David S. McDevitt

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Abstract Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.

Fibroblast growth factor receptors and regeneration of the eye lens

If the eye lens of the adult newt, Notophthalmus viridescens, is removed, a new lens will regenerate and only from the dorsal, not the ventral, iris. The source, pigmented epithelial cells, would normally no longer divide, but upon lentectomy they do re‐enter the cell cycle and form lens. The cause for this capability is unknown, but the mitogenic Fibroblast Growth Factors and their receptors may be involved. We have demonstrated that FGF receptors are present and operative in lens regeneration, since receptor‐directed mitotoxins inhibit regeneration; heterogeneity and differential density in FGF‐binding and receptor localization in iris sectors is also present. We propose that the spatial distribution of FGF receptors, especially the amphibian homolog of FGFR‐3, is important in initiation of regeneration of eye lens. Dev. Dyn. 208:220–226, 1997.

Ontogeny and localization of the α, β, and γ crystallins in newt eye lens development☆

The alpha-, beta-, and gamma-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the alpha-, beta-, and gamma-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. beta-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. gamma-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and alpha-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for beta-crystallins until late in lens morphogenesis, and alpha- and gamma-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.

On the existence of γ-crystallin in the bird lens

Abstract Sauropsidans until recently were acknowledged to possess the lens-specific proteins, α and β-crystallins, while lacking γ-crystallins. The γ-crystallins have been employed as specific indicators of normal and regenerating lens fiber cell differentiation in several other organisms. It has been reported, however, that indeed, birds do possess γ-crystallins, and that furthermore this protein is not lens-fiber specific, since it could be found in annular pad and epithelium as well as fibers. Several physical characteristics of this protein cited by those suggesting its existence and at odds with those known for γ-crystallins, led the authors to further investigate the putative γ-crystallin. Pigeon “γ”-crystallin was prepared exactly according to previously published methods, and subjected to amino acid and N-terminal analyses, and peptide mapping. The fraction isolated satisfied none of the commonly-accepted criteria for γ-crystallins. Thus the pigeon “γ”-crystallin did not exhibit low values for alanine and lysine; a free N-terminal amino acid could not be detected; and peptide mapping did not reveal the distinctive peptide of γ-crystallins, the fastest moving orange spot in the electrophoresis direction. The bird lens, then, does not contain γ-crystallin; the γ-crystallins, when present, remain as reliable indicators of lens fiber differentiation.

Ontogeny and localization of gamma-crystallins in Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii normal lens development

Rana pipiens lens γ-crystallin antibodies were used in the indirect immunofluorescence staining method to investigate the role of γ-crystallins in the normal lens development of the amphibians Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii Michah. In each case, the fluorescence was first localized in a number of cells in the inner wall of the lens, which was in the vesicle stage. With further differentiation, the intensity of immunofluorescence gradually increased and was restricted only to the fibre cells. These results support the concept that, though the γ-crystallins of different amphibian species studied so far show different numbers of protein components by thin layer isoelectric focusing on polyacrylamide gel, they all have similar immunological properties, and are specific for the lens fibre differentiation.

α-, β- and γ- Crystallins in the regenerating lens of Notophthalmus viridescens

Abstract Removal of the lens from the eye of adult Notophthalmus viridescens, the Eastern spotted newt, is followed by regeneration of another lens from the dorsal iris. This cell-type conversion of iris epithelial cells into lens cells is accompanied by the subsequent synthesis of α-, β- and γ-crystallins, proteins specific for the normal vertebrate lens. The immunofluorescence technique was employed to determine the ontogeny and localization of the crystallin classes in the regenerating lens rudiments. Antibodies specific for α-, β- and γ-crystallins were applied to tissue sections through regenerate Stages III–XI Yamada 1967. The first positive reaction for α-crystallins did not occur until Stage VII, in a few cells of the developing lens fiber region; and in the external layer (presumptive lens epithelium) at Stage VIII, at which time secondary lens fibers have begun to form. β-Crystallins were first detectable in the thickening internal layer of the Stage V lens (vesicle) and in the external layer at Stage VIII. The γ-crystallins also first appeared, albeit erratically, in a few cells of the internal layer of the Stage V regenerate, and were not detectable in the external layer/lens epithelium at any time. Thus the crystallins are not present in the earliest stages, but are indicative of cellular differentiation in succeeding stages of lens regeneration.

