Influence of chromatographic fractions of extracts derived from bovine neural retina on newt (Notophthalmus viridescens) lens regeneration in vitro.

Removal of the ocular lens in adult newts (Notophthalmus viridescens) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131:151-172, 1958; Reyer, Dev. Biol. 14:214-225, 1966) and in vitro (Yamada et al., Differentiation 1:65-82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240:343-351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67:265-269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19:923, 1979; Connelly, Differentiation 16:85-91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230:485-490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.

Role of the neural retina in newt (Notophthalmus viridescens) lens regeneration in vitro.

Removal of the lens from the eye of an adult newt (Notophthalmus viridescens) is followed by regeneration of a new lens from the dorsal iris epithelial cells at the pupillary margin. This process is dependent upon the neural retina for its normal completion in vivo and in vitro. To examine the relationship between the retina and lens regeneration, we have conducted experiments that delimit the time period during which the retinal presence is critical (in vivo) and have investigated the influence of extracts of the retina on the progress of regeneration (in vitro). In vivo, removal of the retina at day 11 seriously retards further progression of regeneration while removal of the retina at day 15 does not retard regeneration significantly. This defines a "critical period" in regeneration of the lens during which the retina is required. Explantation of regenerates 11 or 12 days after lentectomy to organ culture medium enriched with either crude retinal homogenate or extracts prepared from chick or bovine retinas according to Courty et al. ('85, Biochimie, 67:265-269) reveals that the progress of regeneration can be supported in culture by the crude extract. This is the first demonstration of complete iris-lens transformation in culture in the presence of retinal extract. It is possible that the retina acts indirectly by promoting passage of the iris epithelial cells through the critical number of mitoses required before redifferentiation into lens cells can occur (as proposed by Yamada, '77, Monogr. Dev. Biol., 13:126). It is also possible that the retina acts by directly instructing the iris cells to redifferentiate.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues.

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.

Cellular behavior in the anteroposterior axis of the regenerating forelimb of the axolotl, Ambystoma mexicanum.

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.

Microdensitometric analysis of denervation effects on newt limb blastema cells.

This report examines the fate of cycling cells in normal and denervated blastemas of adult newts. Cells are found to accumulate in G1 in blastemas which are nerve independent. No stage specific accumulation different from controls is found in limbs with nerve-dependent blastemas.

The circumoval precipitin reaction on Schistosoma mansoni eggs as seen by scanning electron microscopy.

The circumoval precipitin reaction on Schistosoma mansoni eggs was examined for the first time by scanning electron microscopy. The precipitates appeared as layered segmenters, and apparent fusion of adjacent precipitates was observed. This suggests that the single precipitates seen by light microscopy may on occasion be a collection of several precipitates exuding from nearby pores. The precipitates appeared on all areas of the egg surface except the spine.

The influence of hormones and other substances on lens regeneration in vitro.

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 microgram/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.

Lizard and newt tail regeneration: a quantitative study.

Almost perfect fits of the Gompertz equation to the growth in length of tail regenerates in the lizard, Lacerta lepida, and the newt, Notophthalmus viridescens, were obtained. Comparison of certain parameters of the equation with published mitotic index data suggests that the Gompertz equation characterizes each system at least from the time that significant mitotic activity is first observed histologically. An objective method for comparing the regeneration periods of the two species is described and applied. A unified hypothesis derived from consideration of properties of the Gompertz equation successfully accounts for the following phenomena reported, but previously unexplained, in the literature: (1) proximal amputations result in longer regenerates than do distal amputations; (2) proximal amputations elicit greater absolute rates of elongation (in mm/day) than do distal amputations; (3) the percent replaced of the length removed is rather constant, regardless of the absolute length regenerated; and (4) one of the parameters of the Gompertz equation appears to be lognormally distributed in a regenerating population. (See text for references.) A computerized interactive graphical system for normalizing growth equations of individual regenerates and integrating the mathematical model with potential candidates for biological control factors is briefly described.

A scanning electron microscopic comparison of the development of embryonic and regenerating limbs in the axolotl.

A comparison between the surface features of embryonic limb development and limb regeneration was made in the axolotl. Scanning electron microscopy revealed an overall similarity between embryonic and regenerating limbs. A notable feature was the lack of a morphologically discrete apical epidermal specialization on the surface of any of the limbs. Histological preparations revealed no thickening of the apical epidermis in embryonic limbs. There is a definite thickening of the apical epidermis in regenerating limbs during the stages of dedifferentiation and the early blastemal phases, but the thickening projects into the underlying tissues rather than altering the smooth surface contours of the blastema. Minor differences in epidermal specializations (ciliated cells scattered over the entire body and very irregularly shaped cells with knob-like projections at the tips of digits in embryos) may be attributable to differences in overall activity of the animals at various stages in their life history.

Pituitary enhancement of Wolffian lens regeneration in vitro: spatial and temporal requirements.

When irido-corneal complexes (ICC's) from eyes of adult newts (Notophthalmus viridescens) are cultured on top of pituitary glands they produce advanced lens regenerates in up to 90% of the cases (this report and Connelly et al., '73). To determine if the iris and pituitary gland must be in direct contact with each other in order to produce this enhancement of lens regeneration, ICC's were cultured next to pituitary glands or separated from them by a Nuclepore filter. Cultures behaved as ICC's cultured in the absence of the pituitary gland and produced few advanced lens regenerates. To determine how long the iris and pituitary must be in contact for the enhancement to occur, ICC's were explanted and 5, 10 or 15 days later pituitary glands were placed beneath the dorsal iris pupillary margin. There was a progressive decrease in the number of advanced lens regenerates formed in cultures receiving pituitary glands later than five days after initial explantation of the iris. The conclusions drawn from these experiments are that the iris and pituitary gland must be in extensive direct contact during the first five to ten days in culture in order for pronounced enhancement of lens regeneration to occur.

The relationship between growth, developmental stage and postamputation age of the regeneration blasterma of the newt, Notophthalmus viridescens.

The growth of the regeneration blastema of the newt forelimb has been quantitated and analyzed as a function of postamputation age, developmental stage, animal weight, animal length, and cross sectional diameter of the blastemal base in both the anteroposterior and dorsoventral dimensions. Correlation coefficients computed for these variables show that growth of the regenerate in both length and volume is more closely correlated with developmental stage than postamputation age. In addition, the results show a linear relation between the loge (regenerate length) and developmental stage, and between regenerate length and volume. Thus, length can be used to assess growth of the regenerate according to a developmental rather thana chronological time scale. There were no significant correlations between regenerate length or volume and animal length, animal weight or cross sectional dimensions of the blastemal base. These results show that one can use a randomly selected population of animals and study the growth of the regeneration blastema by relating to a developmental time scale through a logarithmic transformation of the linear growth data.

A staging system for forelimb regeneration in the axolotl, Ambystoma mexicanum.

A staging system has been devised for normal regeneration from the upper arm in the mature axolotl. It consists of seven externally definable stages: (1) Wound healing (WH): (2) Dedifferentiation (DD); (3) Early bud (EB); (4) Medium bud (MB); (5) Late bud (LB); (6) Palette (Pal), and (7) Digital outgrowth (DO). Serial histological sections of 38 regenerating limbs were used to correlate gross stages with microscopic events in the regenerative process.