber-type gap junctions. Although immortalized lens-derived cell lines are amenable to both gene transfection and siRNA-mediated knockdown, to our knowledge none are capable of undergoing appreciable epithelial-to-fiber differentiation. Lens central epithelial explants have the converse limitation. A key advance in the field was the development of a primary embryonic chick lens cell culture system by Drs. Sue Menko and Ross Johnson. Unlike central epithelial explants, these cultures also include cells from the peripheral (preequatorial and equatorial) epithelium, which is the most physiologically relevant population for the study of fiber-type gap junction formation. We have modified the Menko/Johnson system and refer to our cultures as dissociated cell-derived monolayer cultures (DCDMLs). We culture DCDMLs without serum to mimic the avascular lens environment and on laminin, the major matrix component of the lens capsule. Here, I review the features of the DCDML system and how we have used it to study lens gap junctions and fiber cell differentiation. Our results demonstrate the power of DCDMLs to generate new findings germane to the mammalian lens and how these cultures can be exploited to conduct experiments that would be impossible, prohibitively expensive and/or difficult to interpret using transgenic animals in vivo.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage^1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.     

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.