The phosphatidylserine receptor from <Emphasis Type="Italic">Hydra</Emphasis> is a nuclear protein with potential Fe(II) dependent oxygenase activity

Background  

Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.     

Hydra and the evolution of apoptosis

Programmed cell death occurs in most, if not all life forms.It is used to sculpt tissue during embryogenesis, to removedamaged cells, to protect against pathogen infection and toregulate cell numbers and tissue homeostasis. In animals celldeath often occurs by a morphologically and biochemically conservedprocess called apoptosis. A novel group of cysteine proteases,referred to as caspases, constitute the central component ofthis process. Caspases are activated following the inductionof apoptosis and cleave a variety of cellular substrates, thusgiving rise to the characteristic morphological events of apoptosis.Apoptosis is rapid and cell corpses are removed by phagocytosis.Recent work has shown that apoptosis also occurs in Cnidariaand Porifera, thus extending the origin of this evolutionaryinnovation down to the first metazoan animal phyla. Here, wereview several examples of the role of apoptosis in cnidariansand then summarize new results on the subcellular localizationof caspases and the control of apoptosis in Hydra. We show byimmuncytochemistry that caspases in Hydra are localized in mitochondria.Following induction of apoptosis caspases are released frommitochondria as proenzymes and then activated by proteolyticcleavage in the cytoplasm. We also present evidence that apoptosisin Hydra is dramatically stimulated by inhibitors of PI3-kinase.Since PI3-kinase is a central component of growth factor signalingcascades in higher metazoans, this result suggests that controlof apoptosis by growth factors is also evolutionarily conserved.We speculate on the role of growth factors in the evolutionof apoptosis.     

Foot differentiation and genomic plasticity in Hydra: lessons from the PPOD gene family

In Hydra, developmental processes are permanently active to maintain a simple body plan consisting of a two-layered, radially symmetrical tube with two differentiated structures, head and foot. Foot formation is a dynamic process and includes terminal differentiation of gastric epithelial cells into mucous secreting basal disc cells. A well-established marker for this highly specialized cell type is a locally expressed peroxidase (Hoffmeister et al. 1985). Based on the foot-specific peroxidase activity, the gene PPOD1 has been identified (Hoffmeister-Ullerich et al. 2002). Unexpectedly, this approach led to the identification of a second gene, PPOD2, with high sequence similarity to PPOD1 but a strikingly different expression pattern. Here, we characterize PPOD2 in more detail and show that both genes, PPOD1 and PPOD2, are members of a gene family with differential complexity and expression patterns in different Hydra species. At the genomic level, differences in gene number and structure within the PPOD gene family, even among closely related species, support a recently proposed phylogeny of the genus Hydra and point to unexpected genomic plasticity within closely related species of this ancient metazoan taxon. Electronic supplementary material Supplementary material is available in the online version of this article at     

Expression of developmental genes during early embryogenesis of<Emphasis Type="Italic"> Hydra</Emphasis>

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.Edited by D. Tautz     

The Hydra small ubiquitin‐like modifier

SUMO is a protein posttranslational modifier. SUMO cycle components are believed to be conserved in all eukaryotes. Proteomic analyses have lead to the identification a wealth of SUMO targets that are involved in almost every cellular function in eukaryotes. In this article, we describe the characterization of SUMO Cycle components in Hydra, a Cnidarian with an ability to regenerate body parts. In cells, the translated SUMO polypeptide cannot conjugate to a substrate protein unless the C‐terminal tail is cleaved, exposing the di‐Glycine motif. This critical task is done by SUMO proteases that in addition to SUMO maturation are also involved in deconjugating SUMO from its substrate. We describe the identification, bioinformatics analysis, cloning, and biochemical characterization of Hydra SUMO cycle components, with a focus on SUMO and SUMO proteases. We demonstrate that the ability of SUMO proteases to process immature SUMO is conserved from Hydra to flies. A transgenic Hydra, expressing a SUMO‐GFP fusion protein under a constitutive actin promoter, is generated in an attempt to monitor the SUMO Cycle in vivo as also to purify and identify SUMO targets in Hydra. genesis 51:619–629. © 2013 Wiley Periodicals, Inc.     

Human, insect and nematode caspases kill Saccharomyces cerevisiae independently of YCA1 and Aif1p

This study characterised the impact of active metazoan apoptotic proteases (caspases) on Saccharomyces cerevisiae viability. Expression of active caspase-3 or caspase-8 in yeast ruptured plasma and nuclear membranes and dramatically impaired clonogenic survival, but did not damage DNA. Deletion of the proposed yeast apoptosis regulators YCA1 or Aif1p did not affect the ability of human, insect or nematode caspases to kill yeast. These data indicate that expression of active metazoan caspases causes irreversible damage to yeast membranes and organelles, in a manner independent of YCA1 and Aif1p.     

