Ectopic head and foot formation in Hydra: Diacylglycerol-induced increase in positional value and assistance of the head in foot formation

In wild type Hydra magnipapillata, daily application of the protein kinase C activator diacylglycerol (DAG) evokes sprouting of periodically spaced ectopic heads along the body column and leads to loss of the ability to regenerate proximal structures including the foot. The present transplantation studies show that the appearance of ectopic heads is preceded by an early increase in the 'positional value' (P-value) or 'head activation potential' of the gastric column. Long before ectopic head structures emerge, pieces of DAG-treated tissue transplanted into the corresponding positional level of untreated hosts induce head formation instead of being integrated, whereas pieces implanted from untreated donors into DAG-treated hosts form feet. Foot formation implies a decrease in the P-value. This down-regulation is promoted through long-range assistance by the head. Thus, after termination of the DAG treatment ectopic feet are intercalated midway between the periodically spaced heads; moreover, untreated polyps onto which additional distal heads have been grafted regenerate feet faster than do one-headed polyps and may form supernumerary feet. Multiheaded animals can also be produced using two substances (K-252a and xanthate D609) that interfere with signal transduction, but the mode by which secondary heads arise is different from DAG-induced ectopic head formation. Presumably because the assistance by the parental head is impaired, buds fail to form a foot and detach and instead give rise to stable secondary body axes. It is assumed that the P-value along the body varies according to the number of cellular receptors for factors serving as intercellular signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of Shade on the Growth and Sink Activity of Young Flower Heads and Peduncles of White Clover (Trifolium repens L.)

The influence of shade on the growth and sink activity of youngflower heads and peduncles of white clover (Trifolium repensL.) plants was measured in controlled environments. Measurementof the peduncle elongation rate using a linear voltage displacementtransducer showed that when the inflorescence alone was shaded,peduncle elongation was higher than in the light. Translocationstudies using ¹¹C-labelling techniques showed that shading theinflorescence alone had little effect on translocation of assimilatesinto the inflorescence but induced a major change in partitioningof assimilates within it. Peduncle elongation induced by shadeaccompanied an increase in partitioning of photoassimilatesto the peduncle, at the expense of the flower head. Key words: White clover, partitioning, translocation     

The mechanism of floral heliotropism in the snow buttercup, Ranunculus adoneus

We designed field experiments using solar-tracking Ranunculus adoneus flowers to determine where photoreception occurred, which organs responded, and how movement was achieved. Flower peduncles bend eastward in the morning and gradually unbend over the course of the day. Peduncles were found to bend significantly more frequently in the middle region near the floral bracts, 1–3 cm below the flower, than elsewhere on the peduncle. Because the peduncle tip continued to track the sun even after the flower itself was removed, our experiments concentrated on shielding (or conversely, exposing) various portions of peduncles from (or to) sunlight. Photoreception occurred primarily in the portion of the stem just beneath the floral receptacle. By following the position of landmarks applied to the stem, we found that 40% more growth occurred on the shaded side of bent peduncles, compared to the sunlit side. In contrast, top-shielded peduncles did not solar track well and grew only 25% more on the shaded side than on the sunlit side. This growth differential corresponded to differences in cell length on the two sides of bent peduncles, with significantly longer epidermal cells occurring on the shaded side than on the sunlit side.     

Foot formation in hydra: commitment of the basal disk cells in the lower peduncle

Foot regeneration in the freshwater hydra Pelmatohydra robusta was examined using a monoclonal antibody AE03 as a marker. This antibody specifically recognizes mucous-producing ectodermal epithelial cells in the basal disk, but not cells in the peduncle region located just above the basal disk in the foot. When the basal disk was removed by amputation at the upper or lower part of the peduncle, AE03-positive (basal disk) cells always appeared at the regenerating tip of the footless polyp approximately 12-16 h later. When a small piece of tissue was cut out from the upper or lower peduncle region, the tissue invariably turned into a smooth spherical or oblong shape within a few hours. AE03 signal appeared in these spheres variably depending on their origin: when tissue pieces were derived from the lower peduncle, the signal appeared in nearly all pieces and often covered the entire surface of the pieces within 24 h. In contrast, the signal appeared in less than 10% of pieces derived from the upper peduncle. Furthermore, the signal seldom covered more than half of the surface of these pieces. When maintained for many days, pieces derived from the upper peduncle often regenerated tentacles, whereas those from the lower peduncle seldom did. These and other observations suggest that epithelial cells in the peduncle can rapidly differentiate into basal disk cells when the basal tissue is removed. However, cells in the upper peduncle are not irreversibly committed to differentiate into basal disk cells because, when cut out as small tissue pieces, they could remain AE03 negative and become tentacle cells. In contrast, the cells in the lower peduncle apparently are irreversibly committed to differentiate into basal disk cells, as they always turned rapidly into AE03-positive cells once they were physically separated from (and freed from the influence of) the basal disk itself, regardless of the separation methods used.     

