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     

The role of GLWamides in metamorphosis of Hydractinia echinata

 The metamorphosis of many marine invertebrate larvae is induced by environmental signals. Upon reception of the cues, internal signals have to be set in motion to convey information to all cells of the larvae. For hydrozoan larvae it was hypothesised that ectodermal neurosensory cells at the anterior part are those cells receptive of the inducer. Recently, it was shown that novel peptides with a common GLWamide terminus are found in Cnidaria. These peptides are located in a specific subset of the anterior sensory cells. It was hypothesised that the neuropeptides represent an internal signal coordinating the metamorphic process. In the current study we present further evidence for this hypothesis. Induction of metamorphosis is very specific for the GLWamide terminus and amidation is essential. The potency to metamorphose is strongly correlated with the presence of GLWamide-immunoreactive cell bodies. Our data fit our hypothesis about a very important role of GLWamides in the initiation of the morphogenetic processes very well. Received: 16 February 1998 / Accepted: 11 April 1998     

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     

Evidence for an instructive role of apoptosis during the metamorphosis of Hydractinia echinata (Hydrozoa)

Apoptosis is a highly conserved mechanism of cell deletion that destroys redundant, dysfunctional, damaged, and diseased cells. Furthermore, apoptotic cell death is essential during the development of multicellular organisms. However, there are only a few examples where the occurrence of apoptosis has been shown to be a direct prerequisite for developmental processes. As described previously by our group, the degradation of larval tissue during the first half of the metamorphosis of Hydractinia echinata involves extensive cell death. A large number of cells are removed, and we observed several cellular features of apoptotic cell death in the dying tissue, e.g., nucleosomal DNA fragmentation and nuclear condensation. Furthermore, we showed that metamorphosis in the basal cnidarian H. echinata depends on the activity of caspases, the central enzymes of apoptosis. In the present study, we build on these previous investigations of apoptosis in H. echinata by characterising a caspase-3 sequence in this species and placing it in an evolutionary context by performing phylogenetic analyses. Furthermore, we report the successful knockdown of a caspase by RNAi and show that apoptosis plays a role as an instructive mechanism in the metamorphosis of H. echinata.     

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).     

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     

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     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional.Offprint requests to: A. Schlawny     

Autoaggressive,multi-headed and other mutant phenotypes in Hydractinia echinata (Cnidaria: Hydrozoa)

In an inbreeding program conducted with the colonial hydroid Hydractinia echinata, each F1 mating produced up to 50% F2 offspring displaying an aberrant, clone-constant phenotype, hence referred to as mutant strain. In autoaggressive strains, in one or several areas of the colony autoreactive stolons direct their aggressive devices (stolon tips filled with cytotoxic stinging cells), normally used to kill allogeneic competitors for living space, towards neighboring stolons or polyps (hydranths) of their own colony. In these areas tumor-like masses of self-aggressive stolons were formed, in severe cases causing the death of the colony. Based on previous genetic studies, the interpretation proposed here attributes autoaggressive behavior to a mosaic-type alternative expression of arl (allorecognition) alleles in heterozygous individuals. Developmental mutant strains termed He-mh form supernumerary heads during regeneration and normal development as well. Common to all He-mh phenotypes isthe production of additional headsalong the bodycolumn of fully-grown polyps. The heads give rise to complete hydranths connected by a tube that derives from the gastric region of the original polyp and eventually transforms into a stolon. In bastol strains, polyps convert the basal region of their body column into a periderm-covered stolon from which the residual apical hydranth detaches. Colonies expressing both the He-mh and the bastol (bst) phenotype frequently lose detaching multi-headed hydranths and the colony disintegrates. The large number of mutant F2 offspring reveals high genetic variability in Hydractinia.     

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.     

Neuronal cell death during metamorphosis of Hydractina echinata (Cnidaria, Hydrozoa)

