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.

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.

A novel wingless gene was isolated from the marine colonial hydroid Hydractinia echinata. Alignments and Bayesian inference analysis clearly assign the gene to the Wnt5A group. In line with data found for the brachyury ortholog of Hydractinia, He-wnt5A is expressed during metamorphosis in the posterior tip of the spindle-shaped planula larva, suggesting that the tip functions as a putative organizer during metamorphosis. Additionally, the outermost cells of the posterior tip are omitted from apoptosis during metamorphosis. In order to investigate this putative organizer function, we transplanted the posterior tip of metamorphosing animals into non-induced larvae and into primary polyps 24 h and 48 h of age. In larvae, the tip induced formation of a secondary axis. In polyps the building of ectopic head structures was induced. Based on our data on axis formation, on gene expression similar to the organizers of other species, and the absence of regular apoptosis, we conclude that the posterior tip of the Hydractinia larva has organizing activity during metamorphosis.

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.

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.

Although they rank quite low in the evolutionary hierarchy, in cnidarians a high number of chemical mediators and hormones is expected and the data received during the last years point in exactly that way. This text reviews the importance and classification of these signalling molecules in cnidaria, and gives examples of those mediators, concentrating on the following main groups: non-peptidic, non-amidated peptidic, and amidated peptidic reagents.

Programmed cell death is not only known as a mechanism mediating tissue destruction, but also as an organismic tool for body shaping and regulation of morphological events during development. Here we report the tight and vital link of the most prominent form of programmed cell death, apoptosis, to one of the oldest, most basic, and most radical developmental processes, the metamorphosis of the marine hydrozoon Hydractinia echinata. Apoptosis, represented by DNA fragmentation, appears very early during metamorphosis, approximately 20 minutes post induction. It is then executed in a very distinct spatial and temporal pattern, including the removal or phagocytosis of a large number of larval cells prior to the appearance of stolons and tentacles. Our data indicate a developmental program striving to reduce all body parts that are no longer necessary, before reaching a distinct turning point, when the development of adult features is initiated. During these events, morphogenesis of basal and apical structures correlates with recycling of that particular larval region, indicated by the presence of apoptosis. Based on these data, the necessity of apoptosis for normal development of adult patterns is inferred and a fundamental association of apoptosis with developmental processes can be stated.