Trypanosoma cruzi: treatment with the iron chelator desferrioxamine reduces parasitemia and mortality in experimentally infected mice

The effects of prolonged treatment with iron chelator (desferrioxamine) on the development of infection in mice inoculated with Y Trypanosoma cruzi were determined. Infected/treated mice presented lower levels of parasitemia and reduced mortality rate compared with infected/non-treated animals. The five out of twenty infected/treated mice that survived the acute phase of infection showed negative hemoculture and positive ELISA in the acute and chronic phases and positive PCR in the acute phase: in the chronic phase, three of the animals presented negative PCR. The single surviving infected/non-treated animal exhibited positive hemoculture, PCR and ELISA in both phases of infection. Infected groups presented lower levels of iron in the liver compared with treated/non-infected or non-treated/non-infected animals. The serum iron levels of the infected/non-treated group were higher on the 21st day post-infection in comparison with control and infected/treated groups. These results suggest that decrease of iron in the host leads to T. cruzi infection attenuation.     

DETERMINATE GROWTH OF ALLIUM SATIVUM PEDUNCLES: EVIDENCE OF DETERMINATE GROWTH AS A DESIGN FACTOR FOR BIOMECHANICAL SAFETY

Determinate growth in an organ can reduce the mechanical liability of dynamic wind loadings. This hypothesis was examined for the peduncles of Allium sativum grown under protected and unprotected field conditions and in the glasshouse. The extent to which the lengths of peduncles approach their critical buckling lengths lcr was correlated with the extent to which peduncles were perturbed mechanically. Glasshouse-grown plants that were protected from dynamic loadings (wind or handling) produced peduncles with low ratios of critical to actual peduncle lengths (lcr/1 = 1.11 ± 0.07); field-grown, unprotected plants had lcr/1 = 1.85 ± 0.29; while plants grown in the field but protected from the wind had lcr/1 = 1.29 ± 0.15. Statistical analyses indicated that peduncle length was the only morphometric parameter examined to significantly differ among plants grown under the three conditions. The frequency distributions of critical (buckling) stress for each of the three populations of peduncles differed; that of the unprotected population conformed to a three-parameter Weibull distribution. Allium sativum exhibits thigmomorphogenesis, i.e., mechanical forces influence the extent and nature of organogenesis. Unlike other thigmomophogenetic species, however, the determinate growth of peduncles did not influence peduncle girth or the elastic (Young's) modulus of tissues. Thus, the extent to which vertical growth proceeded determined the design factor for dynamic loading in these reproductive organs. The design (“margin of safety”) factors of A. sativum peduncles grown under the three different conditions and calculated from lcr/1 were remarkably similar to those specified for engineered columns of wood subjected to stress durations differing in magnitude.     

Pattern formation in hydra tissue without developmental gradients

Ring-shaped pieces of hydra tissue were excised from a specified position on a body column of 20-30 polyps and grafted together in tandem like a chain of beads. A "tandem graft" prepared in this way has the same basic tissue organization and same tube-like morphology as a normal hydra body column, but lacks the head, foot, and developmental gradients ordinarily present. Three major types of structures were formed along the length of the tandem graft: heads, buds, and feet. The relative number of these structures produced was strongly affected by the origin of the tissue used to prepare the tandem graft. Evidence was obtained which suggests that tissue originally located outside of the budding zone in intact hydra has a strong latent capacity to form a bud, and that the level of this capacity forms a gradient from the budding zone toward the hypostome. Evidence was also obtained which is consistent with the view that the head and foot forming mechanisms cross-react positively, increasing the chances for these two structures to be formed next to each other on a tandem graft.     

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.     

Interactions between the foot and bud patterning systems in Hydra vulgaris.

In the freshwater coelenterate, hydra, asexual reproduction via budding occurs at the base of the gastric region about two-thirds of the distance from the head to the foot. Developmental gradients of head and foot activation and inhibition originating from these organizing centers have long been assumed to control budding in hydra. Much has been learned over the years about these developmental gradients and axial pattern formation, and in particular, the inhibitory influence of the head on budding is well documented. However, understanding of the role of the foot and potential interactions between the foot, bud, and head patterning systems is lacking. The purpose of this study was to investigate the role of the foot in the initiation of new axis formation during budding by manipulating the foot and monitoring effects on the onset of first bud evagination and the time necessary to reach the 50% budding point. Several experimental situations were examined: the lower peduncle and foot (PF) were injured or removed, a second PF was laterally grafted onto animals either basally (below the budding zone) or apically (above the budding zone), or both the head and PF were removed simultaneously. When the PF was injured or removed, the onset of first bud evagination was delayed and/or the time until the 50% budding point was reached was longer. The effects were more pronounced when the manipulation was performed closer to the anticipated onset of budding. When PF tissue was doubled, precocious bud evagination was induced, regardless of graft location. Removal of the PF at the same time as decapitation reduced the inductive effect of decapitation on bud evagination. These results are discussed in light of potential signals from the foot or interactions between the foot and head patterning systems that might influence bud axis initiation.     

