Metamorphosis of Hydractinia echinata (Cnidaria) is caspase-dependent

Apoptotic cell death plays an important role in many developmental pathways in multicellular animals. Here, we show that metamorphosis in the basal invertebrate Hydractinia echinata (Cnidaria) depends on the activity of caspases, the central enzymes in apoptosis. Caspases are activated during metamorphosis and this activity can be measured with caspase-3 specific fluorogenic substrates. In affinity labelling experiments 23/25 kDa bands were obtained, which represented active caspase. Specific inhibition of caspase activity with caspase-3 inhibitors abolished metamorphosis completely, reversibly and in a dose-dependent manner. This suggests that caspase activity is indispensable for metamorphosis in Hydractinia echinata.     

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.     

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.     

An src-related tyrosine kinase activity in the hydroid, Hydractinia

Abstract. The metamorphosis of planula larvae of the hydroid, Hydractinia , into primary polyps can be artificially triggered by Li, Rb, and Cs ions, by tumor-promoting phorbol esters [14], and by diacylglycerols. Metamorphosis implies the transition of morphogenetically quiescent cells to a state of terminal differentiation or transdifferentiation. Among the events which occur during the first 2.5 h after induction is an increase in the level of activity of a protein kinase that is precipitated by antisera against Rous sarcoma virus-derived pp60^src and that phosphorylates tyrosine in the precipitated IgG. Immunoprecipitation using a preimmune serum did not yield any corresponding kinase activity. The antigenic relationship between the Hydractinia protein and vertebrate pp60^c-src"' was demonstrated by applying an immune competition assay.     

An src-related tyrosine kinase activity in the hydroid, Hydractinia

Abstract. The metamorphosis of planula larvae of the hydroid, Hydractinia , into primary polyps can be artificially triggered by Li, Rb, and Cs ions, by tumor-promoting phorbol esters [14], and by diacylglycerols. Metamorphosis implies the transition of morphogenetically quiescent cells to a state of terminal differentiation or transdifferentiation. Among the events which occur during the first 2.5 h after induction is an increase in the level of activity of a protein kinase that is precipitated by antisera against Rous sarcoma virus-derived pp60^src and that phosphorylates tyrosine in the precipitated IgG. Immunoprecipitation using a preimmune serum did not yield any corresponding kinase activity. The antigenic relationship between the Hydractinia protein and vertebrate pp60^c-src was demonstrated by applying an immune competition assay.     

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.     

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.     

Homarine (N-methylpicolinic acid) and trigonelline (N-methylnicotinic acid) appear to be involved in pattern control in a marine hydroid

A morphogenetically active compound has been isolated from tissue extract of Hydractinia echinata and identified to be N-methylpicolinic acid (homarine). When applied to whole animals, homarine prevents metamorphosis from larval to adult stage and alters the pattern of adult structures. The concentration of homarine in oocytes is about 25 mM. During embryogenesis, metamorphosis and early colony development the overall homarine content does not change. Adult colonies contain a fourfold lower homarine concentration than larvae. The polyp's head contains twofold more homarine than the gastric region and the stolons. A second, similarly active compound, N-methylnicotinic acid (trigonelline), has also been identified in Hydractinia tissue at concentrations about one-third that of homarine. Incubation of larvae in 10 to 20 microM-homarine or trigonelline prevents head as well as stolon formation. If the compounds are applied in a pulse during metamorphosis, a large part of the available tissue forms stolons. Since microM concentrations of homarine and trigonelline are morphogenetically active, whereas mM concentrations are present in the tissue it appears that both substances are stored within the tissue.     

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     

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     

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.     

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.     

Reorganization of the nervous system during metamorphosis of a hydrozoan planula

Abstract. Laser scanning confocal microscopy is used to reveal the changes that occur in the RFamide-positive nerve net as a free-swimming, solid hydrozoan planula larva is transformed into a sessile, hollow, young polyp. Seven stages of development in Pennaria tiarella are described: planula competent to metamorphose, attaching planula, disc, pawn, crown, developing polyp, and developed primary polyp. The RFamide-positive nervous system undergoes dramatic reorganization during metamorphosis: (1) larval neurons degenerate; (2) new neurons differentiate and reform a nerve net; and (3) the overall distribution pattern of the nervous system changes. This study confirms earlier observations on RFamide-positive neurons of Hydractinia which also show the loss of these cells after the onset of metamorphosis.     

