Inhibition of metamorphosis by RFamide neuropeptides in planula larvae of Hydractinia echinata

The primitive nervous system in planula larvae of Hydractinia echinata (Cnidaria) has sensory neurons containing LWamide or RFamide neuropeptides. LWamides have been shown to induce metamorphosis of planula larvae into adult polyps. We report here that RFamides act antagonistically to LWamides. RFamides inhibit metamorphosis when applied to planula larvae during metamorphosis induction by treatment with LWamides (or other inducing agents such as CsCl ions, diacylglycerol and bacterial inducers). Our results show further that RFamides act downstream of LWamide release, presumably directly on target cells mediating metamorphosis. These observations support a model in which metamorphosis in H. echinata is regulated by sensory neurons secreting LWamides and RFamides in response to environmental cues.Edited by D. Tautz     

Heat shock as inducer of metamorphosis in marine invertebrates

Summary In most sessile marine invertebrates, metamorphosis is dependent on environmental cues. Here we report that heat stress is capable of inducing metamorphosis in the hydroid Hydractinia echinata. The onset of heat-induced metamorphosis is correlated with the appearance of heat-shock proteins. Larvae treated with the metamorphosis-inducing agents Cs⁺ or NH₄ ⁺ also synthesize heat-shock proteins. In heat-shocked larvae, the internal NH₄ ⁺-concentration increases. This fits the hypothesis that methylation plays a central role in control of metamorphosis. In the tunicate Ciona intestinalis, a heat shock is able to induce metamorphosis too. Offprint requests to: M. Walther     

Necessity of protein synthesis for metamorphosis in the marine hydroidHydractinia echinata

Summary In the marine colonial hydroidHydractinia echinata metamorphosis from the larval to the adult (polyp) stage is induced by various agents, including CsCI and dioctanoylglycerol (diC8). Induction is prevented when the inhibitors of protein synthesis cycloheximide or ementine were applied simultaneously with the metamorphosis-inducing agents. With diC8 treatment, the inhibitors caused most animals to transform into mosaics consisting of larval and polyp body parts instead of normal shaped polyps. In contrast, treatment with cycloheximide or ementine just before or after incubation with the metamorphosis-inducing agents did not prevent larvae from metamorphosis. No substantial quantitative changes in protein synthesis occur during induction of metamorphosis, however, the protein pattern is changed upon induction. The most prominent new polypeptides (25 and 73 kD) were observed when CsCI was used to trigger metamorphosis. In addition, both in CsCl- and in diC8-treated larvae, the synthesis of a new 23 kD protein occurred, whilst synthesis of others ceased (41 and 44 kD).     

Metamorphosin A is a neuropeptide

A novel biologically active peptide (metamorphosin A, MMA, pEQPGLW.NH₂) has recently been described. It was isolated from Anthopleura elegantissima and triggers metamorphosis in Hydractinia echinata. Antibodies directed against the C-terminal part of the molecule immunohistochemically stain neurosensory cells and processes in the anterior part of larvae of H. echinata. We assume that in metamorphosis MMA (or a closely related LW-amide) is an internal signal transmitted from the anterior to the posterior body parts. Immunoreactivity is also found in ectodermal nerve processes — but not cell bodies — in the tentacles and in the basal disk of the foot of Hydra magnipapillata. This is, to our knowledge, the first report of LW-amide(s) as (a) neuropeptide(s).     

Stimulation of metamorphosis in Hydractinia echinata involves generation of lysophosphatidylcholine

