Mechanisms of arm-tip regeneration in the sea star,Leptasterias hexactis

Summary Wound healing and regeneration following amputation of arm-tips of the sea star, Leptasterias hexactis, are described using light microscopy, SEM, TEM, and [³H] thymidine autoradiography. The process can be divided into a number of stages. Initially, the wound is closed by contractions of the stump-tip. Re-epithelialization then occurs through migration of epidermal cells from adjacent areas over the wound to form a thin wound epidermis. This is converted into a thicker, permanent covering in concurrence with the onset of cell cycle activity in the wound epidermis and adjacent epidermal regions. Histolysis and phagocytosis of damaged tissues occur beneath the new epidermis and a small connective tissue scar develops at the wound site within which muscle differentiates. At this time, elevated levels of [³H]thymidine incorporation are initiated in the sub-epidermal tissues of the arm-tip. A variety of differentiated cell types enter the cell cycle including cells of the parietal peritoneum, lining of the radial water canal, and the dermis. Cell division is accompanied by the development of a small new arm-tip complete with terminal ossicle, terminal tentacle, and optic cushion. The radial water canal, radial nerve, and perivisceral coelom extend by outgrowth into this newly developing tip. Accelerated growth of the regenerate then occurs in a zone just proximal to the new tip. There is no evidence of a blastema-like mass of rapidly dividing undifferentiated cells at the tip of regenerating arms. Arm-tip regeneration in this sea star may therefore be best described as a morphallactic-like process in which a true blastema is not formed, but in which scattered cell proliferation plays an essential role.     

Cell Proliferation in the Terminal Regions of the Internodes and Stolons of the Colonial Hydroid Campanularia flexuosa

Pattern formation and cell proliferation have been believedto have a causative relationship in the colonial hydroids. Herelinear stolon growth was assumed to be dependent on polarizedcell proliferation restricted to the extreme end of the stolon.The popularity of the concept that pattern formation was a resultof restricted meristematic cell proliferation was based on acorrespondence between the observed growth patterns and theconcept. Nevertheless, there is little direct knowledge of cellproliferation in the stolons of hydroids. The primary objective of this investigation was to determinethe sites of cell proliferation in the terminal growth zonesof the colonial hydroid Campanularia flexuosa in order to evaluatethis concept. The results give evidence that cell proliferationis more or less uniformly distributed throughout the terminalstolon and the internodes of upright stems, and not restrictedto the tips of the stolons and internodes as has been previouslypostulated. Cell proliferation occurs in both the epidermis and gastrodermisof the stolon in a ratio of 3 to 2. The formation of a new uprightis accompanied by increased cell proliferation localized atthe site of initiation. The uniform distribution of cell proliferation in the stolonsupports the hypothesis that growth is via intercalary cellproliferation, which was proposed eailier on the basis of evidenceobtained from the displacement of vital stained bands of tissuein the stolon. Stolon growth via intercalary cell proliferation requires areconsideration of the factors which control morphogenesis.Wolpert has pointed out the importance of determining the mitoticpattern in a developing system before an understanding of positionalinformation within the system may be studied. Thus, informationfor greater insight into the study of positional informationin a developing hydroid has been obtained.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

AN AUTORADIOGRAPHIC ANALYSIS OF THE ROOT EPIDERMIS OF THE SWITCH GRASS (PANICUM VIRGATUM)

Analyses of ³H-uridine, ³H-thymidine, and ³H-lysine incorporation in the root epidermis of Panicum virgatum were undertaken. Highly significant differences between the mean incorporation of ³H-uridine and ³H-lysine in epidermal and adjacent cortical cells were observed. While the cortex exhibited a steady decrease of precursor incorporation with distance from the apex, the epidermal cells exhibited differential incorporation. These results were regarded as further evidence for the hypothesis that cells of two maturation potentials exist in the epidermis of this panicoid grass. Treatment with 20.0 μg/ml of actinomycin D resulted in a differential inhibition in the epidermal-cortical incorporation of ³H-uridine. The possibility of endopolyploidy in the epidermis was suggested by the observation that root hairs, hair initials, and some epidermal cells incorporated two to four times more ³H-thymidine than meristematic cells. Neither puromycin nor actinomycin D treatment affected the protein-positive particles present in the cytoplasm of epidermal cells in this grass. Similarly, RNase did not affect their structural integrity. Attempts to clarify the significance of these inclusions and their possible role, if any, in the differentiation of the epidermis are now in progress.     

A study of regression and budding in Perophora viridis

1. A method has been devised for studying the regression of the zooid of Perophora into a stolon and the subsequent differentiation of a new zooid from this stolon. 2. Circulatory cells of the stolon resulting from regression will aggregate into masses larger than the minimal size necessary for differentiation of a zooid, but fail to differentiate into a zooid. 3. The cells of a zooid after staining with neutral red appear in the stolon during regression and finally come to lie in the newly formed zooid. 4. During the cycle of adult zooid to stolon to newly formed zooid, there is no evidence for cell division from studies with tritiated thymidine. 5. It is concluded that under conditions of starvation, an adult zooid furnishes all the cells for the formation of a stolon and the subsequent zooids without cell division.     