In planula larvae of the invertebrate Hydractinia echinata (Cnidaria, Hydrozoa), peptides of the GLWamide and the RFamide families are expressed in distinct subpopulations of neurons, distributed in a typical spatial pattern through the larval body. However, in the adult polyp GLWamide or RFamide-expressing cells are located at body parts that do not correspond to the prior larval regions. Since we had shown previously that during metamorphosis a large number of cells are removed by programmed cell death (PCD), we aimed to analyze whether cells of the neuropeptide-expressing larval nerve net are among those sacrificed. By immunohistochemical staining and in situ hybridization, we labeled GLWamide- and RFamide-expressing cells. Double staining of neuropeptides and degraded DNA (TUNEL analysis) identified some neurosensory cells as being apoptotic. Derangement of the cytoplasm and rapid destruction of neuropeptide precursor RNA indicated complete death of these particular sensory cells in the course of metamorphosis. Additionally, a small group of RFamide-positive sensory cells in the developing mouth region of the primary polyp could be shown to emerge by proliferation. Our results support the idea that during metamorphosis, specific parts of the larval neuronal network are subject to neurodegeneration and therefore not used for construction of the adult nerve net. Most neuronal cells of the primary polyp arise by de novo differentiation of stem cells commited to neural differentiation in embryogenesis. At least some nerve cells derive from proliferation of progenitor cells. Clarification of how the nerve net of these basal eumetazoans degenerates may add information to the understanding of neurodegeneration by apoptosis as a whole in the animal kingdom.     

Protein kinase C in hydrozoans: involvement in metamorphosis of Hydractinia and in pattern formation of Hydra

A wealth of information has suggested the involvement of protein kinase C (PKC) in metamorphosis of Hydractinia echinata and in pattern formation of Hydra magnipapillata. We have identified a Ca²⁺- and phospholipid-dependent kinase activity in extracts of both species. The enzyme was characterized as being similar to mammalian PKC by ion exchange chromatography. Gel filtration experiments revealed a molecular weight of about 70 kD. In phosphorylation assays of endogenous Hydractinia proteins, a protein with a molecular weight of 22.5 kD was found to be phoshorylated upon addition of phosphatidylserine. Bacterial induction of metamorphosis of Hydractinia echinata caused an increase in endogenous diacylglycerol, the physiological activator of PKC, suggesting that the bacterial inducer acts by activating receptor-regulated phospholipid metabolism. Exogenous diacylglycerol leads to membrane translocation of PKC, indicative of an activation. On the basis of our results and those of Freeman and Ridgway (1990) a model for the biochemical events during metamorphosis is presented.     

Induction of metamorphosis by bacteria and by a lithium-pulse in the larvae ofHydractinia echinata (Hydrozoa)

Summary The planulae ofHydractinia, the metamorphosis of which normally is induced by certain bacteria, will undergo transformation into polyps also when exposed to a lithiumpulse. The optimal concentration and incubation period for rapid and complete transformation have been determined at 24 mM Li⁺ and 2 hrs respectively. 96 mM K⁺ applied for 2 hrs will also result in some induction. The possible mode of action exerted by the Li-ion as compared with induction caused by bacteria is discussed.

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Zusammenfassung Die Planulae vonHydractinia, deren Metamorphose normalerweise durch bestimmte marine Bakterien induziert wird, wandeln sich ebenso in Primärpolypen um, wenn sie einem Li⁺-Puls ausgesetzt werden. Zur schnellen, vollzähligen und vollständigen Metamorphose müssen Konzentration und Behandlungszeit im optimalen Verhältnis gehalten werden. Als optimal erwies sich eine Dosis von 24 mM Li⁺ appliziert für 2 Std. Eine schwache Wirkung erzielt auch ein K⁺-Puls in der optimalen Konzentration von 96 mM. Die mögliche gemeinsame Wirkungsweise des bakteriellen Induktors und der Ionen wird diskutiert.

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We are deeply indebted to Dr. W. Gunkel, Biologische Anstalt Helgoland, for his kindness in placing at our disposal the equipment of the microbiological laboratory. Also the support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

The role of alpha-amidated neuropeptides in hydroid development--LWamides and metamorphosis in Hydractinia echinata

Peptides are increasingly attracting attention as primary signals in the control of development. Even though a large number of peptides have been characterized in cnidarians, little experimental evidence addresses their endogenous role. The life cycle of Hydractinia echinata includes metamorphosis from planula larva into the adult stage of the polyp. This process of stage conversion includes internal signalling, which controls cell cycle activity, cell differentiation, cell death and proportion-controlled morphogenesis. LWamide peptides are considered to be part of the control system. We implemented methods to silence gene activity by dsRNAi in Hydractinia and show a substantial knock-down of LWamide gene activity. In addition, LWamide function was knocked-out pharmacologically by targeting the biosynthesis of amidated peptides and thus preventing functional LWamides. Here we show that extinction of bioactive LWamides from planulae causes loss of metamorphosis competence, a deficiency which can be rescued by synthetic LWamide peptides. Thus, it is shown that LWamides are indispensable and act by conveying outer metamorphosis stimuli to target cells within the animal. Considering non-availability of genetic analysis and the so-far limited success in expressing transgenes in hydroids, gene functions are difficult to analyse in hydroids. The approach as outlined here is suitable for functional analysis of genes encoding amidated peptides in hydroids.     