Summary Whilst the significance of the phosphoinositide cycle in the activation of developmental events by extra-cellular signals is well established, the involvement of the phosphatidylcholine (PC) cycle is a matter just emerging. In the present study, the metabolism of phosphatidylcholine in early metamorphosis of Hydractinia echinata (Coelenterata; Hydrozoa) was investigated by incubation of planula larvae with ³H-choline, extraction of the metabolites and isolation of the metabolites by thin-layer chromatography (TLC). Phosphatidylcholine (PC), lysophosphatidylcholine (LPC), acetylcholine and glycerophosphocholine were the labelled metabolites. Induction of metamorphosis did not stimulate an increased incorporation of choline into PC. In larvae preincubated with ³H-choline to a steady state level of incorporation, a significant transient elevation of the radioactive label in LPC was observed 90 min after addition of metamorphosis stimulating agents. LPC probably derived from PC by the action of a phospholipase A₂ (PLA₂). LPCs from bovine and soybean origin as well as isolated larval LPC did not influence metamorphosis. PLA₂ from bee venom promoted Cs⁺-induced metamorphosis but did not influence phorbol ester-induced metamorphosis. The data suggest that a PLA₂ is activated during metamorphosis. This PLA₂ activation does not occur in those putative receptor cells which receive the primary external inducing stimulus but in the many larval cells which resume proliferation or differentiation in response to a second, internally propagated signal. Offprint requests to: T. Leitz     

Induction of metamorphosis from the larval to the polyp stage is similar in Hydrozoa and a subgroup of Scyphozoa (Cnidaria, Semaeostomeae)

Larvae of cnidarians need an external cue for metamorphosis to start. The larvae of various hydrozoa, in particular of Hydractinia echinata, respond to Cs⁺, Li⁺, NH₄ ⁺ and seawater in which the concentration of Mg²⁺ ions is reduced. They further respond to the phorbolester, tetradecanoyl-phorbol-13-acetate (TPA) and the diacylglycerol (DAG) diC8, which both are argued to stimulate a protein kinase C. The only well-studied scyphozoa, Cassiopea spp., respond differently, i.e. to TPA and diC8 only. We found that larvae of the scyphozoa Aurelia aurita, Chrysaora hysoscella and Cyanea lamarckii respond to all the compounds mentioned. Trigonelline (N-methylnicotinic acid), a metamorphosis inhibitor found in Hydractinia larvae, is assumed to act by delivering a methyl group for transmethylation processes antagonising metamorphosis induction in Chrysaora hysoscella and Cyanea lamarckii. The three species tested are scyphozoa belonging to the subgroup of semaeostomeae, while Cassiopea spp. belong to the rhizostomeae. The results obtained may contribute to the discussion concerning the evolution of cnidarians and may help to clarify whether the way metamorphosis can be induced in rhizostomeae as a whole is different from that in hydrozoa and those scyphozoa belonging to the subgroup semaeostomeae. Electronic Publication     

Metamorphosis of <Emphasis Type="Italic">Hydractinia echinata</Emphasis>—natural versus artificial induction and developmental plasticity

Many marine invertebrates reproduce through a larval stage. The settlement and metamorphosis of most of the species are synchronised and induced by environmental organisms, mainly bacteria. The hydrozoan Hydractinia echinata has become a model organism for metamorphosis of marine invertebrates. In this species, bacteria, e.g. Pseudoalteromonas espejiana, are the natural inducers of metamorphosis. Like in other species of marine invertebrates, metamorphosis can be induced artificially by monovalent cations, e.g. Cs⁺. In this study, we present systematic data that metamorphosis—with both inducing compounds, the natural one from bacteria and the artificial one Cs⁺—are indeed similar with respect to (a) the morphological progression, (b) the localisation of the primary induction signal in the larva, (c) the pattern of apoptotic cells occurring during the initial 10 h of metamorphosis and (d) the disappearance of RFamide-dependent immunocytochemical signals in sensory neurons during this process. However, a difference occurs during the development of the anterior end, insofar as apoptotic cells and settlement appear earlier in planulae induced with bacteria. Thus, basically, Cs⁺ may be used as an artificial inducer, mimicking the natural process. However, differences in the appearance of apoptotic cells and in settlement raise the question of how enormous developmental plasticity in hydrozoans actually can be, and how this is related to the absence of malignant devolution in hydrozoans.     