The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos

Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [³H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [³H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [³H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [³H]thymidine, 63% incorporate [³H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule.     

The mouse epidermis as a model in skin pharmacology: influence of age and sex on epidermal metabolic reactions and their circadian rhythms

The influence of age and sex on epidermal metabolism and histology was studied in mice (NMRI). Within a few weeks postpartum, thymidine triphosphate incorporation into DNA and amino acid incorporation into epidermal protein decreased. Differences between tail epidermis and other skin regions (back, abdomen, ear) were found. Sex dependent differences could not be demonstrated. A circadian rhythm of the studied epidermal metabolic processes was shown (artificial 12L:12D light/dark cycle). Maximum incorporation of thymidine triphosphate and of amino acids was found at the end of the light period at 1800 h, while a minimum incorporation was demonstrated at the end of the dark period. Considering described biological variations, the mouse tail assay seems to be suited to study influences of drugs in epidermal metabolism and histology.     

Yield and persistency of contrasting white clover populations grown in pure swards and in mixed swards with S.23 perennial ryegrass

Ten contrasting white clover populations were grown as spaced plants, in pure clover swards and in mixed swards with S.23 perennial ryegrass. Four of the populations were also tested for tolerance of low temperatures. In the establishment year, the autumn yields of populations were correlated with leaf size. However, during the severe winter which followed, populations with large leaves, particularly those of Mediterranean origin, suffered extensive stolon kill. This winter damage reduced the spring yields and total annual yields of populations with large leaves, so that leaf size and total annual yield were not correlated in the year after sowing. Stolon kill was positively correlated with autumn growth activity as measured by leaflet size and stolon internode length in October. Stolon kill during winter was correlated with cold tolerance of naturally-hardened stolon apices, assessed in artificial cold tests.     

Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis

The discovery of long-lived epithelial stem cells in the bulge region of the hair follicle led to the hypothesis that epidermal renewal and epidermal repair after wounding both depend on these cells. To determine whether bulge cells are necessary for epidermal renewal, here we have ablated these cells by targeting them with a suicide gene encoding herpes simplex virus thymidine kinase (HSV-TK) using a Keratin 1-15 (Krt1-15) promoter. We show that ablation leads to complete loss of hair follicles but survival of the epidermis. Through fate-mapping experiments, we find that stem cells in the hair follicle bulge do not normally contribute cells to the epidermis which is organized into epidermal proliferative units, as previously predicted. After epidermal injury, however, cells from the bulge are recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a marked radial pattern. Notably, although the bulge-derived cells acquire an epidermal phenotype, most are eliminated from the epidermis over several weeks, indicating that bulge stem cells respond rapidly to epidermal wounding by generating short-lived 'transient amplifying' cells responsible for acute wound repair. Our findings have implications for both gene therapy and developing treatments for wounds because it will be necessary to consider epidermal and hair follicle stem cells as distinct populations.     

The nature and construction of skeletal spines inPocillopora damicornis (Linnaeus)

Extreme variability in the size, shape and spacing of skeletal spines ofPocillopora damicornis has been demonstrated both within single colonies and also between colonies from different environments. Preliminary studies indicated that the majority of spines from branch tips at the apex of the colony display a ‘fasciculate’ growth surface in contrast to partly fasciculate or ‘smooth’ growth surfaces exhibited by spines from branch tips at the base of the colony. No significant differences in the height and width of costal spines from apical and basal branch tips within a single colony were observed, although spines from colonies exposed to strong wave action tended to be significantly shorter and narrower than those from more sheltered environments. Both costal and coenosteal spines from wave-exposed colonies displayed branching and divided extremities while those from sheltered environments consisted of simple cones. Spines develop as an outgrowing of the calicoblastic ectoderm which secretes the skeleton. Growing costal and coenosteal spines are enveloped by a layer of calicoblastic ectoderm which penetrates through mesogloea, aboral gastroderm, coelenteron, oral gastroderm, mesogloea and finally oral ectoderm. Spines within the corallite are surrounded by calicoblastic ectoderm, mesogloea and aboral gastroderm only. A scheme for the growth of the spines is discussed.     

The tumor promoter 12-0-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca²⁺. In contrast, the induction in cells grown in low calcium medium was 2–10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca²⁺ medium were switched to 1.2 mM Ca²⁺, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca²⁺ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.     

