The embryonic development of the cnidarian Hydractinia echinata.

With the rapid increase of the quantity of molecular data, many animals joined the ranks of the so-called 'emerging models' of Evo-Devo. One of the necessary steps in converting an emerging model into an established one is gaining comprehensive knowledge of its normal embryonic development. The marine colonial hydrozoan Hydractinia echinata - an excellent model for research on stem cells, metamorphosis, and allorecognition - has been studied for decades. Yet knowledge of its embryonic development remains fragmentary and incomplete. Here we provide a detailed account of H. echinata embryonic development using in vivo observations, histology, immunohistochemistry, and electron microscopy. Furthermore, we propose a model describing the cellular basis of the morphogenetic movements occurring during development and also reveal a functional link between canonical Wnt signaling and regional differences in the morphology of the embryo. Hydractinia embryogenesis is an example of the diversity and plasticity of hydrozoan development where multiple routes lead to the same result - the formation of a normal planula larva.

Hydractinia, a pioneering model for stem cell biology and reprogramming somatic cells to pluripotency.

Hydractinia, a representative marine colonial hydroid, was the first organism in the history of biology in which migratory precursors of germ cells were described and termed "stem cells" (Weismann, 1883). These stem cells, now known as interstitial cells (i-cells), are thought to remain pluripotent throughout their life. Using animals depleted of their own stem cells and repopulated with allogeneic mutant donor stem cells, it was shown that Hydractinia i-cells differentiate into any cell type including epithelial cells and germ cells that express germ line markers such as Vasa, Piwi and Nanos. In Hydra, i-cells also provide germ cells and somatic cells with the exception of epithelial cells. The latter derive from two subpopulations of differentiated epithelial cells with self-renewal capacity. In Hydractinia, forced expression of the Oct4-like transcription factor, Polynem (Pln), in epithelial cells transforms them into stem cells that develop neoplasms. I-cells express the Wnt-receptor Frizzled and are Wnt responsive. Activation of Wnt signaling induces the production of numerous nematocytes (stinging cells) and nerve cells. In parallel, supernumerary tentacles develop. I-cells also express Myc and Nanos. Their misexpression causes severe developmental defects. Hydractinia polyp buds arise from aggregating stem cells, in contrast to Hydra buds, which derive from evaginating epithelial cells. Wnt activation increases budding frequency and the emergence of ectopic head structures. The potential of stem cells to invade neighbors may have provided selection pressure for the evolution of allorecognition and histo-incompatibility. Hence, Hydractinia have now attained the position of a powerful model in stem cell research, axis formation and allorecognition.

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.

A polymorphic, thrombospondin domain-containing lectin is an oocyte marker in Hydractinia: implications for germ cell specification and sex determination.

We have identified a novel, multidomain, polymorphic lectin in the marine cnidarian Hydractinia echinata. The gene is expressed in oocytes and was therefore named CEL for cnidarian egg lectin. The predicted protein has an unusual domain architecture, consisting of variable numbers of thrombospondin type 1 domains, flanked by one N-terminal and two C-terminal galactose binding lectin domains. The diversity of the gene's transcripts results from allelic polymorphism as well as alternative splicing. Hydractinia is dioecious and its sex has been reported previously to be genetically determined. We found intersexual colonies that were functional males, but had immature CEL-positive oocytes alongside mature sperm in the same gonads. Intersexuality was observed to be common in one population but not found in others. Hermaphroditic, self-fertile colonies were found in one locality; however, in these cases gonads contained either male or female gametes without mixed ones. Intersexuality that was considered to be a very rare event is apparently a more common phenomenon, at least in some populations. True hermaphroditism also occurs in this species. CEL can be considered as a marker for early oocyte differentiation and may play a role in germ cell specification and sex determination in cnidarians.

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.

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.

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.

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors.

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.

Wnt signaling in hydroid development: formation of the primary body axis in embryogenesis and its subsequent patterning.

We have studied the role the canonical Wnt pathway plays in hydroid pattern formation during embryonic development and metamorphosis. Transcripts of Wnt and Tcf were asymmetrically deposited in the oocyte and subsequent developmental stages, marking the sites of first cleavage, posterior larval pole and the upcoming head of the metamorphosed polyp. To address the function of these genes, we activated downstream events of the Wnt pathway by pharmacologically blocking GSK-3beta. These treatments rendered the polar expression of Tcf ubiquitous and induced development of ectopic axes that contained head structures. These results allow concluding that Wnt signaling controls axis formation and regional tissue fates along it, determining one single axis terminus from which later the mouth and hypostome develop. Our data also indicate Wnt functions in axis formation and axial patterning as in higher metazoans, and thus point to an ancestral role of Wnt signaling in these processes in animal evolution.

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.

An evolutionary conserved role of Wnt signaling in stem cell fate decision.

Wnt/Frizzled/ss-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ss-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ss-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination.

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.

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.

Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea.

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a λ gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.

Analysis of pattern formation during embryonic development of Hydractinia echinata.

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.

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

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.

Low-molecular-weight factors from colonial hydroids affect pattern formation.

Two morphogenetic factors have been isolated from tissue of colonial hydroids. Both exert strong effects on pattern formation during metamorphosis, regeneration and colony development. Polyp-inhibiting factor (PIF) is a bivalent inhibitor which strongly affects head and bud formation but acts weakly on stolon branching. Proportion-altering factor (PAF) is a distalizing factor. It counteracts the formation of stolon and promotes the formation of head structures during metamorphosis and regeneration. PIF and PAF antagonistically influence the spatial arrangement of polyps within a colony. They are capable of dislocating structures and thus appear to interfere with or are even part of the pattern-controlling mechanism. Both factors are of low molecular size (about 500 daltons), hydrophilic and probably not peptides.

Quantitative analysis of an inhibitory gradient field in the hydrozoan stolon.

An inhibitory field which emanates from the mobile tips of elongating stolons of colonial hydroids has been identified and analyzed. It extends proximally with decreasing intensity for about 400-700 µm and ensures that branching sites occur at appropriate distances along the stolon. The local strengths of inhibition within the field have been measured with a new method which permits high temporal and spatial resolution. Kinetic studies reveal three characteristics. First, inhibition decays rather rapidly after removal of its source. The half life is about 30 min. Second, loss of inhibition immediately triggers initiation of future tip formation. Third, restoration and spreading of inhibition are slow processes which take 8-24 h to recover 90% of the original inhibitory levels.