Membrane Conductances and Spectral Sensitivities of Pecten Photoreceptors

The electrical and spectral properties of depolarizing (proximal) and hyperpolarizing (distal) photoreceptors in the eye of the scallop, Pecten irradians, were examined. Both depolarizing and hyperpolarizing responses are associated with an increase in membrane conductance; in addition, the depolarizing response is characterized by a secondary decrease in conductance at light intensities which inactivate the response. Both responses can be reversed in polarity by applied current across the cell membrane. The depolarizing response has a reversal potential of approximately +10 mv, whereas the estimated reversal potential for the hyperpolarizing response is near -70 mv. The two responses have the same spectral sensitivity function, which agrees with a Dartnall nomogram for a rhodospin with a λ_max at 500 nm. It is suggested that the photochemical reactions produce different end products which give responses of opposite polarity in proximal and distal cells, or alternatively, that the reactions of the respective cell membranes to the same end product are different.     

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.     

The retina of the phalangid,Opilio ravennae,with particular reference to arhabdomeric cells

Summary The retina of the phalangid, Opilio ravennae, consists of retinula cells with distal rhabdomeres, arhabdomeric cells, and sheath cells. The receptive segment of retinula cells shows a clear separation into a Proximal rhabdom, organized into distinct rhabdom units formed by three or four retinula cells, and a Distal rhabdom, consisting of an uniterrupted layer of contiguous rhabdomeres. One of the cells comprising a retinula unit, the so-called distal retinula cell (DRC), has two or three branches that pass laterally alongside the rhabdom, thereby separating the two or three principal retinula cells of a unit. The two morphologically distinct layers of the receptive segment differ with respect to the cellular origin of rhabdomeral microvilli: DRC-branches contribute very few microvilli to the proximal rhabdom and develop extremely large rhabdomeres in the distal rhabdom only, causing the rhabdom units to fuse. Principal retinula cells, on the other hand, comprise the majority of microvilli of the proximal rhabdom, but their rhabdomeres diminish in the distal rhabdom. It is argued that proximal and distal rhabdoms serve different functions in relation to the intensity of incident light.In animals fixed 4 h after sunset, pigment granules retreat from the distal two thirds of the receptive segment. A comparison of retinae of day- and night-adapted animals shows that there is a slight (approximately 15%) increase in the cross-sectional area of rhabdomeral microvilli in dark-adapted animals, which in volume corresponds to the loss of pigment granules from the receptive segment. The length of the receptive segment as well as the pattern and shape of rhabdom units, however, remain unchanged.Each retinula unit is associated with one arhabdomeric cell. Their cell bodies are located close to those of retinula cells, but are much smaller and do not contain pigment granules. The most remarkable feature is a long, slender distal dendrite that extends up to the base of the fused rhabdom where it increases in diameter and develops a number of lateral processes interdigitating with microvilli of the rhabdom. The most distal dendrite portion extends through the center of the fused rhabdom and has again a smooth outline. All dendrites end in the distal third of the proximal rhabdom and are never present in the layer of the contiguous distal rhabdom. Arhabdomeric cells are of essentially the same morphology in day- and night-adapted animals. They are interpreted as photoinsensitive secondary neurons involved in visual information-processing that channel current collected from retinula cells of the proximal rhabdom along the optic nerve. A comparison is made with morphological equivalents of these cells in other chelicerate species.     

An IQGAP-related gene is activated during tentacle formation in the simple metazoan Hydra

Differentiation of body column epithelial cells into tentacle epithelial cells in Hydra is accompanied by changes in both cell shape and cell-cell contact. The molecular mechanism by which epithelial cells acquire tentacle cell characteristics is unknown. Here we report that expression of a Hydra homologue of the mammalian IQGAP1 protein is strongly upregulated during tentacle formation. Like mammalian IQGAP, Hydra IQGAP1 contains an N-terminal calponin-homology domain, IQ repeats and a conserved C terminus. In adult polyps a high level of Hydra IQGAP1 mRNA is detected at the basis of tentacles. Consistent with a role in tentacle formation, IQGAP1 expression is activated during head regeneration and budding at a time when tentacles are emerging. The observations support the previous hypothesis that IQGAP proteins are involved in cytoskeletal as well as cell-cell contact rearrangements. Received: 25 January 2000 / Accepted: 2 May 2000     

