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In this review, we will explore several studies where stem cells from neural, non-neural and even embryonic cells have been used as potential sources to repair the damage retina. In addition, we will also discuss the possibility of inducing retina regeneration by transdifferentiation of cells present in existing eye tissues, such as, the Retinal Pigmented Epithelium (RPE), the Pigmented Ciliary Margin (PCM) and Müller glia cells.

Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.

In this review we present a synthesis on the potential of vertebrate eye tissue regeneration, such as lens and retina. Particular emphasis is given to two different strategies used for regeneration, transdifferentiation and stem cells. Similarities and differences between these two strategies are outlined and it is proposed that both strategies might follow common pathways. Furthermore, we elaborate on specific clinical applications as the outcome of regeneration-based research.

In this paper we describe the basic process of lens regeneration in adult newt and we pinpoint several issues in order to obtain a comprehensive understanding of this ability, which is restricted to only a few salamanders. The process is characterized by dynamic changes in the organization of the extracellular matrix in the eye, re-entering of the cell cycle and dedifferentiation of the dorsal iris pigment epithelial cells. The ability of the dorsal iris to contribute to lens regeneration is discussed in light of iris-specific gene expression as well as in relation to factors present in the eye.

Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.

We have cloned and characterized the expression, during spermatogenesis, of three novel zinc finger genes (Zfp94, Zfp95, Zfp96). Analysis of the deduced protein sequences reveals that all three molecules belong to the LeR family (leucine-rich zinc fingers) and that ZFP95 contains a domain homologous to the Krüppel-associated box. All three genes were found expressed at high levels in testis among other tissues, but testis-specific transcripts were observed for Zfp95 and Zfp96. Northern blot analyses of the testis-specific transcripts of Zfp95 and Zfp96 were performed using whole testis RNA as well as RNA isolated from enriched populations of specific spermatogenic cell types. The testis-specific transcript of Zfp95 showed the highest expression in pachytene spermatocytes, while that of Zfp96 was highly expressed in pachytene spermatocytes, in round spermatids and residual bodies. Northern blot analysis of RNA from the testis of mice carrying the atrichosis mutation further validated these expression patterns. In particular, the testis-specific transcripts of Zfp95 and Zfp96 were greatly reduced in heterozygous, and completely absent in homozygous testis RNA from atrichosis mutant mice, further defining the germ cell specificity of these transcripts.

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.

Based on the role of retinoblastoma (Rb) in lens development and in the cell cycle reentry of muscle cells during limb regeneration, we have analyzed expression or Rb patterns in intact and lens regeneration-undergoing newt eyes. We find that in intact newt eye Rb is expressed in the retina as a gradient with higher levels in the photoreceptor layer and virtually no expression in the ganglion layer. In addition, a second gradient was detected within the photoreceptor layer with expression diminishing at the dorsal and ventral regions. In the intact lens, Rb is expressed in the lens epithelium and in the differentiating lens fibers at the bow region. During lens regeneration, Rb is expressed very strongly in the differentiating lens fibers, but not in the lens epithelium. Using an antibody specific to the hyperphosphorylated form of Rb, we detected the inactive protein only in the pigment epithelial cells of the iris. These distinct patterns might be related to the regenerative potential of the lens in the newt., (Copyright 2003 Wiley-Liss, Inc.)

Eye tissues such as the lens and the retina possess remarkable regenerative abilities. In amphibians, a complete lens can be regenerated after lentectomy. The process is a classic example of transdifferentiation of one cell type to another. Likewise, retina can be regenerated, but the strategy used to replace the damaged retina differs, depending on the animal system and the age of the animal. Retina can be regenerated by transdifferentiation or by the use of stem cells. In this review, we present a synthesis on the regenerative capacity of eye tissues in different animals with emphasis on the strategy and the molecules involved. In addition, we stress the place of this field at the molecular age and the importance of the recent technologic advances., (Copyright 2003 Wiley-Liss, Inc.)

The role of retinoids in eye development has been well studied. Retinoids and their receptors regulate gene expression and morphogenesis of the eye. In this study, a highly specific antagonist of retinoic acid receptor (RAR)-alpha was used in an attempt to study its function in lens regeneration. It was found that this antagonist inhibited lens regeneration and lens fiber differentiation. It was also shown that RAR-alpha is expressed in the lens during the process of regeneration. These results indicate that different RAR might have unique as well as redundant effects and patterns of expression in the regenerating lens.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

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., (Copyright 1998 Academic Press Limited.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

9-cis-retinoic acid has recently been found to be a high affinity ligand for the retinoic X receptor (RXR). RXRs are believed to be involved in metabolic activities rather than in morphogenetic ones. Interestingly, RXR has been found to form heterodimers involving other receptors from the steroid family, such as the thyroid hormone receptor, vitamin D receptor or retinoic acid receptors (RARs). In this paper we examined whether or not 9-cis-retinoic acid had any morphogenetic properties on the regenerating axolotl limb. It is shown that 9-cis-retinoic acid proximalized regenerating limbs and was somewhat more potent in this action than all-trans-retinoic acid. Based on these observations, the possible roles of other receptors during pattern formation is discussed.

We describe a detailed histological analysis of the affected organs in the Short toes (s) mutation of the Mexican axolotl, Ambystoma mexicanum. The s mutant animals displayed a variation in the response to the mutation such as the time of death and the degree of abnormalities of the affected organs (limbs, kidney, ducts, and reproductive structures). In addition to the numerous histological abnormalities characterized here, we also examined the presence of laminin-related molecules in s mutant kidney tissues to elucidate further the nature of the renal aplasia these animals encounter as a result of this recessive lethal mutation. Our data suggest that there may be a disruption in tissue interactions necessary for proper development and functioning of the affected tissues. © 1993 Wiley-Liss, Inc., (Copyright © 1993 Wiley-Liss, Inc.)

Although regeneration-competent newts like Notophthalmus viridescens have been reported to be resistant to carcinogenesis, we have been able to induce transplantable epidermal squamous cell carcinomas with 10-20% incidence by implanting 20-methylcholanthrene s.c. into the scapular region, a tissue that cannot regenerate. As soon as 1 week after exposure to this carcinogen, malignant cells were present in the treated skin, and after 4 weeks, macroscopic tumors of infiltrating squamous carcinoma cells positive for Type IV collagenase and/or rasHa p21 had dissolved areas of the epidermal basement membrane and colonized the dermis. Analysis of Ki-67 expression revealed that these tumors grow via a high growth fraction rather than a short cell cycle time. Morphological and immunohistochemical analyses showed that these tumors caricature the biology of the renewing epidermis: the presence of basal-like cells; differentiating cells; apoptotic cells; and keratinized horn pearls with an exaggerated or overabundant stem cell compartment as compared to the differentiated cell compartment. Immunochemical analyses indicated that the squamous carcinomas arose from the epidermis rather than the mucous glands. Thus, the principle that malignant tumors caricature the process of tissue renewal originally established in rodent tumors appears to be valid down the phylogenetic tree at least to regeneration-competent amphibia. Such a broad conservation indicates that the caricature principle also holds in human tumors.

The nervous system plays an important role during the process of amphibian limb regeneration. However, the molecules that are involved in such a control of regeneration are largely unknown. We have attempted to map protein synthesis in the brains of intact newts and from newts undergoing limb or tail regeneration. Our results show unique protein synthesis in the brain of newts undergoing limb regeneration. Such an analysis can lead to the identification and characterization of these proteins.

Mesenchymal cells from the wing buds of stage 24 chick embryos undergo differentiation to cartilage when plated at high density. Treatment of these cultures with phospholipase D resulted in inhibition of chondrogenesis. Phospholipase D treatment (which produces phosphatidic acid from membrane phospholipids) was found to affect cell proliferation and to dramatically increase intracellular free calcium levels and inositol phosphate production. Intracellular free Ca2+, mobilized as a result of phosphatidylinositol phosphate hydrolysis, may therefore inhibit chondrogenesis in embryonic mesenchymal cells.

Cases of spontaneous neoplasms in amphibia are updated and the rarity of spontaneous neoplasia is considered as a result of either inadequate research or the regenerative capacity of these animals.