A unique aged human retinal pigmented epithelial cell line useful for studying lens differentiation in vitro.

Lens regeneration occurs in some urodeles and fish throughout their adult life. Such an event is possible by the transdifferentiation of the pigment epithelial cells (PECs) from the dorsal iris. Studies of this event at the cellular level have been facilitated owing to the ability of PECs to become lens cells even when they are placed in culture, outside of the eye. In fact, PECs possess the capacity for transdifferentiation regardless of the origin of species or age. However, studies at the molecular level are still hindered by the intrinsic problems of primary cultures, namely storage, reproducibility and genetic manipulation. In an attempt to establish an ideal model system for lens transdifferentiation, we have analyzed the ability of a human dedifferentiated PEC line to differentiate into lens. We have found that this cell line can indeed be induced to synthesize crystallin and morphologically differentiate to three-dimensional structures resembling lentoids under controlled treatment in vitro. Gene expression studies also provided important insights into the role of key genes. This human cell line can be used for detailed genetic studies in order to identify the key factors involved in lens transdifferentiation from PECs.

FGF: an autocrine regulator of human lens cell growth independent of added stimuli.

PURPOSE: Posterior capsule opacification (PCO) arises because of a persistent growth of lens epithelial cells. Cultured human lens cells residing on their native collagen capsule and maintained in serum-free medium actively grow and thus show an intrinsic capacity for regulation. In the present study, the authors investigated the role of the putative FGF autocrine system in human capsular bags. METHODS: Capsular bags were prepared from human donor eyes and maintained in a 5% CO(2) atmosphere at 35 degrees C. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence cytochemistry. De novo protein synthesis was determined by the incorporation of 35S-methionine. Basic fibroblast growth factor (FGF) and FGF receptor (R)-1 were detected using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) techniques. FGFR-1 inhibition was achieved using the specific antagonist SU5402. RESULTS: Human lens epithelial cells can maintain metabolic activity for more than 1 year in a protein-free medium. Basic FGF was shown to be present in capsular bags throughout culture and also in capsular bags removed from donor eyes that had previously undergone cataract surgery. Furthermore, FGFR-1 was identified. Inhibition of FGFR-1 caused a significant retardation of growth on the posterior capsule. On no occasion did any treated bag reach confluence, whereas all match-paired control samples did. CONCLUSIONS: The results provide evidence that FGF plays an integral role in the long-term survival and growth of human lens epithelial cells, independent of external stimuli. Inhibition of FGFR-1 by specific synthetic molecules, such as SU5402, could provide a potential therapeutic approach to resolving PCO.

Role of retinoic acid in lens regeneration.

Prompted by the actions of retinoids and their receptors in gene regulation, in the developing eye and especially in the lens, we have undertaken a detailed study to examine the effects of retinoids on urodele lens regeneration. First, we examined the effects of exogenous retinoids. It was found that exogenous retinoids had no significant effect on lens regeneration. However, when synthesis of retinoic acid was inhibited by disulfiram, or when the function of the retinoid receptors was impaired by using a RAR antagonist, the process of lens regeneration was dramatically affected. In the majority of the cases, lens regeneration was inhibited and lens morphogenesis was disrupted. In a few cases, we were also able to observe ectopic lens regeneration from places other than the normal site, which is from the dorsal iris. The most spectacular case was the regeneration of a lens from the cornea, an event possible only in premetamorphic frogs. These data show that inhibition of retinoid receptors is paramount for the normal course and distribution of lens regeneration. We have also examined expression of RAR-delta during lens regeneration. This receptor was expressed highly in the regenerating lens only. Therefore, it seems that this receptor is specific for the regeneration process and consequently such expression correlates well with the effects of RAR inhibition observed in our studies.

Regulation of Prox 1 during lens regeneration.