Pulses of ammonia and methylamine induce down-regulation of nematocyte and nerve cell populations in Hydrozoa (Hydra; Hydractinia)

Summary Hydrozoa replace used-up nematocytes (cnidocytes) by proliferation and differentiation from interstitial stem cells (i cells). Repeated pulsed exposure ofHydra to elevated levels of unprotonated ammonia leads to successive loss of the various types of nematocytes: first of the stenoteles, then of the isorhizas and finally of the desmonemes. The loss is due to deficits in supply; the number of nematoblasts and differentiating intermediates is reduced. In the hydroidHydractinia the main process leading to numerical reduction was observed in vivo: mature nematocytes as well as precursors emigrate from their place of origin into the gastrovascular channels where they are removed by phagocytosis. This is a regular means by which these animals down-regulate an induced surplus of nematocytes. With lower effectiveness, pulses of methylamine, trimethylamine and glutamine also induce elimination of the nematocyte lineages. In the long term the population of nerve cells, which are permanently but slowly renewed from interstitial neuroblasts, decreases, too. After 2 months of daily repeated treatment the density of the Arg-Phe-amide-positive nerve cells was reduced to 50% of its normal level. Thus, ammonia induces down-regulation of all interstitial cell lineages. The temporal sequence of the ammonia-induced loss reflects the diverse rates with which the various i cell descendants normally are renewed.     

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     

Ammonia induces metamorphosis of the oral half of buds into polyp heads in the scyphozoan Cassiopea

Summary The scyphozoan polyp Cassiopea forms vegetative free swimming buds that metamorphose into sessile polyps. In sterile sea water metamorphosis does not take place. Buds keep swimming for weeks. Application of millimolar quantities of NH ₄ ⁺ causes the buds to metamorphose within one day. The resulting animals bear hypostome and tentacles, however, only occasionally peduncle and foot. Almost all transform either completely into solitary polyp head or only the oral half of the bud developes into a head while the aboral half remains bud tissue which becomes constricted off. Under suited conditions this small bud is able to transform into a normal shaped polyp.     

A substance released by metamorphosing larvae and young polyps ofHydractinia echinata induces metamorphosis in conspecific larvae

Summary A metamorphosis-inducing factor was isolated from medium conditioned by either metamorphosing larvae or 3-day postmetamorphic primary polyps. The factor has a molecular weight 8 kDa and is heatlabile. It does not induce metamorphosis of isolated posterior fragments and is therefore not identical to the internal signal described by Schwoerer-Böhning et al. (1990). The biological significance of the substance is currently unclear, therefore its inducing activity may be a side effect.     

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern

Summary During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.     

Ammonia,tetraethylammonium, barium and amiloride induce metamorphosis in the marine hydroid Hydractinia

Summary In Hydractinia metamorphosis from the swimming larval stage to the sessile polyp stage has been found to be inducible by several agents, including Li⁺, K⁺, Cs⁺, Rb⁺, diacylglycerol (DG), tetradecanoyl-phorbol-acetate (TPA) and some other tumour-promoting phorbol esters. Induction is antagonized by ouabain and compounds which are able to increase the internal level of S-adenosylmethionine (SAM). Based on the finding that Hydractinia larvae contain such compounds in a stored form, including N-methylpicolinic acid, N-methylnicotinic acid and N-trimethylglycine, as well as on the results of experiments with antagonists of SAM production and transmethylation, it has been argued that regulation of the internal SAM level plays a key role in the control of metamorphosis. However, it remains to be clarified whether the inducing agents act by decreasing the SAM level or by via different pathways. In the present study, substances chemically related to the substances known to induce or inhibit metamorphosis were tested for their metamorphosis-inducing abilities. Some were found to be effective, including NH₄⁺, methylamine, tetraethylammonium ions (TEA⁺), ethanolamine, Ba²⁺, Sr²⁺ and the diuretic, amiloride. It is of particular interest that in many organisms TPA and DG increase cytoplasmic pH while amiloride prevents a rise in pH_i. Several of the substances known to trigger metamorphosis may increase the internal NH₄⁺ concentration by hindering the export of the constantly produced NH₄⁺ through K⁺ channels or through the Na⁺-H⁺ antiport. Treatment with Cs⁺ for 1 h increases the internal level of NH₄⁺. Produced and applied ammonia, as well as applied methylamine and ethanolamine, may act by accepting methyl groups, thus reducing the SAM level.