Günter Plickert

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.

Wnt signaling promotes oral but suppresses aboral structures in Hydractinia metamorphosis and regeneration

We studied the role of Wnt signaling in axis formation during metamorphosis and regeneration in the cnidarian Hydractinia. Activation of Wnt downstream events during metamorphosis resulted in a complete oralization of the animals and repression of aboral structures (i.e. stolons). The expression of Wnt3, Tcf and Brachyury was upregulated and became ubiquitous. Rescue experiments using Tcf RNAi resulted in normal metamorphosis and quantitatively normal Wnt3 and Brachyury expression. Isolated, decapitated polyps regenerated only heads but no stolons. Activation of Wnt downstream targets in regenerating animals resulted in oralization of the polyps. Knocking down Tcf or Wnt3 by RNAi inhibited head regeneration and resulted in complex phenotypes that included ectopic aboral structures. Multiple heads then grew when the RNAi effect had dissipated. Our results provide functional evidence that Wnt promotes head formation but represses the formation of stolons, whereas downregulation of Wnt promotes stolons and represses head formation.

Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor

The evolutionary origin of stem cell pluripotency is an unresolved question. In mammals, pluripotency is limited to early embryos and is induced and maintained by a small number of key transcription factors, of which the POU domain protein Oct4 is considered central. Clonal invertebrates, by contrast, possess pluripotent stem cells throughout their life, but the molecular mechanisms that control their pluripotency are poorly defined. To address this problem, we analyzed the expression pattern and function of Polynem (Pln), a POU domain gene from the marine cnidarian Hydractinia echinata. We show that Pln is expressed in the embryo and adult stem cells of the animal and that ectopic expression in epithelial cells induces stem cell neoplasms and loss of epithelial tissue. Neoplasm cells downregulated the transgene but expressed the endogenous Pln gene and also Nanos, Vasa, Piwi and Myc, which are all known cnidarian stem cell markers. Retinoic acid treatment caused downregulation of Pln and the differentiation of neoplasm cells to neurosensory and epithelial cells. Pln downregulation by RNAi led to differentiation. Collectively, our results suggest an ancient role of POU proteins as key regulators of animal stem cells.

Automated in situ detection (AISD) of biomolecules

Abstract In order to facilitate in situ detection of biomolecules in large sample series the processing of whole-mount specimens has been automated. A freely programmable liquid handling system is described by which embryos or similar biological materials are processed. Possible applications include in situ hybridization (ISH), immunocytochemistry (ICC) or reporter gene assays. Process times required for the preparation of whole-mount in situ hybridizations in Drosophila, Xenopus, Gallus and in hydroids were – in part – significantly reduced as compared with manual processing. Application of automated in situ detection (AISD) in random screening is demonstrated in hydroids. Potential further applications are discussed.

Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras

To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system.

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

SummaryDuring 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.

Neuropeptides and photic behavior in Cnidaria

Peptides of the RFamide family occur in neurosecretory cells of all nervous systems of Cnidaria so far studied. Photoreceptive organs – if evolved in a cnidarian species – are always associated with neural cells showing RFamide immunoreactivity. Experimental evidence for the function of RFamides and other neuropeptides in nervous systems and photoreceptive organs is, however, scarce or lacking. RFamide and LWamide immunoreactivity were surveyed in photoreceptive organs of the hydromedusa Cladonema radiatum, in rhopalia of the scyphozoan Aurelia aurita, and in rhopalia of the cubomedusa Tripedalia cystophora. A possible function of neuropeptides in transmission of photic stimuli was assayed by analysing photic behavior in Tripedalia, which has highly developed eyes, and in the simply constructed planula of the hydroid Hydractinia echinata, in which the mode of light perception is unknown. In both species, light orientation was effectively prevented by RFamides administered to the animals in micromolar concentration. In contrast, among four other neuropeptides occurring in the larva of Hydractinia, only one interfered with phototaxis and then only at 10× higher concentrations. Planulae depleted of bioactive peptideamides also lost phototaxis while still locomotorily active. The results support the hypothesis that one possible function of RFamides in Cnidaria is to transmit photic stimuli to epitheliomuscular targets.

Cnidarian Interstitial Cells: The Dawn of Stem Cell Research

The first stem cells described in the biological literature were those of hydroid cnidarians; their detection by Weismann in 1883 gave rise to his germ line and “germ plasm” theory (with “germ plasm” meaning what is called genome today). Somatic cells preserving properties of eggs (called Stammzellen, i.e. stem cells, by him) were considered by him to be the cellular source of regeneration and reproduction. Weismann’s studies have been the foundation of modern cnidarian stem cell research. In the latter, hydroid stem cells have been referred to as interstitial cells (shortly i-cells), and have mostly been studied in two cnidarian genera: the freshwater polyp Hydra and the colonial marine hydroid Hydractinia. In these animals, i-cells constitute a complex system of multipotent (in Hydra) or totipotent (in Hydractinia) stem cells and their derivatives. I-cells’ potencies have been investigated by specific elimination of stem cells and reintroduction of i-cells from donors. The complement of stem cells confers potential immortality to the genetic individual. Cnidarians’ cells in general have an unmatched capability of re- and transdifferentiation. Isolated, fully differentiated striated muscle cells of hydroid medusae may resume features of multipotent stem cells and give rise to almost all cell types including germ cells. Reverse development of adult stages back into juveniles is a further manifestation of cnidarian developmental plasticity. Typical i-cells have not been described in other cnidarian groups. In these taxa the source of new nematocytes nerve and germ cells may be differentiated cells that preserve plasticity. Following a historical perspective we review recent advances in hydroid i-cell research, and discuss the potential of invertebrate stem cell work.

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.

Analysis of pattern formation during embryonic development of Hydractinia echinata

SummaryPatterning 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.