Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

Mechanoreception and synaptic transmission of hydrozoan nematocytes

Mechanoelectric transduction and its ultrastuctural basis were studied in the cnidocil apparatus of stenotele nematocytes of marine and freshwater Hydrozoa (Capitata and Hydra) as a paradigm for invertebrate hair cells with concentric hair bundles. The nematocytes respond to selective deflection of their cnidocil with phasic-tonic receptor currents and potentials, similar to vertebrate hair cells but without directional dependence of sensitivity. Ultrastructural studies and the use of monoclonal antibodies allowed correlating the mechanoelectric transduction with structural components of the hair bundle. Two other types of depolarising current and voltage changes in nematocytes are postsynaptic, as concluded from their ionic and pharmacological characteristics. One of these types is induced by mechanical stimulation of distant nematocytes and sensory hair cells. It is graded in amplitude and duration, but different from the presynaptic receptor potential. Adequate chemical stimulation of the stenoteles strongly increases the probability of discharge of their cnidocyst, if the chemical stimulus precedes the mechanical one. Simultaneously, the probability of synaptic signalling induced by mechanical stimulation is increased, reaching nearly 100%. The chemoreception of the phospholipids used could be localized in the shaft of the cnidocil, because of the water-insolubility of the stimulant. This chemical stimulation itself does not cause a receptor potential; its action is classified as a modulatory process. Electron microscopy of serial sections of the tentacular spheres of Coryne revealed synapses that are efferent to nematocytes and hair cells besides neurite–neurite synapses, each containing 3–10 clear and/or dense-core vesicles of 70–150 nm diameter. The only candidates to explain the graded afferent signal transmission of nematocytes and hair cells are regularly occurring cell contacts associated with 1(–4) clear vesicles of 160–1100 nm diameter. Transient fusion and partial depletion of stationary vesicles are discussed as mechanisms to reconcile functional and structural data of many cnidarian synapses. Review contributed to the Symposium on Neuro-Anatomy and -Physiology of Coelenterates; 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA; July 6–11, 2003.     

Structure of the mouth of Hydra spp. A breach in the epithelium that disappears when it closes

Summary When the mouth of a hydra is not open, the ectodermal and endodermal epithelia are each continuous over the oral surface. All cells are joined to all neighboring cells by septate junctions. A mouth forms when muscles stretch the center of the oral region and pull several cells into a thin lamella. Finally, cell attachments break. Rapidly (within one second) this breach expands as a round opening until it is approximately half the diameter of the surrounding column. Considerable rearrangment of the central cells, and therefore of their septate junctions, likely takes place rapidly as the mouth expands. A few cells may be lost from the epithelia. Closure of the mouth is slow and results in irregular thickening and wrinkling of the tissue. Final closure to restore histological continuity resembles wound healing more than occlusion typical of the mouths of other animals. The lack of a permanent mouth may be a primitive character or an adaptation to a simple body plan, and is shared by many hydrozoan polyps but not by more complex cnidarians.     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.     

Characterization of a RFamide-positive subset of ganglionic cells in the hydrozoan planular nerve net

Summary The complexity of the hydrozoan planular nervous system was examined. Using a whole-mount technique with indirect immunofluorescence, the spatial pattern of ganglionic cells showing RFamide-like immunoreactivity was visualized. RFamide antiserum bound a subset of ganglionic cells in the anterior and upper middle regions of the planula and a few ganglionic cells in the upper tail region. Labeled cells consisted of bipolar and multipolar neurons. Stained processes from these cells formed a three-dimensional nerve net that followed the contour of the mesoglea; such fibers were striking in terms of their large numbers, long lengths, and organization into distinct bundles. Labeled fibers were seen to contact other ganglionic cells, sensory cells, epitheliomuscle cells, the mesoglea, and the outside free surface. All stained cell bodies and fibers were found in the ectoderm. Using the same technique the reappearance of RFamide-positive ganglionic cells in epithelial tissue of chimeric grafts of planulae was observed. Interstitial cells capable of forming RFamide-positive ganglionic cells underwent extensive anterior-posterior migrations in the grafts, moved into the epithelial tissue, and differentiated into RFamide-positive ganglionic cells. Stained repopulated ganglionic cells always formed in the same position in the epithelial tissue as was observed in control planulae suggesting that the expression of RFamide-like substances may be position dependent in the planula.     

