Uri Frank

ALLOIMMUNE MATURATION IN THE CORAL STYLOPHORA PISTILLATA IS ACHIEVED THROUGH THREE DISTINCTIVE STAGES, 4 MONTHS POST-METAMORPHOSIS

Adult colonies of the reef–building coral Stylophora pistillata discriminate precisely between ‘self’ and ‘non–self’ attributes and respond selectively against specific allogeneic challenges. We studied the ontogeny of these allospecific responses on newly settled polyps by establishing allogeneic contacts within groups of 2–6 siblings or non–related offsprings. Interactions were observed for up to 8 months. Three types of responses, depending on the age of the interacting partners, were documented. The first was tissue fusion and the formation of a stable chimera, observed in partners less than 2 months old. The second was observed in contacts of partners 2–4 months old. It started with tissue fusion and transitory chimera since separation of the chimera–partners or polyp death resulted when the oldest partner in the chimera reached the age of 4 months. The third type was the regular histoincompatibility response, as documented in allogeneic interactions of adult colonies, recorded here in all encounters with S. pistillata partners over 4 months old. Maturation of allorecognition in this species was therefore achieved through three time–dependent stages, 4 months following metamorphosis. Combinations of siblings or genetically unrelated partners did not affect the results. We propose that the coral alloimmune maturation system may be used as a new evolutionary model scheme for studying tissue transplantation and tolerance.

In vitro establishment of continuous cell cultures and cell lines from ten colonial cnidarians

Continuous cell cultures from the ten taxa of sedentary colonial marine cnidariansStylophora pistillata, Porites lutea, Favia favus (Anthozoa, Hexacorallia, Madeporaria),Parerythropodium fulvum fulvum, Dendronephthya hemprichi, Nephthya sp.Heteroxenia fuscescence (Anthozoa, Octocorallia, Alcyonacea),Clathraria rubrinoides, Plexaura A (Anthozoa, Octocorallia, Gorgonacea) andMillepora dichotoma (Hydrozoa) were established in vitro. Primary cultures of various cell types (5 to 20 μm) were obtained from colony fragments and/or planula larvae (collected in 1993 from coral reefs at Eilat, Red Sea and from the San Blas islands, Panama) using three dissociation approaches: a mechanical approach, chemical approach and a novel approach, spontaneous dissociation. Cells were cultured in a modified Leibowitz L15 medium, with 5 to 10% heat-inactivated fetal bovine serum, diluted in seawater. Cell proliferation was observed in primary cultures within 7 to 20 d following dissociation. When developed to secondary cultures, spindle-shaped cells (5 to 10 μm) gradually replaced all other cell types seen in primary cultures ofS. pistillata, C. rubrinoides, P.f. fulvum andM. dichotoma. The spindle-shaped cells differentiated to several cell types when the diluted medium was replaced with a concentrated one, or when microbial contamination occurred. Secondary cultures of spindle-shaped cells from three species (S. pistillata, C. rubrinoides andM. dichotoma) were cloned. They gave rise to continuously proliferating cell lines (NIO-SPP-1, NIO-CR-1 and NIO-MD-1, respectively). A clone fromPlexaura planula, originated from one rounded cell, started to differentiate and gave rise to a heterogeneous culture. Samples of the above four cultures were frozen for future work. Rounded cells of various sizes (5 to 30 μm) dominated secondary cell cultures ofH. fuscescence, D. hemprichi, Nephthya sp.,P. f. fulvum andF. favus. Several cell cultures fromP. lutea developed flattened cell interconnected by ectoplasmic networks characteristic of the eukaryotic unicellular organisms of the phylum Labyrinthulomycota. We propose that this culture methodology may be used as a ubiquitous protocol for producing tissue cultures from other cnidarians. The established cell lines may be used in a variety of disciplines in cnidarian biology and ecology.

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.

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.

Distinct mechanisms underlie oral vs aboral regeneration in the cnidarian Hydractinia echinata

Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1+ stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology. DOI: http://dx.doi.org/10.7554/eLife.05506.001

Nontransitive patterns of historecognition phenomena in the Red Sea hydrocoral Millepora dichotoma

Allogeneic assays were conducted in situ on the Red Sea hydrocoral Millepora dichotoma. Forty-five pairwise combinations among ten colonies and ten control isografts were set up in four replicates each (180 and 40 pairs, respectively) and followed for up to 8 mo in the Gulf of Eilat, Red Sea in 1992–1993. In 42 allogeneic combinations we recorded a reproducible and unilateral tissue and skeleton overgrowth which developed within the first 10 wk up to 20 mm. Following the development of these primary overgrowths, four types of secondary responses were observed among most incompatible combinations: reversals in overgrowth directionality, tissue necroses, stand-offs and abnormal growth patterns. Secondary responses within a given set of replicates of most allogeneic combinations were characterized by high variability in type and intensity of response. Based on the outcomes of primary overgrowths, a complex nontransitive hierarchy was constructed for the ten colonies. All isografts and three allogeneic combinations fused within 3 wk. Fusion pattern between the three allogeneic combinations was nontransitive: one M. dichotoma colony repeatedly fused with two other colonies (four assays each). However, these two colonies not only did not fuse with each other, but one of them repeatedly overgrew its confrére. In the third compatible combination, the most superior and the most inferior colonies in the network of hierarchies among the ten colonies, fused in all tested assays. These results are compared with allogeneic outcomes in other enidarians, where nontransitive fusion between allogenic colonies have never been recorded.

The transcriptome of the colonial marine hydroid Hydractinia echinata

An increasing amount of expressed sequence tag (EST) and genomic data, predominantly for the cnidarians Acropora, Hydra and Nematostella, reveals that cnidarians have a high genomic complexity, despite being one of the morphologically simplest multicellular animals. Considering the diversity of cnidarians, we performed an EST project on the hydroid Hydractinia echinata, to contribute towards a broader coverage of this phylum. After random sequencing of almost 9000 clones, EST characterization revealed a broad diversity in gene content. Corroborating observations in other cnidarians, Hydractinia sequences exhibited a higher sequence similarity to vertebrates than to ecdysozoan invertebrates. A significant number of sequences were hitherto undescribed in metazoans, suggesting that these may be either cnidarian innovations or ancient genes lost in the bilaterian genomes analysed so far. However, we cannot rule out some degree of contamination from commensal bacteria. The identification of unique Hydractinia sequences emphasizes that the acquired genomic information generated so far is not large enough to be representative of the highly diverse cnidarian phylum. Finally, a database was created to store all the acquired information (http://www.mchips.org/hydractinia_echinata.html).

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.

SCYPHOZOAN JELLYFISH'S MESOGLEA SUPPORTS ATTACHMENT, SPREADING AND MIGRATION OF ANTHOZOANS' CELLS IN VITRO

Mechanically and enzymatically dissociated cells from five anthozoan species were laid on seven substrates in vitro. Cells were taken from two sea anemones (Aiptasia sp. and Anemonia sulcata), a scleractinian coral (Stylophora pistillata) and two alcyonacean corals (Heteroxenia fuscescence andNephthea sp). Substrates tested: glass (coverslips), plastic (uncoated tissue culture plates), type IV collagen, gelatin, fibronectin, mesoglea pieces from the scyphozoan jellyfish Rhopilema nomadica and acetic acid extract of jellyfish mesoglea. Except for the mesoglea pieces, cells did not respond to any one of the other substrates, retaining their rounded shape. Following contact with mesoglea pieces, cells attached and spread. Subsequently they migrated into the mesogleal matrix at a rate of 5–10μm/h during the first 2–5h. No difference was found between the behavior of cells from the five different cnidarian species.