Cytokinin activation and redistribution of plasma-membrane ion channels in Funaria

I have investigated changes in electrical current across the plasma membrane that occur during cytokinin-induced bud formation in Funaria hygrometrica Hedw., using a non-intrusive vibrating microelectrode. Before cytokinin treatment the target caulonema cells have maximal inward current at the nuclear region. After cytokinin treatment inward current increases twofold along the length of the cell. Within minutes, however, current decreases at both the nuclear zone and the proximal end while increasing at the distal end of target cells, preceding and predicting the presumptive division site. Inward current at the distal end falls to resting levels after establishment of a bulging growth zone, and remains low around developing buds. This current is blocked by gadolinium nitrate, a Ca²⁺-uptake inhibitor, indicating a Ca²⁺ component of the current. The polarity of the target cells can be disrupted by microfilament inhibitors and cytokinin-induced buds form over the nucleus, halfway along the length of the cell. I suggest that cytokinin activates plasma-membrane ion channels which are subsequently redistributed to the distal ends of target cells by a microfilament-dependent process. Cytokinin-induced concentration of ion channels over presumptive bud sites may be envisioned to exert spatial control of cytoplasmic ion concentrations and stimulate bud formation by establishing a new growth zone, directing nuclear migration, and stimulating cell division.Abbreviations BA 6-benzyladenine - [Ca²⁺]_i intracellular calcium-ion concentration     

Nerve commitment in Hydra: I. Role of morphogenetic signals

The kinetics of nerve commitment during head regeneration in Hydra were investigated using a newly developed assay for committed cells. Committed nerve precursors were assayed by their ability to continue nerve differentiation following explanation of small pieces of tissue. Committed nerve precursors appear at the site of regeneration within 6 hr after cutting and increase rapidly. The increase is localized to the site of regeneration and does not occur at proximal sites in the body column of the regenerate. The increase is delayed about 8–12 hr when regeneration occurs at sites lower in the body column. The results show, furthermore, that redistribution of committed precursors does not play a major role in the pattern of nerve differentiation during regeneration. Since the increase in committed nerves coincides with the increase in morphogenetic potential of the regenerating tissue, the results strengthen the idea that morphogenetic signals are involved directly in the control of nerve commitment in Hydra.     

Tentacle regeneration in hydra: A quantitative methodological approach

A quantitative method is proposed for the evaluation of distal regeneration in Hydra attenuata; it is based on estimates of tentacle elongation during 10 days of regeneration, determination of a Tentacle Regeneration Index, and a statistical analysis of profiles obtained from various samples in different experiments. The results show that: polyps under normal conditions have similar regeneration patterns, regardless of individual variability; and ATxII, a neurotoxin of cnidarian origin, produces a statistically significant increase in the Tentacle Regeneration Index. The results are discussed in relation to pattern formation and growth in Hydra.     

REGENERATION IN THE COENOCYTIC MARINE ALGA,CAULERPA, WITH RESPECT TO GRAVITY

Small pieces of the green algal coenocyte Caulerpa are capable of regenerating complete new plants. This study investigated the effect of gravity on the site of differentiation of newly forming organs during regeneration. Pairs of 3.0-cm blade segments from C. prolifera and C. mexicana, as well as 3.0-cm rhizome segments of C. prolifera, were randomly assigned to either upright or inverted positions. This orientation was maintained throughout each experiment. Results revealed that the blade segments maintained a strict polarity of regeneration regardless of inversion and/or centrifugation. Rhizoids and rhizomes formed at the original basal end of the segment, while blades formed at the apical end. This polarity was seen throughout the length of long blades. Rhizome segments, on the other hand, failed to exhibit a strict polarity of regeneration with respect to gravity.     