Evolution of astacin-like metalloproteases in animals and their function in development

Astacin-like metalloproteases are ubiquitous in the animal kingdom but their phylogenetic relationships and ancient functions within the Metazoa are unclear. We have cloned and characterized four astacin-like cDNAs from the marine hydroid Hydractinia echinata and performed a database search for related genes in the draft genome sequence of the sea anemone Nematostella vectensis. These sequences and those of higher animals' astacins were subjected to phylogenetic analysis revealing five clusters within the Eumetazoa. The bone morphogenetic protein-1/tolloid-like astacins were represented in all eumetazoan phyla studied. The meprins were only found in vertebrates and cnidarians. Two clusters were taxon-specific, and one cluster represented astacins, which probably evolved after the split of the Cnidaria. Interestingly, grouping of astacins according to the protease catalytic domain alone resulted in clusters of proteins with similar overall domain architecture. The Hydractinia astacins were expressed in distinct cells during metamorphosis and some also during wound healing. Previously characterized cnidarian astacins also act during development. Based on our phylogeny, however, we propose that the developmental function of most of them is not homologous to the developmental function assigned to higher animals' astacins.     

Serotonin Plays an Early Role in the Metamorphosis of the HydrozoanPhialidium gregarium

Hydrozoan larvae normally metamorphose in response to an obligate external environmental cue. Application of certain artificial chemical stimuli will also induce metamorphosis. These chemicals and their inhibitors have been used to define and order some of the signal transduction events involved in this process. Results from this study show that exogenous application of serotonin (5-HT) will induce metamorphosis and that 5-HT immunoreactive cells are present in larvae when they are competent to metamorphose. The 5-HT inhibitors ketanserin, clozapine, and 5,7-DHT prevent metamorphosis from occurring as a response to a natural inducing stimulus. Additionally, 5-HT signaling occurs prior to both an influx of external Ca²⁺from seawater and activation of protein kinase C, two other steps in the metamorphic signal transduction pathway. The neuropeptide LWamide, previously shown to induce metamorphosis in a related hydrozoan,Hydractinia echinata,also induced metamorphosis inPhialidium.When larvae were cotreated with LWamide and the 5-HT antagonist ketanserin, settlement occurred but was not followed by polyp morphogenesis. These results are used to present a model for the action of 5-HT during metamorphosis inPhialidium gregarium.     

Morphological chimeras of larvae and adults in a hydrozoan--insights into the control of pattern formation and morphogenesis

In the marine hydroid Hydractinia echinata, metamorphosis transforms the spindle-shaped larva into a primary polyp. It bears a hypostome with a ring of tentacles at its apical end, a gastric region in the middle and stolons at the base. In nature, metamorphosis is induced in response to external stimuli provided by bacteria. These stimuli can be replaced by artificial inducers, one of which is heat shock. Among heat shock treated stages are those undergoing complete metamorphosis but also specimens forming chimeras of different developmental stages. In the chimeric larvae, the posterior is transformed into the apical hypostome of the adult polyp while the anterior part of the larva persists as larval tissue. After transverse sectioning, these stage chimeras regenerate the missing body parts with respect to the nature of the tissue at the wound surface. This shows that the decision to make larva or polyp morphology depends not on the majority of the tissue in the original body section, but on stage specificity within the regenerating animal part. Single cells can escape from this general rule, since RFamide nerve cells which usually differentiate in polyp tissue appear in regenerated larval tails of sectioned stage chimeras. The results indicate that the pattern-forming system of the larva and of the adult have features in common. The primary signals controlling patterning along the anterior-posterior axis in larvae and the apical-basal axis in polyps arethus likelyto be the same while the interpretation of these primary signals by the individual cells changes during metamorphosis.     

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.     

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     

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.     

The Effect of a DNA Damaging Agent on Embryonic Cell Cycles of the Cnidarian Hydractinia echinata

The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria.