The chick's marginal zone and primitive streak formation. II. Quantification of the marginal zone's potencies--temporal and spatial aspects

When a posterior fragment of the chick's marginal zone (PM) was exchanged with equal sized lateral marginal zone fragment (LM), of the same blastoderm, its capacity to initiate an ectopic primitive streak (PS) was found to be both size and stage dependent. Good correlation was demonstrated between the areas of PM fragments and the number of cells they contained. In stage X blastoderms, PM fragments containing less than 1200 cells were incapable of initiating an ectopic PS. Transplanted PMs containing between 1200 and 1500 cells initiated a lateral ectopic PS in 50% of the cases, while in the other 50% a posterior PS developed from the original posterior side. PMs containing 1500 cells or more in all cases initiating an ectopic PS and inhibited the formation of a posterior PS. At stage XI, laterally transplanted PMs containing less than 1800 cells were not effective. Stage XI PMs containing 1800-2300 cells in some cases succeeded in initiating a lateral ectopic PS, in addition to the posterior one. Stage XI PMs containing 2300 cells or more invariably promoted the development of an ectopic PS, but were unable to suppress the formation of a posterior PS, so that two PSs developed in the same blastoderm, one posterior and one ectopic. When a stage XI PM fragment was laterally transplanted into a younger, stage X blastoderm, the minimal effective cell number needed to initiate an ectopic PS increased to at least 3000 cells, again without inhibiting the formation of a posterior PS. The inductive potential of a stage X PM is therefore at least twice that of a stage XI PM. The marginal zone belt of stage X blastoderms was checked for the decrease in its developmental potential from the posterior to the lateral side by evaluating its effect on the developmental expression of two competing stage X PMs, one located posteriorly and the other inserted laterally. The developmental expression of the laterally inserted PM was consistently inferior to that of the posterior PM. The developmental expression of each PM was not related to absolute size, but depended on the size ratio of lateral PM/posterior PM. When the ratio was 1.2 or less, only posterior PSs developed. When the ratio was 1.3-1.4, three different results were encountered: (1) only a posterior PS, (2) posterior plus lateral, and (3) only lateral PS. When the ratio was 1.5 or more, only a lateral PS developed, which suppressed the posterior PS.     

The influence of cell interactions and tissue mass on differentiation of sea urchin mesomeres

The developmental potential of different blastomeres of the sea urchin embryo was re-examined. We have employed a new method to isolate substantial numbers of different kinds of blastomeres from 16-cell-stage embryos, and we have used newly available molecular markers to analyze possible vegetal differentiation. We have found that, while isolated mesomere pairs behave according to the classical expectations and develop into ectodermal vesicles, there is a clear effect of reaggregating two or more mesomere pairs. They survive better in long-term culture and, after prolonged periods, they display an astonishing ability to express vegetal differentiation. We also combined mesomeres with stained micromeres or macromeres from the vegetal hemisphere. Although induction of guts and spicules was observed, there was little if any effect of varying the ratio of different blastomeres on the kinds of differentiation obtained.     

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.     

Variation in anuran embryogenesis: differences in sequence and timing of early developmental events

Comparative embryology of closely related species can shed light on the evolution of developmental processes. An important mechanism in the evolution of developmental processes, which can lead to significant changes in larval or adult form, is variation in the sequence and timing of developmental events. We compared the development of 12 species of anurans, including a wide taxonomic range as well as a number of congeneric species. The comparison consisted of monitoring a series of external morphological markers and histological markers. For each species we noted the timing of each of the markers, using a uniform parameter of normalized time. We compared the normalized time of each of these events among the species, as well as the sequence of the events. Our analysis revealed many differences in sequence and in timing of developmental events. We mapped these differences on a cladogram of the studied species, using sequence units as discrete characters. The differences do not seem to be connected to the phylogenetic relations between the species or to any obvious ecological factors. We suggest a hypothetical ancestral sequence of developmental events, and discuss the possible factors that could have caused the observed variations from the ancestral sequence.     

The chick's marginal zone and primitive streak formation. I. Coordinative effect of induction and inhibition

A variety of transplantation experiments of posterior and lateral marginal zone fragments at stages X, XI, and XII have been carried out in order to test their relevance to the development of a primitive streak (PS). At the stages studied the marginal zone (MZ) was shown to behave as a ring-like gradient field, the maximal value of which was at the posterior end (PM). The PM was found to be capable at the same time of promoting the development of a PS and of suppressing the inductive potential of other regions of the MZ. By systematically evaluating inductive and suppressive capacities of PMs, at different developmental stages, it was found that both features are maximal at stage X. During stages XI and XII, both properties gradually decrease in the MZ and build up in the forming hypoblast.     