PURPOSE: To determine the expression pattern of Prox 1 during the process of lens regeneration in the urodele Notophthalmus viridescens. METHODS: Polymerase chain reaction was performed to amplify a partial newt Prox 1 sequence. In situ hybridization and immunodetection methods were used to detect the Prox 1 mRNA and the Prox 1 protein, respectively. RESULTS: Prox 1 mRNA was present in the retina and in the lens (in the epithelium and bow region) of the intact eye. Prox 1 protein was found to be predominantly present in the lens and dorsal iris of the intact eye, although some trace levels of Prox 1 protein were detected in the ventral iris as well. After lentectomy, expression of the mRNA was also pronounced in the dorsal dedifferentiating iris and the regenerating lens. The ventral iris also expressed Prox 1 but seemingly at lower levels. Although Prox 1 protein showed upregulation in the dorsal iris during the process of lens regeneration, trace levels were also detected in the ventral iris. In the retina, Prox 1 protein was distributed in horizontal cells of the inner nuclear layer, whereas the mRNA was expressed in all layers of the retina. CONCLUSIONS: Prox 1 was unevenly distributed in the intact cells of the newt iris, with significantly higher levels of Prox 1 protein present in the dorsal versus the ventral margin. This protein was differentially regulated during the process of lens regeneration, with obvious upregulation in the dorsal iris. Prox 1 is the first transcriptional factor to be shown to be regulated in the dorsal versus ventral iris during the process of lens regeneration.

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles.

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.

Regulation of lens regeneration by fibroblast growth factor receptor 1.

Lens regeneration in vivo is restricted to some urodeles only. After removal of the lens, this remarkable event is initiated from the dorsal iris. The pigmented epithelial cells from the dorsal iris dedifferentiate and subsequently transdifferentiate to form the regenerating lens. This property of the dorsal iris implies specific regulation along the dorsal-ventral axis. To date, no known genes are known to be specifically expressed in the dedifferentiating cells and to be involved in lens regeneration. In this paper, we show that FGFR-1 expression and function is correlated with the process of lens regeneration from the dorsal iris. Following lentectomy, FGFR-1 protein is specifically present in the dedifferentiating pigment epithelial cells in the dorsal iris, but is absent from the ventral iris. Subsequently, FGFR-1 protein is present throughout the process of lens regeneration and fiber differentiation. Furthermore, we show that an FGFR-1-specific inhibitor is able to inhibit the process of transdifferentiation and lens regeneration. In this sense, FGFR-1 can be regarded as the first known lens regeneration-associated factor.

Regulation of homeobox-containing genes during lens regeneration.

In this study, the expression of homeobox-containing genes was evaluated after lentectomy in the newt, which is competent for lens regeneration, and in the axolotl which is not. Such a comparison was designed to offer insights about possible regulation due to regenerative abilities. Six homeobox-containing genes were examined: NvHox A4, NvHox B1, NvHox 7, NvHox X, Nvmsx-1 and Xbr1. For all genes examined, it was found that soon after lentectomy in the newt there was a general down-regulation in the retina. This down-regulation varied among the Hox genes with NvHox 7 and NvHox B1 being virtually absent in the initial stages; their expression was re-established to the original levels after the reappearance of lens. The expression patterns, for NvHox 7 and NvHox B1 were the same when the lens was removed and then displaced. However, in axolotl, down-regulation was not observed. These data suggest that the observed regulation is related to the process of lens regeneration and provide the first molecular evidence that lens regeneration could be dependent on retina and underline the importance of this tissue in lens regeneration. Such patterns link expression of homeobox-containing genes and lens regeneration and can be now used to understand the underlying mechanisms of lens regeneration and transdifferentiation.

Graded expression of Emx-2 in the adult newt limb and its corresponding regeneration blastema.