The adaptedness of the floral phenotype in a relict endemic, hawkmoth-pollinated violet. 1. Reproductive correlates of floral variation

This paper examines the relationship between quantitative variation in floral morphology (sizes of petals, spur and peduncle) and maternal reproductive success (seed production) in Viola cazorlensic (Violaceae), a narrowly endemic violet of south-eastern Spain pollinated by day-flying hawkmoths (Sphingidae). This plant is characterized by broad intraspecific variation in size and proportions of floral parts. Floral morphology does not influence significantly the probability of fruit set. Among flowers setting fruit, spur length and size of petals have no significant effect on seed production, but capsules from long- and short-peduncted flowers contain significantly more seeds than capsules from flowers with intermediate peduncles. Individual plants differ significantly in average floral characteristics. Plants with comparatively long and short peduncles tend to produce more seeds than those with intermediate ones, even after accounting statistically for individual differences in flower production. These findings are interpreted as evidence of disruptive selection on peduncle length during the study season. Floral variability in this species may be explained by the combined action of disruptive selection on peduncle length (the character most variable among individuals) and little, if any, stabilizing selection on spur length and size of petals     

Formation of supernumerary structures by the embryonic chick wing depends on the position and orientation of a graft in a host limb bud

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.     

Growth of rose flower peduncles and effects of applied plant growth regulators

Removal of flower buds results in abscission of peduncles of the rose cv. Nubia and cessation of peduncle growth in cv. Mercedes. Peduncle growth was inhibited when pistils and stamens were removed, but was not affected by removal only of sepals and petals. Growth of the decapitated peduncles of Mercedes was partially restored by the application of auxin in lanolin paste on the base of the peduncle and was completely restored by the application of gibberellin, while the combined application of auxin and gibberellin was the most effective for growth restoration. Growth of non-decapitated Nubia peduncles was promoted by application of gibberellin or gibberellin and auxin but not auxin alone.Peduncle elongation of both cultivars was not affected by application of cytokinin and the effect of gibberellin was antagonized by combined application with cytokinin. The peduncles strength (resistance to bending) was affected more strongly by auxin than by gibberellin, and most strongly when auxin and gibberellin were combined. The effect of auxin on the strength of peduncles, but not of gibberellin, was antagonized by application of cytokinin. Excised, non-decapitated flowering stems of Nubia treated with gibberellin and auxin in situ, showed, recovery of the peduncles from wilting after exposure to heat conditions.     

Increasing in ROS levels and callose deposition in peduncle vascular bundles of wheat (Triticum aestivum L.) grown under nitrogen deficiency

Nitrogen availability is closely related to crop senescence and productivity, but its associated effect on reserve remobilization is not yet fully understood. In this study, we observed that nitrogen deficiency (N^?) led to significant decreases in the activities of superoxide dismutase (SOD) (P<0.05), guaiacol peroxidase (P<0.05), and catalase (P<0.05) as well as a higher concentration of reactive oxygen species (ROS) (P<0.05) in wheat (Triticum aestivum L.) peduncles during the middle grain-filling compared with the application of 225 kg N ha^?1 (N⁺). Callose concentration showed the same trend of temporal changes as ROS. Histochemical staining revealed that both ROS and callose predominantly occurred in vascular bundles of peduncles. Ultimately, grain filling rates and grain weight in N^? wheat were reduced compared with N⁺ plant. These data suggest that the grain yield decline in N^? wheat may be at least partially attributed to the higher callose deposition in peduncle vascular bundles and ROS level is closely associated with the increase in the callose deposition in wheat peduncle vascular bundles.     

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.     

In the multiheaded strain (mh-1) of Hydra magnipapillata the ectodermal epithelial cells are responsible for the formation of additional heads and the endodermal epithelial cells for the reduced ability to regenerate a foot

Hydra consist of three self-renewing cell lineages: the ectodermal epithelial, endodermal epithelial and interstitial cell lineages. The role of these cell lineages in head formation and foot regeneration in Hydra magnipapillata was studied by comparing the multiheaded strain mh-1 with the wild-type. Adult polyps of this strain show a reduced ability to regenerate a foot in the apical body half several days before additional heads are formed there. Cell lineage chimeras were produced, and it was found that in mh-1, the ectodermal epithelial cell lineage is responsible for the formation of additional heads, whereas the endodermal epithelial cell lineage and, to a lesser extent, the derivatives of the interstitial cell lineage, are responsible for the reduced ability of foot regeneration.     

