Evaluation of tentacle regeneration as a biological assay inHydra

Summary Tentacle number in non-buddingHydra attenuata, randomly selected from mass culture varies <0.5 tentacles over a 3 month period. Replicate samples of untreated regenerates (n=50–60), however, show some variability in mean tentacle number regenerated (S _x0.13–0.15). The variability is similar whether experiments are performed using randomly selected animals or animals with identical tentacle numbers. The variability is, further, not the result of profound differences in the time of tentacle initiation in individual animals.Addition of 10^–5 M glutamate or a methanol extract to the assay medium results in both an earlier appearance of tentacles and in more tentacles being regenerated during early time periods. The mean tentacle number of methanol extract-treated animals is significantly higher than the mean tentacle number of either control or glutamate-treated animals at all time periods examined.The distribution of tentacle number classes among regenerates is normal in control and glutamate-treated animals but nonparametric in methanol extract-treated animals, making statistical analysis of the data using Student'st-test in-appropriate. The usefulness of the Mann WhitneyU and Kruskal-Wallis tests is discussed, as is the appropriateness of tentacle regeneration as an assay forhydra morphogens.     

Analysis of early stages of budding inHydra by means of an endogenous inhibitor

Summary Buds originate inHydra attenuata at a position 1/3 of the body length from the basal disc. The position with respect to the vertical axes is determined first and the position of the bud on the circumference of this budding region is specified later.Bud formation in hydra is reversibly prevented by pre-treatment with an inhibitor purified from hydra tissue (Berking, 1977). Some hours after the end of the treatment with the inhibitor, bud formation is resumed. From the starting or restarting point of development after the inhibitory treatment to the visible beginning of bud formation, 4 intermediary stages were distinguished on the basis of different responses to a second treatment with inhibitor. The pre0treatment is followed immediately by a period of maximal sensitivity to the inhibitor, which varies in length. At the conclusion of this phase the time interval required for the visible appearance of buds is fixed (12 h). In this and the following phase another application of inhibitor can cancel the entire preparatory process from the pre-treatment onwards. A transition to near complete resistance to inhibitor is the basis for defining a third phase. In a fourth phase, immediately before the evagination of the bud starts, the proesence of the inhibitor will again hinder the development. Upon removal of the inhibitor the suppressed buds will appear.     

Head regeneration inHydra is initiated release of head activator and inhibitor

Summary Hydra regenerating heads release at least two substances into the surrounding medium: one stimulates and one inhibits head formation. The inhibitor is released mainly during the first hour after cutting, the activator is released more slowly with a maximum in the second hour and with substantial release still during the following six hours. The release of both substances seems to be specific for head regeneration: it is not found in animals regenerating feet. The sequential release of these substances leads to the early changes observed at the cellular level during head regeneration inhydra: the inhibitor produces a decrease, the activator an increase in the mitotic activity of interstitial and epithelial cells, if assayed on intact animals. Head regeneration is blocked, if the release of the head activator is prevented. It is therefore suggested that these substances are necessary to initiate head regeneration inhydra.     

An electron microscopic and radioautographic study of hypostomal regeneration inHydra viridis

Summary The gastrodermal secretory cells inHydra viridis are limited to specific regions in the body column. There are two types of mucous cells present, and they are limited to the hypostome. The zymogen cells are absent from the hypostome, but they extend along the body column from the tentacles to the peduncle. Transection beneath the tentacles produces a proximal portion of the hydra devoid of mucous cells. This piece regenerates new tentacles and a normal hypostome, filled with mucous cells, within four days.The following events were observed during regeneration. The zymogen cells formed an aggregate within twenty-four hours in the region of the presumptive hypostome. These cells organized and formed lobes of zymogen cells that were positioned similarly to the arrangement of mucous cells in the normal animal. Sparsely distributed small basophilic cells were also present in the reforming hypostome. Using corresponding thick and thin sections we identified the cells incorporating radiosulfate: 1) The zymogen cells in the distal aggregate. 2) Small basophilic cells, some filled with free ribosomes, and others with a well-developed E. R. 3) Secretory cells containing both mucous and serous granules. 4) Secretory cells with granules similar to the granules in mouse Paneth cells.The fate of the secretory granules in the zymogen cells in the distal aggregate is unknown. Some are autolysed within the cell, and others are extruded. However, some observations suggest that there may be a direct transformation of some of the serous granules to mucous granules. The E. M. observations, the radiosulfate incorporation data, and the migrations of cells to the wound site, suggest that both the zymogen cells and basophilic cells transform to mucous cells. Identification of the early stages of mucous synthesis in these basophilic cells enabled us to study the sequence of mucous granule maturation of both the hypostomal mucous cells.The two most significant questions which we feel remain unaswered are: 1) What are the ultrastructural events during the zymogen cell transformation to a mucous cell ? 2) What is the origin of the small gastrodermal basophilic cells ?