Upregulation of a Hydra vulgaris cPKC gene is tightly coupled to the differentiation of head structures

 Two different cDNA clones from Hydra (HvPKC1a and HvPKC1b) were characterized, which encode members of the cPKC family of protein kinase Cs (PKCs). The two predicted proteins differ only in their amino-terminal sequences and thus probably represent the products of alternatively spliced mRNAs from a single gene. In situ hybridization with a probe recognizing sequences in common between the two mRNAs detects HvPKC1 RNA in all parts of the adult polyp except the foot. The mRNA is contained in ecto- and endodermal epithelial cells as well as a certain subset of gland cells and pairs of interstitial cells. During head and foot formation, induced by either regeneration, budding, lithium treatment or repeated application of a diacylglycerol, HvPKC1 expression is upregulated immediately prior to the evagination of tentacles and downregulated by foot formation. Although PKC activity is clearly inducible in vitro by diacylglycerol and a tumour promoting phorbol ester, structural features detected in the regulatory domains of HvPKC1a and 1b indicate that endogenous activators for Hydra PKC might differ from those of other organisms. The results corroborate the hypothesis that signal transduction systems using protein kinase C are key elements controlling the formation of head structures in Hydra. Received: 2 May 1997 / Accepted: 4 December 1997     

An osmoregulatory basis for shape oscillations in regenerating hydra

The freshwater polyp Hydra has considerable regeneration capabilities. A small fragment of tissue excised from an adult animal is sufficient to regenerate an entire Hydra in the course of a few days. During the initial stages of the regeneration process, the tissue forms a hollow sphere. Then the sphere exhibits shape oscillations in the form of repeated cycles of swelling and collapse. We propose a biophysical model for the swelling mechanism. Our model takes the osmotic pressure difference between Hydra's inner and outer media and the elastic forces of the Hydra shell into account. We validate the model by a comprehensive experimental study including variations in initial medium concentrations, Hydra sphere sizes and temperatures. Numerical simulations of the model provide values for the swelling rates that are in agreement with the ones measured experimentally. Based on our results we argue that the shape oscillations are a consequence of Hydra's osmoregulation.     

Limits of the distal inversion in the t complex of the house mouse: Evidence from linkage disequilibria

The suppression of crossing-over and the consequent linkage disequilibrium of genetic markers within the t complex of the house mouse is caused by two large and two short inversions. The inversions encompass a region that is some 15 centiMorgans (cM) long in the homologous wild-type chromosome. The limits of the proximal inversions are reasonably welldefined, those of the distal inversions much less so. We have recently obtained seven new DNA markers (D17Tu) which in wild-type chromosomes map into the region presumably involved in the distal inversions of the t chromosomes. To find out whether the corresponding loci do indeed reside within the inversions, we have determined their variability among 26 complete and 12 partial t haplotypes. In addition, we also tested the same collection of t haplotypes for their variability at five D17Leh, Hba-ps4, Pim-1, and Crya-1 loci. The results suggest that the distal end of the most distal inversion lies between the loci D17Leh467 and D17Tu26. The proximal end of the large distal inversion was mapped to the region between the D17Tu43 and Hba-ps4 loci, but this assignment is rather ambiguous. The loci Pim-1, Crya-1, and the H-2 complex, which have been mapped between the Hba-sp4 and Grr within the large distal inversion, behave as if they recombine from time to time with their wildtype homologs.     

SELECTIVE INHIBITION OF REGENERATION IN HYDRA VULGARIS BY ACTINOMYCIN D

Twelve-hour continuous pretreatment of regenerating Hydra with 60 μg/ml actinomycin D inhibited the synthesis of RNA by 98%. In such Hydra, hypostome regeneration was found more affected than basal disc regeneration since a complete blockage of development of oral structures occurred. It is assumed that the hypostome regeneration requires new DNA-dependent RNA synthesis. Better differentiation of the basal disc is explained on the basis of a stable variety of messenger RNA (mRNA), which would become activated at the time of determination. The formation of mesoglea and the development of fibrous materials in the basal disc are attributed to new DNA-dependent RNA synthesis.     