Amputation of a newt limb causes stump cells to organize the reformation of the missing structures. The phenomenon is remarkably precise in that the regeneration is perfect. During the first few days following amputation, the tissue proximal to the plane of amputation gives rise to the blastema, an area of growth composed of mesenchymal cells covered by a single epithelium. The blastema possesses a morphogenetic potential characteristic of the structures that have been amputated. Looking for control genes putatively involved in regeneration, we cloned the newt version of the mouse and human Emx-2. Its expression is restricted to the skin of the regeneration territories and is graded along the proximal-distal axis of both forelimb and hindlimb, with higher levels in distal regions. The regeneration blastema also show this proximal-distal graded level of expression with distal blastemas (mid-radius and ulna) showing higher levels of expression when compared to blastemas of more proximal origin (mid-humerus). Finally, retinoic acid proximalizes both the level of Emx-2 expression and the positional memory of the blastema suggesting Emx-2 may participate in pattern formation by specifying positional information.

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Conservation of fibroblast growth factor function in lens regeneration.

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.

Regulation of the Wilms' tumor gene during spermatogenesis.

Spermatogenesis is the process by which male germ cells develop and mature, a pathway that includes a transition from a mitotic to a meiotic cell cycle. Throughout this pathway, the germ cells are in close contact with their nurturing cells, the Sertoli cells. Sertoli-germ cell interactions are difficult to study in mammals due to the complex cellular organization of their seminiferous tubules. The urodele amphibian testis, however, provides a unique system to study the process of germ cell maturation; it is organized in a gradient-like cystic structure, in which synchronized germ cells can be found within the same cyst. The Wilms' tumor gene (WT1) has been shown to be an essential gene for the formation of the gonads in mice, and it has been implicated in a variety of differentiation processes. The WT1 gene is thus a good candidate for the study of the differentiation processes involved in the maturation of the male germ cells. By using a probe for the urodele WT1 homologue in in situ hybridization studies, as well as an antibody against the WT1 protein in immunohistochemistry studies, we determined that WT1 gene expression in Sertoli cells depends on the stage of maturation of the associated germ cell. Thus, WT1 mRNA was detected only in Sertoli cells of cysts that contained early spermatogonia. No mRNA expression was observed in cysts containing late spermatogonia, germ cells undergoing meiosis, or germ cells going through spermiogenesis. Immunohistochemistry studies confirmed that WT1 protein was strongly expressed in Sertoli cells associated with early spermatogonia but not in late ones. The protein was also found in Sertoli cells associated with germ cells that undergo the subsequent stages of meiosis and spermiogenesis. These results suggest that WT1 could be involved in the regulation by Sertoli cells of germ cell maturation and possibly in the progression from a mitotic to a meiotic cell cycle.

9-cis retinoic acid antagonizes the stimulatory effect of 1,25 dihydroxyvitamin D3 on chondrogenesis of chick limb bud mesenchymal cells: interactions of their receptors.

Retinoids or vitamin D have been found to profoundly affect pattern formation and chondrogenesis in the developing limb. These substances mediate their actions through their nuclear receptors. In the present investigation, we present data showing that 9-cis RA, the ligand for RXR can stimulate chondrogenesis of chick limb bud mesenchymal cells, however, in combination, it antagonizes the stimulatory effect of vitamin D in the same system. The receptors for 9-cis RA (RXR) and vitamin D (VDR) were also shown to be present in the mesenchymal cells and to form heterodimers. These results implicate these receptors in cartilage differentiation during limb development.

Can insights into urodele limb regeneration be achieved with cell cultures and retroviruses?

Recent advances in the field of amphibian limb regeneration have provided insights into its cellular and molecular events. This review summarizes the development of cell lines from limb tissues and their application to the study of transdifferentiation and limb regeneration. In addition, the availability of suitable retroviral vectors for salamanders is discussed for it has opened new avenues for experimentation at the molecular level.

Cloning of homoeobox sequences expressed in the intact and the regenerating newt eye.

In this report we present the sequences of four different homeoboxes cloned from the intact and regenerating eye of the newt Notophthalmus viridescens. All these homeoboxes are novel for this species.

Transdifferentiation as a basis for amphibian limb regeneration.