Anti-Ulcerogenic Effect of Methanolic Extracts from Enicosanthellum pulchrum (King) Heusden against Ethanol-Induced Acute Gastric Lesion in Animal Models

A natural source of medicine, Enicosanthellum pulchrum is a tropical plant which belongs to the family Annonaceae. In this study, methanol extract from the leaves and stems of this species was evaluated for its gastroprotective potential against mucosal lesions induced by ethanol in rats. Seven groups of rats were assigned, groups 1 and 2 were given Tween 20 (10% v/v) orally. Group 3 was administered omeprazole 20 mg/kg (10% Tween 20) whilst the remaining groups received the leaf and stem extracts at doses of 150 and 300 mg/kg, respectively. After an additional hour, the rats in groups 2–7 received ethanol (95% v/v; 8 mL/kg) orally while group 1 received Tween 20 (10% v/v) instead. Rats were sacrificed after 1 h and their stomachs subjected to further studies. Macroscopically and histologically, group 2 rats showed extremely severe disruption of the gastric mucosa compared to rats pre-treated with the E. pulchrum extracts based on the ulcer index, where remarkable protection was noticed. Meanwhile, a significant percentage of inhibition was shown with the stem extract at 62% (150 mg/kg) and 65% (300 mg/kg), whilst the percentage with the leaf extract at doses of 150 and 300 mg/kg was 63% and 75%, respectively. An increase in mucus content, nitric oxide, glutathione, prostaglandin E2, superoxide dismutase, protein and catalase, and a decrease in malondialdehyde level compared to group 2 were also obtained. Furthermore, immunohistochemical staining of groups 4–7 exhibited down-regulation of Bax and up-regulation of Hsp70 proteins. The methanol extract from the leaves and the stems showed notable gastroprotective potential against ethanol.     

Vegetation succession in lakes of West Connemara,Ireland: comparing predicted and actual changes

Abstract. Changes in the vegetation of lakes and wetlands were investigated over a period of 18 years. It was assumed that changes in vegetation were related to changes in agricultural land use resulting in increased phosphate levels in surface waters. Data were collected in 1975, 1988 and 1993. Multivariate techniques were used to relate changes in vegetation to changes in environmental factors. With the use of a Markovian chain model, vegetation development was projected into the future. Projections based on vegetation dynamics between 1975 and 1988 were compared with actual changes in the vegetation. The vegetation dynamics appeared stable on a regional scale but quite dynamic on a local scale. A continuous decline in species diversity was noted as well as an overall increase of phosphate level. However, only minor changes in vegetation could be attributed to this increase of phosphate. Major changes were a result of fluctuations in water level. These changes coincide with periods of drier and wetter climate. Because of the fluctuating nature of these changes, predicted vegetation change did deviate from the observed change.     

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.     

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.     

Mint essential oil can induce or inhibit potato sprouting by differential alteration of apical meristem

Sprouting of potatoes during storage, due to tuber dormancy release, is associated with weight loss and softening. Sprout-preventing chemicals, such as chlorpropham (CIPC), can negatively impact the environment and human health. Monthly thermal fogging with mint (Mentha spicata L.) essential oil (MEO) inhibited sprouting in eight potato cultivars during large-volume 6-month storage: the tubers remained firm with 38% lower weight loss after 140 days of storage. The sprout-inhibitory action may be nullified: treated tubers washed with water resumed sprouting within days, with reduced apical dominance. MEO application caused local necrosis of the bud meristem, and a few weeks later, axillary bud (AX) growth was induced in the same sprouting eye. MEO components analysis showed that 73% of its content is the monoterpene R-carvone. Tubers treated with synthetic R-carvone in equivalent dose, 4.5 μl l⁻¹, showed an inhibitory effect similar to that of MEO. Surprisingly, 0.5 μl l⁻¹ of MEO or synthetic R-carvone catalyzed AX sprouting in the tuber. To the best of our knowledge, this is the first report of an essential oil vapor inducing early sprouting of potato tubers. R-carvone caused visible damage to the meristem membrane at sprout-inhibiting, but not sprout-inducing doses, suggesting different underlying mechanisms. After 5 days’ exposure to R-carvone, its derivatives transcarveol and neo-dihydrocarveol were found in buds of tubers treated with the inhibitory dose, suggesting biodegradation. These experiments demonstrate the potential of MEO vapor as an environmentally friendly alternative to CIPC in stored potatoes and as a research tool for the control of sprouting in plants.