THE FERTILE SPIKE OF OPHIOGLOSSUM PETIOLATUM. I. MECHANISM OF ELONGATION

The frond of Ophioglossum consists of a sterile segment and a fertile segment or spike. An investigation of fertile spike elongation reveals that growth of the spike proceeds by activity of an intercalary meristem located in the most distal region of the peduncle subtending the sporangial area. Anatomical comparisons of all regions of developing spikes, counts of mitotic figures along the length of spikes of various ages, determination of cell lengths of peduncle ground parenchyma cells, and historadioautography of spikes treated with H³-thymidine confirms the presence in the apical portion of the peduncle of a region of frequently dividing cells intercalated between two regions of more mature tissues. Marking experiments indicate that the petiole of the sterile segment of the frond elongates in a similar fashion. Although this type of intercalary meristem is rather common in angiosperm flower scapes and peduncles, this is the first detailed analysis of this type of growth in a pteridophyte genus.     

Effects of applied auxin,gibberellin and cytokinin on the activity of peroxidases in the peduncles of rose flowers

The activity of peroxidases in the proximal part of the flower peduncle of rose cv. Nubia was promoted by exogenous application of auxin but not by gibberellin or cytokinin. In cv. Mercedes the activity was promoted also by gibberellin and cytokinin. In the distal parts of the peduncles of both cultivars, peroxidase activity was not affected by any of the applied growth regulators. In young flowers of cv. Nubia the protein content of the penduncles was affected only by cytokinin, and in aged flowers only by auxin, while in Mercedes peduncles the content of protein was not affected by any of the applied growth regulators. The specific activity of peroxidases was promoted by auxin in peduncles of Nubia and by both auxin and cytokinin in peduncles of Mercedes flowers.     

Effect of cetrorelix on sperm morphology during migration through the epididymis in the cynomolgus macaque (Macaca fascicularis)

The importance of the cynomolgus monkey as a model for human reproductive medicine prompted this examination of epididymal sperm morphology. Computer-aided sperm morphological analysis was used for the first time to provide morphometric data on sperm heads as they traversed the epididymal duct of Macaca fascicularis. The duct was divided into six regions, starting close to the testis (proximal) and ending close to the vas deferens (distal). To determine the androgen-dependence of the changes, one group of animals received a GnRH-antagonist (Cetrorelix, Asta Medica, Frankfurt, Germany) to induce testicular regression and lower epididymal androgens, while a control group received only vehicle. Epididymides were removed 16 and 25 days after treatment, and sperm heads were analysed by a computer-assisted morphometric analyser. Cluster analysis revealed swollen sperm head cells in proximal regions 1 and 2 of the epididymis, but fewer such forms distally. Normal head shapes became the majority in region 4 and these underwent a gradual but statistically significant decrease in size (area, perimeter, length, width) and shape as they reached the distal regions. In the animals given Cetrorelix, sperm with swollen heads were found more distally than in the controls, although they were also never present in the distal cauda (region 6). Normal heads still became predominant in region 4 after 16 days treatment, and in region 6 after 25 days. The normal forms in the cauda epididymidis of treated animals were significantly larger than cells from control animals. We conclude that epididymal sperm maturation in the monkey is characterised by both a loss of sensitivity to distortion on air-drying, and by a decrease in sperm head size. The former, but not the latter, is attained by sperm in androgen-deficient epididymides from GnRH-antagonist-treated monkeys.     

Developing fruit direct post-floral morphogenesis in Helleborus niger L

In fertilized flowers of Helleborus niger L., the sepals (the showy elements of the perianth at anthesis) grow, spread, and turn green, and the peduncles elongate. These processes did not proceed to completion when the pistils were removed at the bud stage, but could be restored by the application of plant growth regulators. Cytokinins and gibberellins stimulated the formation of well-developed chloroplasts in, and spreading of, the sepals; the gibberellin, GA3, and the auxin, 4-chloroindole-3-acetic acid, promoted peduncle elongation. In fruit-bearing flowers, on the other hand, paclobutrazol, an inhibitor of gibberellin biosynthesis, reduced chlorophyll formation in the sepals, reversed sepal spreading, and inhibited peduncle elongation. Of the endogenous growth regulators in developing fruit, the following cytokinins were identified: zeatin, dihydrozeatin, N6-(2-isopentenyl)adenine and their ribosides and 9-glucosides. Zeatin riboside drastically increased in abundance (about 200 times), shortly after fertilization, when chlorophyll accumulation in the sepals occurred at the fastest rate, and remained the most prominent identified cytokinin until seed ripening.