Localization of dopamine in the freshwater hydrozoanHydra attenuata

Summary High performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.     

Anchorage and retraction of nematocytes in the tentacles of the cubopolyp Carybdea marsupialis are mediated by a species-specific mesogleal support

Each tentacle of the cubopolyp Carybdea marsupialis is armed with only a single nematocyte at its tip. The correct position of the nematocyte is maintained by a crown-shaped cup formed by the mesoglea. In maximally contracted tentacles, the nematocyte and 7–10 surrounding accessory cells are completely retracted into an ectodermal invagination. A belt of muscle cells revealing a distinct cross-striation in specimens labelled with fluorescein-isothiocyanate-phalloidin is located around the basal part of the nematocyte. These muscle cells are linked both to the mesogleal cup and to the nematocyte by specialized desmosomal contact zones. An additional set of long slender muscle strands runs through the complete length of the tentacles. Their myofibrils reveal only a weak striation pattern. Whereas the contraction of the tentacles seems to depend on the slender muscle strands, the retraction of the apical cell complex is thought to be mediated by the cross-striated muscle belt.     

Scanning electron microscopy of the muscle system of Hydra magnipapillata

The fine structure of the ectodermal and endodermal muscle layers of Hydra magnipapillata has been analyzed by scanning electron microscopy after hydrolytic removal of the mesoglea with NaOH and subsequent exposure of the basal and lateral aspects of the layers by mechanical dissection. The ectodermal muscle layer consists of fibrous processes of epithelial cells extending longitudinally to the body axis, whereas the endodermal muscle layer comprises cells with hexagonal bases and several strands of myonemes oriented circularly. In each layer, the muscular elements tightly interdigitate, extending a continuous muscle sheet along the mesoglea. The ectodermal and endodermal muscle sheets communicate with each other via foliate microprojections penetrating the mesoglea. On the lateral aspect of the ectodermal epithelium, spiny nerve fibers run along the upper surface of the muscle processes. The spines are often attached to muscle processes, suggesting that the former monitor muscle contraction. Nerve fibers occasionally come into contact with the mesoglea through narrow gaps between the muscle processes. In the hypostomal ectoderm, a small spindle-shaped cell, probably sensory in nature, extends an apical cilium and a long basal process.     

Boundary cells of endodermal origin define the mouth of Hydra vulgaris (Cnidaria)

We investigated morphology, dynamics and origin of cells surrounding the mouth of Hydra vulgaris using the monoclonal antibody L96. This antibody recognises a one cell-thick ring of endodermal epithelial cells exactly at the boundary between endoderm (gastrodermis) and ectoderm (epidermis). L96⁺ cells can stretch considerably without any cell rupture during mouth opening. Thus, our data prove the existence of a distinct cell population defining hydra's mouth. A model for mouth opening is proposed and the significance of L96⁺ cells for boundary formation between ectoderm and endoderm is discussed.     

Immunocytochemical evidence for an NMDA1 receptor subunit in dissociated cells of<Emphasis Type="Italic"> Hydra vulgaris</Emphasis>

We have previously reported immunocytochemical, biochemical, behavioral, and electrophysiological evidence for glutamatergic transmission through (±)--amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)/kainate receptors in hydra. We now report specific localization of the N-Methyl-D-aspartic acid receptor subunit 1 (NMDAR1) in epithelial, nerve, nematocytes, and interstitial cells of hydra. Macerates of tentacle/hypostome pieces of Hydra vulgaris were prepared on agar-coated slides, fixed with buffered formaldehyde/glutaraldehyde, and fluorescently labeled with monoclonal antibodies against mammalian NMDAR1. Negative controls omitted primary antibody. Digital images were recorded and analyzed. Specific localized and intense labeling was found in ectodermal battery cells, other epithelial cells, nematocytes, interstitial cells, and sensory and ganglionic nerve cells, and in battery cells was associated with enclosed nematocytes and neurons. The labeling of myonemes was more diffuse and less intense. In nerve and sensory cells, punctate labeling was prominent on cell bodies. These results are consistent with our earlier evidence for glutamatergic neurotransmission and kainate/NMDA regulation of stenotele discharge. They support other behavioral and biochemical evidence for a D-serine-sensitive, strychnine-insensitive, glycine receptor in hydra and suggest that the glutamatergic AMPA/kainate-NMDA system is an early evolved, phylogenetically old, behavioral control mechanism.     