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Zusammenfassung Die gastrodermalen sekretorischen Zellen von Hydra viridis kommen nur in spezifischen Regionen der Körpersäule vor. Es gibt zwei Typen muköser Zellen, und diese findet man ausschließlich im Hypostom. Zymogene Zellen gibt es nicht im Hypostom, aber erstrecken sich längs der Körperachse von den Tentakeln zum Pedunkulus. Sektion unterhalb der Tentakel produziert eine proximale Region von Hydra ohne muköse Zellen. Dieses Stück regeneriert neue Tentakel und ein normales Hypostom mit mukösen Zellen innerhalb vier Tagen.Die folgenden Vorgänge wurden beobachtet während der Regeneration. Die Zymogenzellen bildeten ein Aggregat in der Gegend des präsumptiven Hypostoms innerhalb 24 Std. Diese Zellen bildeten Lappen von Zymogenzellen in ähnlicher Anordnung wie die mukösen Zellen im Normaltier. Ebenfalls vorhanden im neu sich bildenden Hypostom waren locker verteilte, kleine basophile Zellen. Durch Verwendung alternierender dicker und dünner Schnitte identifizierten wir die Zellen, die radioaktives Sulfat einbauten: 1. Zymogenzellen im distalen Aggregat. 2. Kleine basophile Zellen, einige mit freien Ribosomen angefüllt, andere mit gut entwickelten endoplasmatischen Retikulum. 3. Sekretorische Zellen mit mukösen und serösen Granula. 4. Sekretorische Zellen mit Granula, die ähnlich aussehen wie die Granula von Maus Paneth-Zellen. Das Schicksal der sekretorischen Granula in den Zymogenzellen des distalen Aggregates ist unbekannt. Einige werden innerhalb der Zellen autolysiert, andere ausgestoßen. Es scheint aber, daß einige der seriösen Körner sich direkt in muköse umwandeln. Die elektronenoptischen Bilder, die Ergebnisse des Sulfat-Einbaus, und die Wanderung von Zellen zur Wunde weisen darauf hin, daß sowohl Zymogenzellen, als auch basophile Zellen sich in muköse Zellen verwandeln. Identifikationen der Frühstadien von Mukus-Synthese in diesen basophilen Zellen erlaubte uns, die Sequenz der Reifung der Mukus-Granula beider hypostomalen Mukuszellen zu studieren.Die zwei wichtigsten, noch unbeantworteten Fragen verbleiben: 1. Was für feinstrukturelle Veränderungen finden statt während der Transformation von Zymogenzellen in muköse Zellen ? 2. Woher stammen die kleinen gastrodermalen, basophilen Zellen ?

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This paper was prepared from a thesis submitted in partial fulfillment for the degree of Master of Arts.

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This work was supported by the National Institutes of Health grant No GM-11218 to Dr. A. L.Burnett.     

Head regeneration and polarity reversal inHydra attenuata can occur in the absence of DNA synthesis

Summary Regeneration in hydra is considered to be morphallactic because it can occur in the absence of cell division. Whether DNA synthesis is required for regeneration or other repatterning events is not known. The question was investigated by blocking DNA synthesis with hydroxyurea and examining several developmental processes. Head regeneration, reversal of regeneration polarity and battery cell differentiation all took place in the absence of DNA synthesis. Hence, morphallactic regulation in hydra is independent of both DNA synthesis and mitosis.     