Highly Conserved Caspase and Bcl-2 Homologues from the Sea Anemone Aiptasia pallida: Lower Metazoans as Models for the Study of Apoptosis Evolution

Key insight into the complexities of apoptosis may be gained from the study of its evolution in lower metazoans. In this study we describe two genes from a cnidarian, Aiptasia pallida, that are homologous to key genes in the apoptotic pathway from vertebrates. The first is a novel ancient caspase, acasp, that displays attributes of both initiator and executioner caspases and includes a caspase recruitment domain (CARD). The second, a Bcl-2 family member, abhp, contains a BH1 and BH2 domain and shares structural characteristics and phylogenetic affinity with a group of antiapoptotic Bcl-2s including A1 and Bcl-2L10. The breadth of occurrence of other invertebrate homologues across the phylogenetic trees of both genes suggests that the complexity of apoptotic pathways is an ancient trait that predates the evolution of vertebrates and higher invertebrates such as nematodes and flies. This paves the way for establishing new lower metazoan model systems for the study of apoptosis. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stuart Newfeld]     

Evolving visual pigments: Hints from the opsin-based proteins in a phylogenetically old “eyeless” invertebrate

Visual pigments are photosensitive receptor proteins that trigger the transduction process producing the visual excitation once they have absorbed photons. In spite of the molecular and morpho-functional complexity that has characterized the development of animal eyes and eyeless photoreceptive systems, opsin-based protein family appears ubiquous along metazoan visual systems. Moreover, in addition to classic rhodopsin photoreceptors, all Metazoa have supplementary non-visual photosensitive structures, mainly located in the central nervous system, that sense light without forming an image and that rather regulate the organism's temporal physiology. The investigation of novel non-visual photopigments exerting extraretinal photoreception is a challenging field in vision research. Here we propose the cnidarian Hydra as a useful tool of investigation for molecular and functional differences between these pigment families. Hydra is the first metazoan owning a nervous system and it is an eyeless invertebrate showing only an extraocular photoreception, as it has no recognized visual or photosensitive structures. In this paper we provide an overview of the molecular and functional features of the opsin-based protein subfamilies and preliminary evidences in a phylogenetically old species of both image-forming and non-visual opsins. Then we give new insights on the molecular biology of Hydra photoreception and on the evolutionary pathways of visual pigments.     

Molecular and biological characterization of a zonula occludens-1 homologue in Hydra vulgaris, named HZO-1

Zonula occludens-1 (ZO-1) is one of the earliest identified molecular components of tight junctions. Sequence analysis has placed ZO-1 into the broader membrane-associated guanylate kinase (MAGUK) protein family that contains such diverse members as postsynaptic density 95 (PSD-95), Drosophila discs large tumor suppressor gene product (dlg-A), p55, and TamA. Studies in both vertebrates and invertebrates have established that the MAGUK family is involved in a wide variety of cellular functions. These functions involve the regulation of such cellular processes as: (1) tight junction formation, (2) cell proliferation, (3) cell differentiation, and (4) neuronal synapse transmission. Extending these studies, we report the presence of a ZO-1 homologue in Hydra vulgaris, a member of the Cnidaria, the second oldest phylum of the animal kingdom. Hydra ZO-1 (HZO-1) is encoded by a single messenger RNA (mRNA) of approximately 6.0 kb that contains an open reading frame of 5,085 bp. The 191 kDa predicted protein consists of a characteristic MAGUK domain structure, including three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH₃) domain, and a guanylate kinase (GUK) domain. Western blot analysis using an antibody generated from a synthetic peptide designed from the HZO-1 sequence confirmed the presence of a Hydra protein of the appropriate mass. While whole mount in situ hybridization determined that HZO-1 mRNA was expressed along the entire longitudinal axis of Hydra, cross-sectional analysis established that HZO-1 mRNA expression was restricted to the ectoderm or outer cell layer of the organism’s epithelial bilayer. Consistent with this mRNA expression pattern, immunofluorescence studies localized HZO-1 protein to the apical plasma membrane of ectodermal cells. It is unclear what role HZ0-1 has in the cellular physiology of Hydra; however, immunolocalization studies indicate a conserved plasma membrane-associated function(s), as reported for its counterparts in other invertebrate and vertebrate species. These studies establish that the MAGUK family of proteins with a membrane-associated function arose early during metazoan evolution, even before the divergence of protostomes and deuterostomes. Received: 11 May 2000 / Accepted: 26 July 2000     

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8–10 m) containing about 3×10⁹ bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5–10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.Abbreviations CT Chromosome territory - BrdU Bromodeoxyuridine - IdU Iododeoxyuridine - CldU Chlorodeoxyuridine Communicated by E.A. Nigg     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.     