The Hydra small ubiquitin‐like modifier

SUMO is a protein posttranslational modifier. SUMO cycle components are believed to be conserved in all eukaryotes. Proteomic analyses have lead to the identification a wealth of SUMO targets that are involved in almost every cellular function in eukaryotes. In this article, we describe the characterization of SUMO Cycle components in Hydra, a Cnidarian with an ability to regenerate body parts. In cells, the translated SUMO polypeptide cannot conjugate to a substrate protein unless the C‐terminal tail is cleaved, exposing the di‐Glycine motif. This critical task is done by SUMO proteases that in addition to SUMO maturation are also involved in deconjugating SUMO from its substrate. We describe the identification, bioinformatics analysis, cloning, and biochemical characterization of Hydra SUMO cycle components, with a focus on SUMO and SUMO proteases. We demonstrate that the ability of SUMO proteases to process immature SUMO is conserved from Hydra to flies. A transgenic Hydra, expressing a SUMO‐GFP fusion protein under a constitutive actin promoter, is generated in an attempt to monitor the SUMO Cycle in vivo as also to purify and identify SUMO targets in Hydra. genesis 51:619–629. © 2013 Wiley Periodicals, Inc.     

The fine structure of the compound eye of a damsel-fly

Summary The compound eyes of two species of damsel-flies, Ishunura senegalensis and Cersion calamorum, were examined by electron microscopy. Each ommatidium is composed of eight retinula cells which are semistratified in the receptor layer. The retinula cells are divided into four types from the difference of levels in the rhabdom formation; one distal large cell having the rhabdomere only in the distal layer, four middle cells forming the rhabdom in the middle layer, two proximal cells making up the rhabdom in the proximal layer and one distal small cell having no rhabdomere in any layers. In addition, the lamina ganglionaris was partly observed. Some retinula axons terminate at an different level from the other axons. The functional differentiation among these different types of cells is discussed with relation to the analysis of the polarized light and the discrimination of the diffraction images.This work is supported by a grant from the U.S. Army Research and Development Group (Far East), Department of the Army (DA-CRD-AG-S29-544-67-G61).The authors wish to express their gratitude to Drs. H. Morita and H. Tateda for their helpful discussions throughout this study.     

The level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

 Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms. Received: 10 July 1997 / Accepted: 8 November 1997     

Stress-induced localization of HSPA6 (HSP70B′) and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B′) and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.     

Resilience to aging in the regeneration‐capable flatworm Macrostomum lignano

Animals show a large variability of lifespan, ranging from short‐lived as Caenorhabditis elegans to immortal as Hydra. A fascinating case is flatworms, in which reversal of aging by regeneration is proposed, yet conclusive evidence for this rejuvenation‐by‐regeneration hypothesis is lacking. We tested this hypothesis by inducing regeneration in the sexual free‐living flatworm Macrostomum lignano. We studied survival, fertility, morphology, and gene expression as a function of age. Here, we report that after regeneration, genes expressed in the germline are upregulated at all ages, but no signs of rejuvenation are observed. Instead, the animal appears to be substantially longer lived than previously appreciated, and genes expressed in stem cells are upregulated with age, while germline genes are downregulated. Remarkably, several genes with known beneficial effects on lifespan when overexpressed in mice and C. elegans are naturally upregulated with age in M. lignano, suggesting that molecular mechanism for offsetting negative consequences of aging has evolved in this animal. We therefore propose that M. lignano represents a novel powerful model for molecular studies of aging attenuation, and the identified aging gene expression patterns provide a valuable resource for further exploration of anti‐aging strategies.     

DNA synthesis and mRNA accumulation during germination of embryos of the orchidSpathoglottis plicata

Summary Germination of embryos of the orchid,Spathoglottis plicata Blume involves the formation of a protocorm in which DNA synthesis and cell divisions are confined to the proximal end whereas cells at the distal end undergo enlargement. Although³H-thymidine was incorporated into the distal cells of the embryo during the early period of germination, DNA synthesis was not followed by mitosis and cytokinesis. Poly(A)-RNA detected by in situ hybridization of sections to³H-poly-(U) was present uniformly in all cells of the embryo of the dry seed. However, coincident with the formation of the apical meristem, there was an increase in the density of auto-radiographic silver grains in the cells of the embryo, with a concentration of silver grains in the proximal part. The results indicate that regulatory events in the embryo prior to seed maturity determine the fate of its proximal and distal parts during germination.Dedicated to the memory of Professor John G. Torrey