Metamorphosis inHydractinia: Studies with activators and inhibitors aiming at protein kinase C and potassium channels

Summary Metamorphosis of planula larvae involves an activation of morphogenetically quiescent cells. The present work extends a previous study [Leitz T and Müller WA (1987) Dev Biol 121:82–89] on the participation of the phosphatidylinositol/diacylglycerol/protein kinase C system. Metamorphosis is stereospecifically induced by diacylglycerols, 1,2,-sn-dioctanoylglycerol (diC₈) being by far the most effective substance. K-252a and sphingosine, inhibitors of mammalian protein kinases C, profoundly inhibited metamorphosis. Phorbolester-binding studies and the corresponding Scatchard plots revealed a specific and saturable binding of [³H]phorbol 12,13-dibutyrate to a single site of particulate fractions ofHydractinia with a specific binding affinityK _d = 50 nM. K⁺ ionophores stimulated Cs⁺ — but inhibited diC₈-induced metamorphoses, K⁺-channel blockers enhanced the inducing action of Cs⁺ or diC₈. On the basis of these data and observations of others we propose that the activation ofHydractinia larvae takes place in some cells at the anterior end as a result of activation of a kinase-C-like enzyme, which directly or indirectly leads to the closure of K⁺ channels. Closure of these channels then causes depolarisation and, thus, release of an internal signal. This hypothesis unifies notions about the role of K⁺ channels and of the phosphatidylinositol system in initiation of metamorphosis inHydractinia.     

Catecholamines induce metamorphosis in the hydrozoan Halocordyle disticha but not in Hydractinia echinata

Summary Planula larvae of the marine hydroids Halocordyle disticha and Hydractinia echinata were treated with the catecholamines epinephrine, norepinephrine and dopamine, as well as with certain of their precursors and agonists. Norepinephrine, l-dopa, dopamine and the dopamine agonist ADTN at concentrations ranging from 0.1 to 0.001 mM induced metamorphosis within 24 h in Halocordyle disticha, with no observable morphogenetic abnormalities. Epinephrine, the adrenergic agonists phenylephrine, isoproterenol and methoxyamine, and the catecholamine precursors phenylalanine and tyrosine were found not to induce metamorphosis at the concentrations employed. None of the compounds was effective in inducing metamorphosis in Hydractinia echinata. A model is presented for neural control of metamorphosis in Halocordyle disticha     

Modulation of Protein Phosphorylation by M r 25,000 Protein Partially Overlapping Phosvitin and Lipovitellin 2 in Xenopus laevis Vitellogenin B1 Protein

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35–45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.     

Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs⁺ or tumor-promoting phorbol esters (W. A. Müller (1985) Differentiation 29, 216–222). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [³H]-inositol. Upon exposure of the larvae to Cs⁺ the label in inositol trisphosphate (InsP₃) increased twofold as early as 15 sec after addition of Cs⁺. Within the first 60 sec the levels of inositol monophosphate (InsP₁) and inositol bisphosphate (InsP₂) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs⁺, only the label in InsP₂ was increased. When applied to saponin-permeabilized larvae, InsP₃ did not induce metamorphosis. But 1,2-dioctanoyl-rac-glycerol (diC₈) was effective in inducing metamorphosis with a half-maximal effective concentration of 9 μM. The percentage of metamorphosed animals after the application of 5 μM diC₈ (30 mM Cs⁺) was increased by the simultaneous application of 1 μM (0.1 μM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata.     

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 cDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.     

On natural metamorphosis inducers of the cnidarians Hydractinia echinata (Hydrozoa) and Aurelia aurita (Scyphozoa)

 Hydractinia echinata and Aurelia aurita produce motile larvae which undergo metamorphosis to sessile polyps when induced by external cues. The polyps are found at restricted sites, A. aurita predominantly on rocks close to the shore, H. echinata on shells inhabited by hermit crabs. It has been argued that the differential distribution of the polyps in their natural environment largely reflects the distribution of the natural metamorphosis-inducing cues. In the case of H. echinata, bacteria of the genus Alteromonas were argued to meet these conditions. We found that almost all substrates collected in the littoral to induce metamorphosis in H. echinata, and several bacterial strains isolated from the sea, including the common E. coli, induce metamorphosis efficiently. In A. aurita metamorphosis may be induced by the water–air interface, whereby metamorphosis precedes (final) settlement. Received: 7 December 1998 / Accepted: 8 July 1999     