Analysis of complete mitochondrial DNA sequences of three members of the Montastraea annularis coral species complex (Cnidaria, Anthozoa, Scleractinia)

Complete mitochondrial nucleotide sequences of two individuals each of Montastraea annularis, Montastraea faveolata, and Montastraea franksi were determined. Gene composition and order differed substantially from the sea anemone Metridium senile, but were identical to that of the phylogenetically distant coral genus Acropora. However, characteristics of the non-coding regions differed between the two scleractinian genera. Among members of the M. annularis complex, only 25 of 16,134 base pair positions were variable. Sixteen of these occurred in one colony of M. franksi, which (together with additional data) indicates the existence of multiple divergent mitochondrial lineages in this species. Overall, rates of evolution for these mitochondrial genomes were extremely slow (0.03–0.04% per million years based on the fossil record of the M. annularis complex). At higher taxonomic levels, patterns of genetic divergence and synonymous/nonsynonymous substitutions suggest non-neutral and unequal rates of evolution between the two lineages to which Montastraea and Acropora belong.     

Gland cells in the tentacles of the jellyfish Cyanea lamarcki reactive with an antibody against 5-hydroxytryptamine

Summary A lightand electron-microscopic immunocytochemical study on the localization of 5-hydroxytryptamine in Cyanea lamarcki revealed a reaction product in the epidermal gland cells surrounding the nematocyst clusters. The closely related scyphozoan medusa Cyanea capillata lacked a reaction product in the similar mucus-producing gland cells. Earlier conflicting views on the presence and localization of 5-hydroxytryptamine in coelenterates can thus be due to large species differences. It can be stated that 5-hydroxytryptamine is lacking in the venom of Cyanea.     

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions

We have undertaken a study of the structure of the amino-terminal domain of the phytochrome polypeptide purified from Avena sativa L. Amino-acid sequencing was used to indentify arginine 52 as the precise location of a conformation-specific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35–40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility.Abbreviations Da dalton - M_r relative molecular mass - Pr, Pfr red and far-red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986     

Central projections of the pineal complex in the silver lamprey Ichthyomyzon unicuspis

Summary The central projections of the pineal complex of the silver lamprey Ichthyomyzon unicuspis were studied by injection of horseradish peroxidase. The pineal tract courses caudally along the left side of the habenular commissure, and a few fibers penetrate the brain through the caudalmost portion of this commissure. Most of the fibers, however, continue caudally and enter the brain through the posterior commissure. The pineal tract projects bilaterally to the subcomissural organ, the superficial and periventricular pretectum, the posterior tubercular nucleus, the dorsal and ventral thalamus, the dorsal hypothalamus, the optic tectum, the torus semicircularis, the midbrain tegmentum, and the oculomotor nucleus. A few fibers decussate in the tubercular commissure, but the course of these decussate fibers could not be followed owing to the bilateral nature of the projections. No retrogradely labeled cells were found in the brain. With the exception of the projections to the optic tectum and torus semicircularis, the pineal projections in the silver lamprey are similar to those reported in other anamniote vertebrates.     