Characterization of the Hydra Lamin and Its Gene: A Molecular Phylogeny of Metazoan Lamins

We report sequences for nuclear lamins from the teleost fish Danio and six invertebrates. These include two cnidarians (Hydra and Tealia), one priapulid, two echinoderms, and the cephalochordate Branchiostoma. Combining these results with earlier data on Drosophila, Caenorhabditis elegans, and various vertebrates, the following conclusions on lamin evolution can be drawn. First, all invertebrate lamins resemble in size the vertebrate B-type lamin. Second, all lamins described previously for amphibia, birds and mammals as well as the first lamin of a fish, characterized here, show a cluster of 7 to 12 acidic residues in the tail domain. Since this acidic cluster is absent from all invertebrate lamins including that of the cephalochordate Branchiostoma, it was acquired with the vertebrate lineage. The larger A-type lamin of differentiated cells must have arisen subsequently by gene duplication and insertion of an extra exon. This extra exon of the vertebrate A-lamins is the only major change in domain organization in metazoan lamin evolution. Third, the three introns of the Hydra and Priapulus genes correspond in position to the last three introns of vertebrate B-type lamin genes. Thus the entirely different gene organization of the C. elegans and Drosophila Dmo genes seems to reflect evolutionary drift, which probably also accounts for the fact that C. elegans has the most diverse lamin sequence. Finally we discuss the possibility that two lamin types, a constitutively expressed one and a developmentally regulated one, arose independently on the arthropod and vertebrate lineages. Received: 4 February 1999 / Accepted: 1 April 1999     

Plasticity of epithelial cell shape in response to upstream signals: A whole-organism study using transgenic Hydra

Multicellular organisms consist of a variety of cells of distinctive morphology, with the cell shapes often reproduced with astonishing accuracy between individuals and across species. The morphology of cells varies with tissues, and cell shape changes are of profound importance in many occasions of morphogenesis. To elucidate the mechanisms of cell shape determination and regulation is therefore an important issue. One of the simplest multicellular organisms is the freshwater polyp Hydra. Although much is known about patterning in this early branching metazoan, there is currently little understanding of how cells in Hydra regulate their shape in response to upstream signals. We previously reported generation of transgenic Hydra to trace cells and to study cell behavior in vivo in an animal at the basis of animal evolution. Here, we use a novel transgenic line which expresses enhanced green fluorescent protein (eGFP) specifically in the ectodermal epithelial cells to analyze the structure and shape of epithelial cells as they are recruited into specific regions along the body column and respond to upstream signals such as components of the canonical Wnt signaling pathway. As a general theme, in contrast to epithelial cells in more complex animals, ectodermal epithelial cells in Hydra are capable of drastic changes in structure, shape, and cell contact along the body column. The remarkable phenotypic plasticity of epithelial cells in response to positional signals allows Hydra to build its body with only a limited number of different cell types.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.     

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G₁ DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.     

Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

Substance P (SP) and its receptor, the neurokinin‐1 receptor (NK‐1 R), are expressed by human tenocytes, and they are both up‐regulated in cases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt, which has anti‐apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti‐Fas treatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti‐Fas‐induced apoptosis, and by which mechanisms SP mediates an anti‐apoptotic response. Anti‐Fas treatment resulted in a time‐ and dose‐dependent release of lactate dehydrogenase (LDH), i.e. induction of cell death, and SP dose‐dependently reduced the Anti‐Fas‐induced cell death through a NK‐1 R specific pathway. The same trend was seen for the TUNEL assay, i.e. SP reduced Anti‐Fas‐induced apoptosis via NK‐1 R. In addition, it was shown that SP reduces Anti‐Fas‐induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti‐Fas induces cleavage/activation of caspase‐3 and cleavage of PARP; both of which were inhibited by SP via NK‐1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti‐apoptotic effect of SP was, at least partly, induced through the Akt‐dependent pathway. In conclusion, we show that SP reduces Anti‐Fas‐induced apoptosis in human tenocytes and that this anti‐apoptotic effect of SP is mediated through NK‐1 R and Akt‐specific pathways.     

Identification of a Hydra homologue of the β-catenin/plakoglobin/armadillo gene family

The β-catenin/plakoglobin/armadillo gene family encodes a group of highly conserved proteins which play important roles in cadherin-mediated cell adhesion and in signal transduction mechanisms involved in regulating development. This gene family previously had been isolated only from higher metazoans. Here, we describe the isolation and characterization of a β-catenin (βCtn) homologue from Hydra magnipapillata a diploblastic lower metazoan. Comparison of the putative amino acid (aa) sequence of Hydra βCtn, with its homologues in higher metazoans, shows that a repeating 42-aa motif present in its central domain is highly conserved throughout the metazoa. This suggests that βCtn appeared very early in metazoan evolution, possibly when primitive multicellular animals started to form epithelial cell layers.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

The absence of electrically demonstrable polarizing factors in Hydra

Various investigators have shown that in the marine hydroids, Tubularia, Obelia, Eudendrium, and Pennaria, regeneration and polarity is affected by an electrical field applied parallel to the regenerate. Using electrical currents up to the physiological limits for Hydra, no relation between electrical current and regeneration rate or polarity could be demonstrated. This is in spite of the fact that adult Hydra are normally electrically polarized with the distal end approximately–18 mV relative to the proximal end of the animal. When the electrophoretic mobility and isoelectric point of cells from distal, central and proximal thirds of Hydra were measured, a significant difference was found between cells of the two cell layers but not between cells of the three body thirds. These results are discussed in relation to Hydra growth factors described by various other authors.