Morphological stages of regeneration in the planarian Dugesia tigrina: A light and electron microscopic study

A precise sequence of four morphological stages of head regeneration in the planarian Dugesia tigrina has been determined by light and electron microscopy. Each stage is identified by a particular morphogenetic process: I, wound healing; II, blastema development; III, growth; IV, differentiation. A wound epidermis consisting of a thin, sheet-like layer of cells, rapidly forms from undamaged epidermal cells at the wound margin. The early blastema is comprised of neoblasts which mature into regeneration cells. The maturational changes include the appearance of a nucleolus, nuclear pores, and perinuclear dense aggregates of granulofibrillar material in these cells. These elements are not evident in the neoblasts of the younger blastema. No mitotic cells are encountered in the blastema or wound epidermis. Cytoplasmic expansion of the regeneration cells is correlated with the formation of numerous microtubules radiating from a juxtanuclear centrosphere. During differentiation of muscle cells, distended, granule-studded cisternae, having moderately fibrillar contents, are regularly disposed adjacent to small patches of myofilaments. Presumptive epidermal cells are recognized by prominent “islands” of finely fibrillar cytoplasm. These cytoplasmic zones persist for a time during definitive differentiation when Golgi bodies, vacuoles, mucous droplets, and rhabdites become evident. The newly formed epidermal cells become inserted among the cells of the wound epidermis. Thus, cells of both the blastema and of the wound epidermis contribute to the reconstituted epidermis.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Down-regulation of epidermal growth factor receptors in mouse embryos

Previously we have shown (E. D. Adamson, M. J. Deller, and J. B. Warshaw, 1981, Nature (London) 291, 656–659) that epidermal growth factor (EGF) binds specifically to the cells and stimulates the incorporation of tritiated thymidine into DNA of several tissues of mouse embryos in a dose-dependent fashion when tested in vitro. However, in vivo a different response is obtained; exogenous EGF causes reduced incorporation of radiolabeled thymidine compared to buffer-injected control embryos. Several possible explanations are being explored. Here we present evidence that one of the responses of embryonic tissues in vivo to exogenous EGF is “down-regulation” of its receptors.     

A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA

We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.     

Ditylenchus dipsaci Infestation of Trifolium repens. II. Dynamics of Infestation Development

Trifolium repens (white clover) stolons were inoculated with Ditylenchus dipsaci (stem nematode), and the development of resulting infestations was monitored. Nematodes initially remained confined to superficial locations, concentrating in petiole axils near inoculation points. They were able to migrate slowly from the inidal inoculation points and infest adjacent axils, especially in regions near the stolon tip. As time progressed, in some axils, nematodes migrated through the stolon epidermis and colonized slowly expanding subepidermal pockets of host tissue (ca. 0.2-mm length of stolon/day). In these loci nematodes established exponentially increasing populations, but the rates of locus expansion remained constant, indicating that locus expansion was limited by unidentified host-dependent factors. As a result of increasing population pressure within subepidermal loci, J4 entered a "diapause" state and the rate of egg production by adults declined, thereby reducing rate of population growth to more sustainable levels. Typically, these populations peaked at ca. 10,000 individuals in ca. 160 days occupying 3-cm lengths of stolon. Thereafter, heavily infested regions of stolons started to die, leading to the formation of longitudinal splits in their epidermis. In other axils, nematodes did not migrate into the stolons but remained confined to axils. Some of these populations increased a hundred-fold in 95 days, with population growth ending when petioles started to die. Host plant stolon morphology was affected only when subepidermal stolon populations developed high population levels (>100 nematodes) within close proximity (<2 cm) to active terminal meristems. This occurred either when axillary buds became active on previously infested nodes or when nematodes established endoparasitic populations at locations near the stolon tip during winter and spring, when the rate of stolon extension was limited by low light intensity. Affected stolon tips could "escape" from the influence of such infestations when light intensity and temperature increased. Nematode activity was limited by low temperature rather than light intensity. Global warming is likely to lead to greater damage to infested plants during the winter and early spring because the predicted milder winter temperatures will enhance nematode activity but not necessarily promote stolon growth.     

The effects of interferon on epidermal growth factor action

Epidermal growth factor-stimulated thymidine incorporation in human fibroblasts is inhibited more than 80% by human interferon, whereas the stimulation of α-aminoisobutyrate uptake is unaffected. Maximum inhibition of thymidine incorporation is observed after treatment of cells with interferon prior to the onset of DNA synthesis. However, even after the initiation of DNA synthesis, interferon rapidly blocks any further increase in thymidine incorporation. Despite these effects, interferon treatment causes no alterations in epidermal growth factor binding, receptor downregulation or receptor reappearance.     

3H-thymidine incorporation method versus colony forming assay in the determination of anchorage-independent cell growth of rat sarcoma (XC) and Morris hepatoma 7777 (MH) cells.

The modification of 3H-thymidine incorporation method of Tanigawa was used to the estimation of anchorage-independent growth of virally and chemically transformed rat cells. The relationship between colony forming assay and 3H-TdR incorporation test was determined, depends on the composition of culture medium and the period of incubation of rat sarcoma (XC) cells with thymidine. The influence of exogenous mitogens (RFG, TGF beta 1 and insulin) and autocrine factor (at different step of purification) on the growth of Morris hepatoma 7777 (MH) cells was estimated by both methods. Regression analysis comparing the results of colony counting and thymidine incorporation revealed good correlation between the two methods. The modification can be used the determination of growth stimulating or growth inhibiting activity and in multistep purification procedure of autocrine growth factors.