Limb regeneration is a phenomenon occurring only in some urodeles. The process seems to be initiated by the dedifferentiation of the terminally differentiated cells. These cells differentiate, subsequently, to the tissues that comprise the limb, thus reconstructing the pattern of the missing limb part. In this paper we review and present evidence that certain cell types of the limb have the capacity to differentiate to different cell types than their original one by cellular metaplasia. This switch is called transdifferentiation. The focus of this review is the process of dedifferentiation which is the necessary prerequisite for differentiation, and the possible mechanisms involved.

Expression of pax-6 during urodele eye development and lens regeneration.

Regeneration of eye tissues, such as lens, seen in some urodeles involves dedifferentiation of the dorsal pigmented epithelium and subsequent differentiation to lens cells. Such spatial regulation implies possible action of genes known to be specific for particular cell lineages and/or axis. Hox genes have been the best examples of genes for such actions. We have, therefore, investigated the possibility that such genes are expressed during lens regeneration in the newt. The pax-6 gene (a gene that contains a homeobox and a paired box) has been implicated in the development of the eye and lens determination in various species ranging from Drosophila to human and, because of these properties, could be instrumental in the regeneration of the urodele eye tissues as well. We present data showing that pax-6 transcripts are present in the developing and the regenerating eye tissues. Furthermore, expression in eye tissues, such as in retina, declines when a urodele not capable of lens regeneration (axolotl) surpasses the embryonic stages. Such a decline is not seen in adult newts capable of lens regeneration. This might indicate a vital role of pax-6 in newt lens regeneration.

Protein separation techniques in the study of tissue regeneration.

The present report reviews the use of protein separation by means of two-dimensional gel electrophoresis in the study of tissue regeneration. It is shown that such an approach can provide data on protein synthesis in different stages of limb regeneration or comparative data with other regenerative processes such as tail and lens regeneration. Such an approach is more realistic than other methods employing gene cloning or generation of antibodies and can lead to the actual identification and characterization of factors that are involved in these phenomena.

Expression of transforming growth factor-beta 1-4 in the chick limb bud mesenchymal cells: regulation by 1,25-dihydroxyvitamin D3.

When mesenchymal cells from the early chick limb bud (stage 23-24) are plated at high cell density they spontaneously undergo chondrogenesis, implicating extensive cell-to-cell interactions. In the past it has been shown that TGF-beta or 1,25 dihydroxyvitamin D3 influences this process. In the present study we investigated the expression of TGF-beta isoforms in the mesenchymal cells after treatment with 1,25-dihydroxyvitamin D3. We found that TGF-beta 2 was down-regulated due to the treatment. The size of the transcripts was the expected as described for sternum cartilage cells but with differential use preference.

Expression of N-cadherin and alkaline phosphatase in chick limb bud mesenchymal cells: regulation by 1,25-dihydroxyvitamin D3 or TGF-beta 1.

When mesenchymal cells from the early chick limb bud (stage 23-24) are plated at high cell density they spontaneously undergo chondrogenesis implicating extensive cell-to-cell interactions. In the past it has been shown that TGF-beta and vitamin D can influence this process and can stimulate chondrogenesis. Given the importance of cell adhesion molecules during cellular interactions we decided to examine the effects of 1,25-dihydroxyvitamin D3 or TGF-beta on the expression of molecules involved in cell-to-cell adhesion (N-cadherin) or cell-substrate adhesion (alkaline phosphatase). Immunofluorescence demonstrated that N-cadherin was expressed in the mesenchymal cells and in the very early cartilage nodules but it was down-regulated in the older nodules. As shown by Western blotting, the expression of N-cadherin declined as chondrogenesis proceeded and was affected in cultures treated with 1,25-dihydroxyvitamin D3 and TGF-beta 1. Alkaline phosphatase was also expressed in the mesenchymal cells; these cells preferentially use an alternative transcript compared to the cartilage cells of the sternum. Thus, our data suggest that the involvement of 1,25-dihydroxyvitamin D3 in chondrogenesis could be mediated via regulation of cell adhesion.