Neuronal architecture of the central complex in Drosophila melanogaster

Summary On the basis of 1200 Golgi-impregnated brains the structure of the central complex of Drosophila melanogaster was analyzed at the cellular level. The four substructures of the central complex — the ellipsoid body, the fanshaped body, the noduli, and the protocerebral bridge — are composed of (a) columnar small-field elements linking different substructures or regions in the same substructure and (b) tangential large-field neurons forming strata perpendicular to the columns. At least some small-field neurons belong to isomorphic sets, which follow various regular projection patterns. Assuming that the blebs of a neuron are presynaptic and the spines are postsynaptic, the Golgi preparations indicate that small-field neurons projecting to the ventral bodies (accessory area) are the main output from the central complex and that its main input is through the large-field neurons. These in turn are presumed to receive input in various neuropils of the brain including the ventral bodies. Transmitters can be attributed immunocytochemically to some neuron types. For example, GABA is confined to the R1–R4 neurons of the ellipsoid body, whereas these cells are devoid of choline acetyltransferase-like immunore-activity. It is proposed that the central complex is an elaboration of the interhemispheric commissure serving the fast exchange of data between the two brain hemispheres in the control of behavioral activity.     

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

Summary Fertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.     

The bridge-partitioning complex of germ-cell intercellular bridges in the testis of the golden hamster

The bridge-partitioning complex present in pre-existing intercellular bridges of dividing spermatogonia in the juvenile golden hamster testis was studied by electron microscopy. There is a close temporal adjustment in the appearance of this structure to those stages of mitosis during which the cells are without a nuclear membrane, i.e., the bridge-partitioning complex is formed at the transition between prophase and prometaphase and gradually disappears during telophase. In addition, in a certain form of degenerative dividing germ cells, which completely lack a bridge-partitioning complex in pre-existing intercellular bridges, condensed chromatin not surrounded by a nuclear membrane occasionally projects through these open bridges and thus may well change over to a neighboring cell of the same clone. These results strongly indicate an essential barrier function of the bridge-partitioning complex. It temporarily prevents intraclonal exchange of nuclear material during those stages of mitosis where a nuclear membrane is lacking and, thus, maintains genetic integrity of male germ cells during synchronous divisions.     

芒和荻种子对复合盐碱胁迫的生理响应

土壤盐碱化在世界范围内普遍存在,日益严峻的盐碱化形势严重威胁着植物的生长发育。芒(Miscanthus sinensis)和荻(Miscanthus sacchariflora)作为能源植物具有良好的经济效益和生态效益,并且在城市园林中已得到广泛应用。该研究以引种自辽宁省本溪阿家岭的芒和哈尔滨市太阳岛的荻为对象,模拟我国东北大庆盐碱地的低(浓度1、2)、中(浓度3)、高(浓度4、5)浓度土壤环境,分别对芒和荻的种子进行复合盐碱胁迫处理,对芒和荻的种子萌发情况进行研究。结果表明：(1)复合盐碱胁迫处理下,芒种子的发芽率随着复合盐碱浓度的升高而降低,发芽势、活力指数、发芽指数、胚根长度、胚芽长度、胚根鲜重、胚芽鲜重和耐盐碱指数均先升高后降低;荻种子的发芽率、发芽势、活力指数、发芽指数、胚根长度、胚芽长度、胚根鲜重、胚芽鲜重和耐盐碱指数均随着复合盐碱浓度的升高而降低。(2)芒和荻的种子能够抵抗低、中浓度的复合盐碱胁迫处理,当高浓度的复合盐碱胁迫处理时,各项指标均下降明显,且芒种子的各项指标均优于荻种子,说明芒种子比荻种子更耐盐碱。该研究结果基本界定芒荻种子的复合盐碱耐受范围,为今后芒荻类能源植物的耐盐碱性筛选和在园林中的应用提供了理论依据。     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

Acrosome formation and the centriolar complex in the spermatozoa of Sabella penicillum (Polychaeta)

Summary The acrosomal vesicle of Sabella penicillum spermatids consists of an electrondense core and a more transparent surrounding zone. During subsequent differentiation the vesicle membrane forms several invaginations in the juxtanuclear area. These invaginations later establish contact with the core. In the mature spermatozoon the spaces between the invaginations appear as electron-dense tubules; this is probably due to a shift of material from core to periphery. The ultrastructure of the centriolar complex is described in detail.Work supported by the Norwegian Research Council for Science and Humanities (NAVF; Grant Nr. D 61.44) and The Austrian Fonds zur Förderung der wissenschaftlichen Forschung Projekt 2183 and N 39.