GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells.In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises.The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.     

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ , interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.     

INDIVIDUAL VARIATION IN THE DISTRIBUTION OF LENS POTENCY IN WOLFFIAN LENS REGENERATION

After lentectomy in newts, lens regeneration originates from the iris. The regenerant was externally observed with a stereomicroscope as a depigmented area (DA) of the iris, and the extent of DA up to 15 days after lentectomy was measured. The extent of DA was found to differ among individuals, whereas it was the same in both eyes of each animal. In a number of animals one eye was used for lentectomy. After measuring the DA, two groups of animals were selected; a "W-group" with an extremely wide DA that deviated from the standard value, and "N-group", with an extremely narrow DA. Six iris sectors obtained from the animals of the W-group or N-group were implanted into lentectomized eyes of other animals to investigate the difference in the distribution of lens potency in these two groups. Animals of the W-group possessed a wider distribution of lens potency than animals of the N-group. Pulse-labelling with ³H-thymidine on lentectomized eyes of both groups was done 0, 3, 5, 7 and 12 days after lentectomy. DNA-synthesis began earlier and continued longer in the dorsal part of the iris of the W-group than in that of the N-group. The distribution of lens potency in the iris is discussed on the basis of these findings.     

CD44 expression is developmentally regulated in the mouse lens and increases in the lens epithelium after injury

Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of α-smooth muscle actin expression typically used as a marker of epithelial–mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of α-smooth muscle actin.     

Cultivation of the retinal pigment epithelium in the cavity of the lentectomized eye of newts

A study was made of proliferative activity and transdifferentiation of the cells of retinal pigment epithelium (RPE) cultivated in the cavity of the lensectomized eye of adult newt. Implantation of the newt RPE together with vascular membrane and scleral coat resulted in the regeneration of retina. In this process the character of changes in the proliferative activity of RPE and differentiation of retinal cells were the same as in the regeneration of retina in situ. RPE implanted with the vascular membrane alone, despite a high level of proliferation during the first ten days of cultivation, no differentiated retina was formed. Possible causes of these differences are discussed, and the comparison is made of the data obtained with those on RPE cultivation in vitro. After lens removal, with RPE implants present in the eye cavity, in addition to the regenerated lens, 2-3 extra lenses and retina were formed from the cells of the inner layer of the recipient's dorsal iris. Also some cases were revealed of lens formation from the cells of ventral iris. With a complete detachment of the recipient's retina (an after-effect of transplantation) a second differentiated retina regenerated in situ from the recipient's RPE cells.     

Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis

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

Transdifferentiation of Adult Frog Iris in Retina or Lens by Exogeneous Influences

The mechanisms of transdifferentiation of iris epithelial cells of Rana temporaria (Anura) in culture depending on influences from different sources were studied. In terminally differentiated iris cells, the process of transdifferentiation is initiated by dedifferentiation. Melanosomes are shed from iris cells due to cell surface activity. After depigmentation, iris epithelial cells become capable of proliferating and competent to react to the influences of various exogenous factors. Under the influence of retinal factors secreted by lentectomized tadpole eyes, both dorsal and ventral irises are converted to neural retina. Under the influence of factors from eye vesicles, the irises are converted to neural retina as well. Similar results were obtained in transfilter experiments, in which a 3-day period of transfilter interaction between the irises and eye vesicles ensured depigmentation of the iris followed by transdifferentiation into complete NR with visual receptor. Lentoid formation occurred under the influence of adult frog lens epithelium. Immunofluorescent analysis confirmed the lens nature of the lentoids. In control experiments under the conditions of the tadpole eye orbit, in which programming influences were absent, iris epithelial cells remained unaffected.
The problem of true cell-reprogramming to new differentiation in contrast to expression of inherent properties of the iris epithelial cells is discussed.     

Radioautographic study of the cellular proliferation of retinal pigment epithelium in axolotls

The proliferative activity of the pigment epithelium cells in the axolotl eyes was studied using 3H-thymidine in two types experiments: after the removal of lens, iris and retina and upon the cultivation of the pigment epithelium pieces in the cavity of lens-less eye. Irrespective of the operation type, the level of proliferation of the pigment epithelium cells changed regularly with respect to the time of observation. In the intact eye, the level of proliferation of the pigment epithelium cells was not high: the index of labelled nuclei equaled 0.5%, no mitoses were found. The highest values of the index of labelled nuclei (12.6-32.1%) and of the mitotic index (0.54-1.07%) were registered on the 10-20th days after the operation. After 40 days, the indices of proliferative activity of the pigment epithelium cells approached gradually those for the intact eye. The cultivation of the pigment epithelium cells in the cavity of a lens-less eye for 50 days did not result in their transdifferentiation into retina cells. The layered retina found in 7.7% of cases after the removal of lens, iris and retina could regenerate either from the cells of the retina growth zone localized in the region of embryonic split, or due to transdifferentiation of the pigment epithelium cells.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.     

Ultrastructure of traumatic cataractogenesis in the frog: a comparison with mouse and human lens.

