Thomas Leitz

LWamides from Cnidaria constitute a novel family of neuropeptides with morphogenetic activity

Metamorphosin A (MMA) isolated from the anthozoan Anthopleura elegantissima has recently been shown to interfere with developmental control in the colonial hydroid Hydractinia echinata. In order to identify the functional homologue in this species we have cloned cDNAs of the precursor protein from Hydractinia and, for comparison, precursor sequences from two further anthozoans. The deduced preproproteins contain multiple copies of propeptides to be processed into a great variety of novel neuropeptides most of which are N-terminally different from MMA. Original MMA is only contained in the anthozoan precursors. Most of the novel neuropeptides will have the carboxyl terminus LWamide. Therefore, we term this novel neuropeptide family the LWamides. Peptides synthesized according to the precursor sequence of H. echinata and added to planulae trigger metamorphosis. In contrast, none of 11 other known biologically active peptides including carboxamidated neuropeptides were effective. Expression analysis by in situ hybridization and by antibodies against the H. echinata peptide reveals the presence of the gene product in planulae at the proper time and at the due spatial location expected for a natural role in metamorphosis. LWamide transcripts are also observed in nerve cells of primary and adult polyps, suggesting LWamides to be a multifunctional family of neuropeptides.

Induction of settlement and metamorphosis of Cnidarian larvae: Signals and signal transduction

Summary Settlement and metamorphosis are induced by environmental cues in many marine invertebrates. These signals emanate from biotic or abiotic material indicating the presence of a suitable habitat. In Cnidarians, pelagic larvae undergo metamorphosis to a sessile polyp. Environmental signals reactivate the morphogenetically inactive larvae and internal mechanisms are turned onto coordinate the development of the polyp. In recent years developmental biologists, ecophysiologists and ecologists have shown a considerable interest in the metamorphosis of Cnidarians. The purpose of this review is to give a survey on what is known about the natural induction of metamorphosis and to provide information on the biochemical and cellular mechanisms underlying the metamorphic events in Cnidarians. The emphasis is on the external and internal signals and their transduction mechanisms used to control metamorphosis.

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

Abstract 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. Muller (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.

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).

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

SummaryMetamorphosis 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 affinityKd = 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.

Arachidonic acid and the control of body pattern inHydra

SummaryRepeated 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.

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 of the marine Hydrozoan Hydractinia echinata fleming, 1828

The marine hydrozoan Hydractinia echinata Fleming, 1828 develops through a demersal planula larval stage. Recent work on the induction of metamorphosis of the planula has contributed greatly to understanding of the role of cell communication by signals, signal transmission, and signal transduction during metamorphosis of marine invertebrates. The purpose of this paper is to survey what is known about the natural induction of metamorphosis and to provide information on the biochemical and cellular mechanisms underlying the metamorphic events in H. echinata. A critical look at the effect of inhibitors is given, in conjunction with an hypothesis about the sequence of early events in metamorphosis.

Stimulation of metamorphosis in Hydractinia echinata involves generation of lysophosphatidylcholine

SummaryWhilst 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.

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

SummaryA 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.