Presence of Lateral Eye Lens Crystallins in the Median Eye of the American Chameleon

With the use of immunofluorescence techniques, gamma globulin antibody specific for the crystallins of Anolis carolinensis lateral eye lens was applied to sections through the median (parapineal) eye of Anolis carolinensis. Only the median eye lens exhibited fluorescence specific for the crystallins; other structures were negative. These results indicate that the lens of the reptilean median eye shares tissue-specific antigenic determinants with the lens of the lateral eye. This suggests a possible evolutionary relation between the two structures, based on biochemical, as well as previously reported anatomical, criteria.

The lens proteins of eastern North American salamanders, and their application to urodelan systematics

The soluble lens proteins of 12 eastern North American salamander species of seven genera (P. glutinosus, P. cinereus, P. jordani, D quadramaculatus, D. fuscus, D. monticola, D. ochrophaeus, Ps. ruber, G. porphyriticus, E. bislineata, N. viridescens and A. maculatum) were investigated, using cellulose acetate electrophoresis. Each of the species, under the conditions employed, exhibited a characteristic, qualitatively distinct electrophoresis pattern, with the greatest resolution occurring in the γ-crystallin region. It was not possible, however, to distinguish the lens proteins of (a) sub-adults from those of adults (five species), (b) intergrade/subspecies from those of derived species (two species) and (c) a colour-phase from those of normal dorsal colouration (one species). The full species status of all but two of the 12 was confirmed; the separate taxonomic status of D. fuscus and D. ochrophaeus is questionable due to the nearly-identical electrophoresis patterns obtained. Such a discriminative ability in the lens protein system suggests its use as a “phylogenetic fingerprint” in urodelan systematics, with possible application to other problems of speciation in the vertebrates.

Transdifferentiation in Animals A Model for Differentiation Control

Transdifferentiation may be generally defined as the change of one recognizable cell type to another different cell type. The term was first used by Selman and Kafatos (1974) to denote the change of the cuticular cells of the moth larval silk gland to those producing HCO3 during metamorphosis/development and has since been used in many different contexts. So as not to produce a welter of semantics to replace the term already in use, I shall instead categorize the phenomenon of transdifferentiation by levels: primary, secondary, and tertiary transdifferentiation. Primary (or true) transdifferentiation would include the cell-type conversion or cell metaplasia that is so well documented to occur in some amphibian eye tissues in vitro and in amphibian (newt) eye tissues in situ (Fig. 1). This level is characterized by verifiably postmitotic cells, terminally differentiated and producing a specific cell product, transforming into a completely different cell type with differing cell product(s). Secondary transdifferentiation is marked by the conversion of those cells or tissues not definitely demonstrable as terminally differentiated, i.e., from an embryonic or possible stem-cell source. Also included is the concept of transdetermination (Hadorn, 1965), in which certain groups of cells in Drosophila occasionally become determined or committed to a developmental fate different from that expected. Tertiary transdifferentiation would encompass other purported/ reported changes of tissue types, e.g., that of muscle to cartilage (Namenwirth, 1974), and of striated to smooth muscle in Anthomedusa as reported by Schmid and Alder (1984) and Weber et al (1987). The well-known plasticity of plant tissues, especially in vitro, is the rule, rather than the exception, and as a topic of transdifferentiation is beyond the scope of this chapter.

Eye lens regeneration and the crystallins in the adult newt, Notophthalmus viridescens * **

Upon lens removal, the adult Eastern Spotted newt, Notophthalmus viridescens, has the capacity to regenerate an ocular lens. Crystallins, proteins characteristic of the vertebrate lens, were studied from normal and 3-month regenerated adult newt lenses. When separated by high-performance liquid chromatography (HPLC) or Sephadex G-200SF column chromatography, the crystallins from normal and regenerated lenses were fractionated into what appear to be the classical four groups: alpha, beta High, beta Low, and gamma. Upon further examination by immunoelectrophoresis, the first peak contains both alpha and beta crystallins. This study provides evidence that most of the crystallins from the regenerated lenses share biochemical properties with those of the normal lens crystallins based on their native molecular weight, isoelectric point, and the molecular wt of their constituent polypeptides, indicating that the fidelity of gene expression in reactivated iris tissue is high. Some differences are found between normal and regenerated lens crystallins and are most obvious in the beta-crystallin region: the proportion of beta crystallins is decreased in regenerated lenses when the total proteins are fractionated by column chromatography and some of the beta-crystallin polypeptide chains found in normal lenses are missing from regenerated lenses. Iris epithelial cells are normally withdrawn from the cell cycle and are synthesizing a tissue-specific product, melanin. After lentectomy these cells dedifferentiate, redifferentiate into lens cells, and their progeny then synthesize different tissue-specific proteins, crystallins. Little is known about the specific mechanism(s) for the activation of gene expression in eukaryotes, but the regenerating lens suggests itself as a good model in which to study this biological problem.