Plant regeneration from explant and protoplast derived calluses of Medicago littoralis

Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.5 mg l^–1 N⁶-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N⁶-benzyladenine plus -naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine - IAA indole-3-acetic acid - BOA 1,2-benzisoxazole-3-acetic acid - KIN kinetin - MS Murashige & Skoog (1962) - GRFMS growth regulator free MS medium - B5 Gamborg et al. (1968) - RL Phillips & Collins (1979) - KM8 KM8P Kao & Michayluk (1975) - CPW Frearson et al. (1973) - f. wt fresh weight - FDA fluorescoin diacetate     

The fine structure of the hypostome and mouth of hydra

Summary The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed.     

The fine structure of the hypostome and mouth of hydra

The hypostome and mouth of fresh-water Hydra were examined by scanning electron microscopy. The external surface of the hypostome possesses cnidocils, possibly sensory hairs, and small spiny protrusions surrounding the mouth; the internal surface has cylindrical microvilli, free flagella and adherent flagella. The adherent flagella are most numerous close to the mouth where they cause the cell surface to appear smooth when viewed at low magnifications. Free flagella and leaf-like microvilli increase in prominence towards the tentacles and enter on proper. The edge of the mouth has an abrupt boundary marking the apposition of epidermal and gastrodermal cells. A transitional groove occurs at the boundary and the cells underlying the groove are smaller than those on other regions of the hypostome. The transition groove may represent a site of cell loss in normal cell turnover. Some of the small underlying cells may represent nervous elements involved in regulating hypostome activity during the feeding reation.     

Retroviruses have differing requirements for proteasome function in the budding process

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.     

Arachidonic acid and the control of body pattern inHydra

Summary Repeated stimulation ofHydra magnipapillata with the diacylglycerol (DG) 1,2-sn-dioctanoylglycerol (diC₈) induces an increase in positional value and eventually the development of ectopic heads. Upon stimulation, the polyps release [¹⁴C]-arachidonic acid from previously labelled endogenous sources. Arachidonic acid (AA) is not released into the external medium but remains within the animal, AA, linoleic acid and their lipoxygenase products were identified by gas chromatography-mass spectrometry. Several metabolites were found, most abundantly 12-HETE (hydroxy-eicosa-tetraenoic acid), 8-HETE, 9-HODE (hydroxy-octadecadienoic acid), and 13-HODE; this is the first evidence of their presence in coelenterates. Externally applied AA causes ectopic head formation, though less effectively than diC₈. When administered simultaneously, (diC₈) and AA, which both are known to activate protein kinase C (PKC), act synergistically in inducing ectopic head formation. Since released endogenous AA can spread in tissues, it may mediate a temporal and spatial extension of PKC activation and, hence, broaden the range in which positional value increases. However, in addition to the activation of PKC, the generation of AA metabolites appears to be essential for the induction of ectopic head formation, since not only a selective inhibitor of PKC, chelerythrine, but also an inhibitor of lipoxygenases, NDGA (nordihydroguaiaretic acid), significantly reduces the effectiveness of both AA and DG. Correspondence to: W.A. Müller     

Modulatory action of 12-O-tetradecanoylphorbol-13-acetate on bud production inHydra

Summary A low concentration of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.0 ng/ml) induced a transient inhibition of bud production in hydra which were fed daily. However, when hydra were starved following TPA-treatment, they produced further buds. Phorbol (1.0 ng/ml) and dimethyl sulfoxide (0.001%) did not influence bud production under either feeding or starvation conditions. These results indicate that TPA modulates asexual reproduction in hydra.     

Cellular and molecular characterization of transdifferentiation in the process of morphallaxis of budding tunicates

In the budding tunicate,Polyandrocarpa misakiensis, a bud consists of two epithelial sheets, of which the inner, atrial epithelium shows developmental multipotency. It contains pigment granules in the cytoplasm and expresses a few differentiation markers on the cell surface. During bud development, these features are lost and new differentiation markers appear in organ rudiments that arise from the atrial epithelium. This transdifferentiation of the multipotent epithelium requires at least one cycle of cell division. It may be triggered by endogenous retinoids, probably retinoic acid (RA). RA acts on mesenchymal cells, which then secrete proteases that would serve as an actual transdifferentiation factor of the atrial epithelium.