Metamorphosis ofHydractinia echinata Insights into pattern formation in Hydroids

Summary Hydractinia echinata is a marine, colony-forming coelenterate. Fertilized eggs develop into freely swimming planula larvae, which undergo metamorphosis to a sessile (primary) polyp. Metamorphosis can be triggered by means of certain marine bacteria and by Cs⁺. Half a day after this treatment a larva will have developed into a polyp. The induction of metamorphosis can be prevented by addition of inhibitor I, a substance partially purified from tissue ofHydra. The larvae ofH. echinata also appear to contain this substance. Inhibitor I appliedafter the onset of metamorphosis blocks its continuation as long as it remains in the culture medium. Cs⁺ applied within the same period of time also blocks the continuation of metamorphosis. However, these two agents have opposite effects on the body pattern of the resultant polyps. The experiments indicate that application of Cs⁺ triggers the generation of the pre-pattern. Inhibitor I appears to be a factor of this prepattern. A model is proposed which describes the basic features of head and foot/stolon formation not only forHydractinia but also for other related hydroids.     

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     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

Summary

We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34^cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.     

Expression of selected domains of the circumsporozoite antigen ofPlasmodium knowlesi

The circumsporozoite antigen of the simian malarial parasite,Plqsmodium knowlesi, consists of tandemly repeated immunodominant peptide units which may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the whole of the non-repetitive region of this antigen has been expressed inEscherichia coli. The entire amino-terminal region up to the start of the repeats, and the full non-repetitive carboxyl region starting from the end of the repeats up to the termination codon, have been expressed separately, as fusion proteins with a 26 kD glutathione-S-transferase protein ofSchistosomq japonicum. A repeat-less truncated antigen has also been expressed as the same fusion protein. The amino-terminal fusion protein (GST-CSN), is a soluble protein of a molecular weight of 38 kD, which could be purified by affinity chromatography on immobilized glutathione. The carboxylterminal fusion protein (GST-CSC), is insoluble, migrates with an anomalous molecular weight of 32 kD, and binds to the affinity matrix weakly. The truncated repeat-less fusion protein (GST-CSNC) is also, an insoluble protein of molecular weight of 48 kD. Unlike the two separate domains, GST-CSNC is an extremely unstable protein inEscherichia coli.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Taurine found to stabilize the larval state is released upon induction of metamorphosis in the hydrozon Hydractinia

Summary Larvae of Hydractinia echinata, a colonial marine hydroid, can be triggered experimentally to undergo metamorphosis into polyps. The efficiency of induction is density-dependent: a high larval density allows fewer larvae to metamorphose than a low density. Culture medium of metamorphosing larvae was found to contain taurine as the major constituent of its inhibitory activity. The concentration of taurine in larvae is 90 mM which is much higher than that of other free amino acids. Taurine interferes effectively with the onset of metamorphosis if applied externally at a concentration equivalent to 1/1000 of the animal's overall internal concentration. Upon induction of metamorphosis the larva releases three quarters of its taurine into the medium. Taurine may have a function in control of onset of metamorphosis because, applied exogenously in micromolar quantities, it stabilizes the larval state, i.e. the larvae resist metamorphosis-inducing stimuli. The chemically related compounds 4-aminobutyric acid (GABA) and -alanine are much less effective. There exist other larval state stabilizing compounds in Hydractinia including homarine, trigonelline, betaine and methionine. These compounds are though to act by delivering methyl groups leading to the production of S-adenosylmethionine. Taurine is not able to supply methyl groups. Furthermore, in contrast to the four other compounds taurine does not interefere with the advance of metamorphosis when applied after induction, and of these five substances, only taurine is released upon induction of metamorphosis.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.