Normal and needle-punctured lenses of Rana pipiens were examined with the electron microscope in order to characterize the sequence of ultrastructural changes that follow the injury over a 5-month period. Results were compared with those obtained previously in experimentally injured mouse and accidentally injured human lenses. The normal adult frog lens was found to have a morphology similar to that of mammalian lenses. As in the human, frog lens epithelial cells contained scattered microfilaments and were connected by desmosomes and gap junctions. They differed from mouse cells, which had been shown to lack desmosomes and to have microfilaments organized into dense bundles. These differences are postulated to be related to the degree of accommodative deformation of the lens displayed by these species. After injury, cellular debris and fibrin, accumulated in the wound, were phagocytized by extrinsic cells derived from the blood and ocular tissues. Leucocytes, pigmented cells and fibroblasts remained in the wound for eight weeks, along with epithelial cells which proliferated and migrated from the wound margins.Epithelial cells showed an increase in those organelles associated with protein synthesis and transport, and in microfilaments. In cataractous lenses, epithelial cells showed changes in matrix, and lens fibers became organized into smaller, denser compressed units. At five months, considerable healing had taken place, but localized opacities persisted in many frog lenses.     

CETACEAN LENS ZONES OF DISCONTINUITY—INDICES OF HEALTH AND DEVELOPMENT

Details of the lenses of eyes in two living Tursiops truncatus and one Grampus griseus were photographed. In the laboratory the lens cortex could be retro-illuminated by a fundus camera and spatially periodic stria could be visualized, in large part because of the highly developed tapetum of the cetacean eye. Varying spaces were present between the stria. On a three-dimensional basis, the best analogy is the layering of an onion. One photograph was digitized giving good quantification of the sizes of the layers and their number. Similar, but less easily resolved, lens cortex organization has been described frequently in humans as "zones of optical discontinuity." These have been explained as a periodic temporal consequence of normal aging with layer thickness depending on general health. We found that young cetaceans have few "zones" and older cetaceans have many. Lens zones measurement may be developed to Provide objective data on history of cetacean health and age.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

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

GEFT, A Rho family guanine nucleotide exchange factor, regulates lens differentiation through a Rac1-mediated mechanism

The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressed at high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, we demonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and that overexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us to explore the expression of GEFT in other tissues, which share common developmental ancestry to the nervous system, specifically the ocular system. Using immunohistochemical analysis specific for GEFT protein expression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cells induced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strong increase in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFT resulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is in part dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blocked GEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants. These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eye development. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.     

Mechanisms of repression and derepression of artificial transformation of pigmented epithelium into retina inXenopus laevis

Summary The present study shows that pigmented epithelium of tadpoles and adult frogs ofXenopus laevis, like that of the other Anurans and the Cyprinid fishes, cannot transform into retina without the action of retinal factors. Transformation of pigmented epithelium into retina occurs when a sheet of it is implanted into the lensless eye. Transformation of pigmented epithelium also occurs when a sheet of it is wrapped in Bruch's membrane of the adult frog and afterwards implanted into a lensless eye, thus suggesting that Bruch's membrane is permeable to the inducing factors. Bruch's membrane was shown to play a polarizing role in the newly formed retina. Artificial transformation is based on a mechanism involving both the elimination of the repressive action of membranes adjacent to pigmented epithelium and the influence of retinal factors.     

Developmental aspects of galectin-3 expression in the lens

In order to investigate the temporal and spatial expression pattern of the lectin galectin-3 during lens development we performed immunohistochemical studies using monoclonal and polyclonal antibodies against galectin-3 on paraffin sections of human, mouse and rat eyes. Galectin-3 has been shown to be involved in various biological functions related to cell adhesion, proliferation, apoptosis and differentiation in other tissues. In the human lens, galectin-3 shows a selective expression pattern during lens development. It is present in all cells of the early lens vesicle and at later stages it is strongly expressed during the elongation phase in differentiating primary lens fibres. From about 7 weeks onwards the anterior lens epithelium fails to express galectin-3. Adult lenses, however, exhibit immunoreactivity in the anterior epithelial cells and in the early differentiating secondary fibres of the lens' outer cortex prior to the onset of degradation of the nuclei. In contrast to the observed expression pattern in prenatal human lenses, mouse and rat lenses exhibited immunoreactivity for galectin-3 during postnatal and adult stages only. At these stages, the expression pattern closely resembles that seen in the corresponding human lenses. The spatiotemporal pattern of galectin-3 distribution during lens development favours a role of this lectin in adhesion processes and in the regulation of programmed organelle elimination during lens cell differentiation.     

家兔晶状体纤维的超微结构扫描电镜和冰冻断裂的研究

关于脊椎动物眼睛晶状体纤维细胞的研究,已有不少报道,但涉及到其细胞表面结构的有关工作还不多。至于不同部位细胞的结构,特别是晶状体核纤维细胞的构形,报道也不完全一致。本文主要介绍用常规扫描电镜以及冰冻断裂扫描电镜技术,研究家兔晶状体纤维细胞的三维结构。Hansson(1970);Sakuragawa和Kuwabara(1975);Nelson和Rafferty(1976)均以小自鼠为材料。Farnsworth(1974)以及Sakuragawa(1975)分别用大白鼠为研究对象。他们先后开展了晶状体的扫描电镜研究,并对该课题的发展作出了一定贡献。Dickson和Crock(1972;1975)将冰冻断裂扫描电镜技术引入对猴晶状体