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Hydra mesoglea: a model for investigating epithelial cell--basement membrane interactions.

Isolated hydra mesoglea served as a suitable substrate for the attachment and spreading of hydra cells in vitro, irrespective of the species tested. Hydra cells did not attach and spread on substrates typically used for culturing mammalian cells. Mammalian and Drosophila cells attached and spread on plastic culture dishes but not on isolated mesoglea. Xenopus epithelial cells spread on both plastic and mesoglea. Because of the similarities of hydra mesoglea to vertebrate basement membranes, suggestions are offered for using mesoglea to study the interactions of epithelial cells with their basement membranes.

Animals↗

Signalling by the FGFR-like tyrosine kinase, Kringelchen, is essential for bud detachment in Hydra vulgaris.

Signalling through fibroblast growth factors (FGFR) is essential for proper morphogenesis in higher evolved triploblastic organisms. By screening for genes induced during morphogenesis in the diploblastic Hydra, we identified a receptor tyrosine kinase (kringelchen) with high similarity to FGFR tyrosine kinases. The gene is dynamically upregulated during budding, the asexual propagation of Hydra. Activation occurs in body regions, in which the intrinsic positional value changes. During tissue displacement in the early bud, kringelchen RNA is transiently present ubiquitously. A few hours later - coincident with the acquisition of organiser properties by the bud tip - a few cells in the apical tip express the gene strongly. About 20 hours after the onset of evagination, expression is switched on in a ring of cells surrounding the bud base, and shortly thereafter vanishes from the apical expression zone. The basal ring persists in the parent during tissue contraction and foot formation in the young polyp, until several hours after bud detachment. Inhibition of bud detachment by head regeneration results in severe distortion, disruption or even complete loss of the well-defined ring-like expression zone. Inhibition of FGFR signalling by SU5402 or, alternatively, inhibition of translation by phosphorothioate antisense oligonucleotides inhibited detachment of buds, indicating that, despite the dynamic expression pattern, the crucial phase for FGFR signalling in Hydra morphogenesis lies in bud detachment. Although Kringelchen groups with the FGFR family, it is not known whether this protein is able to bind FGFs, which have not been isolated from Hydra so far.

Amino Acid Sequence↗

An ancient Wnt-Dickkopf antagonism in Hydra.

The dickkopf (dkk) gene family encodes secreted antagonists of Wnt signalling proteins, which have important functions in the control of cell fate, proliferation, and cell polarity during development. Here, we report the isolation, from a regeneration-specific signal peptide screen, of a novel dickkopf gene from the fresh water cnidarian Hydra. Comparative sequence analysis demonstrates that the Wnt antagonistic subfamily Dkk1/Dkk2/Dkk4 and the non-modulating subfamily Dkk3 separated prior to the divergence of cnidarians and bilaterians. In steady-state Hydra, hydkk1/2/4-expression is inversely related to that of hywnt3a. hydkk1/2/4 is an early injury and regeneration responsive gene, and hydkk1/2/4-expressing gland cells are essential for head regeneration in Hydra, although once the head has regenerated they are excluded from it. Activation of Wnt/beta-Catenin signalling leads to the complete downregulation of hydkk1/2/4 transcripts. When overexpressed in Xenopus, HyDkk1/2/4 has similar Wnt-antagonizing activity to the Xenopus gene Dkk1. Based on the corresponding expression patterns of hydkk1/2/4 and neuronal genes, we suggest that the body column of Hydra is a neurogenic environment suppressing Wnt signalling and facilitating neurogenesis.

Amino Acid Sequence↗

Expression of Cnox-2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation.

Cnox-2 is a HOM/HOX homeobox gene that we have identified in the simple metazoan Hydra vulgaris (Cnidaria: Hydrozoa). Cnox-2 is most closely related to anterior members of the Antennapedia gene complex from Drosophila, with the greatest similarity to Deformed. The Cnox-2 protein is expressed in the epithelial cells of adult hydra polyps in a region-specific pattern along the body axis, at a low level in the head and at a high level in the body column and the foot. The expression pattern of Cnox-2 is consistent with a role in axial pattern formation. Alteration of hydra axial patterning by treatment with diacylglycerol (DAG) results in an increase of head activation down the body column and in a coordinate reduction of Cnox-2 expression in epithelial cells in 'head-like' regions. These results suggest that Cnox-2 expression is negatively regulated by a signaling pathway acting through protein kinase C (PKC), and that the varying levels of expression of Cnox-2 along the body axis have the potential to result in differential gene expression which is important for hydra pattern formation.

Amino Acid Sequence↗

HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra.

Developmental gradients are known to play important roles in axial patterning in hydra. Current efforts are directed toward elucidating the molecular basis of these gradients. We report the isolation and characterization of HyAlx, an aristaless-related gene in hydra. The expression patterns of the gene in adult hydra, as well as during bud formation, head regeneration and the formation of ectopic head structures along the body column, indicate the gene plays a role in the specification of tissue for tentacle formation. The use of RNAi provides more direct evidence for this conclusion. The different patterns of HyAlx expression during head regeneration and bud formation also provide support for a recent version of a reaction-diffusion model for axial patterning in hydra.

Amino Acid Sequence↗

Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human SPINK1 pancreatic phenotype.

In hydra, the endodermal epithelial cells carry out the digestive function together with the gland cells that produce zymogens and express the evolutionarily conserved gene Kazal1. To assess the hydra Kazal1 function, we silenced gene expression through double-stranded RNA feeding. A progressive Kazal1 silencing affected homeostatic conditions as evidenced by the low budding rate and the induced animal death. Concomitantly, a dramatic disorganization followed by a massive death of gland cells was observed, whereas the cytoplasm of digestive cells became highly vacuolated. The presence of mitochondria and late endosomes within those vacuoles assigned them as autophagosomes. The enhanced Kazal1 expression in regenerating tips was strongly diminished in Kazal1(-) hydra, and the amputation stress led to an immediate disorganization of the gland cells, vacuolization of the digestive cells and death after prolonged silencing. This first cellular phenotype resulting from a gene knock-down in cnidarians suggests that the Kazal1 serine-protease-inhibitor activity is required to prevent excessive autophagy in intact hydra and to exert a cytoprotective function to survive the amputation stress. Interestingly, these functions parallel the pancreatic autophagy phenotype observed upon mutation within the Kazal domain of the SPINK1 and SPINK3 genes in human and mice, respectively.

Amino Acid Sequence↗

Developmental roles of epithelial and interstitial cell lineages in hydra: analysis of chimeras.

Chimeric hydra were prepared by recombining epithelial and interstitial cells between 3 strains of hydra of different sizes (maxi, normal, and mini strains). The resulting chimeras generally resembled the epithelial cell parent more than the interstitial cell parent in size, budding rate, tentacle number, and form. This suggests that epithelial cells normally exert considerable influence over hydra morphogenesis. However, the chimeras show some differences ascribable to interstitial cell origin. Furthermore, the 3 original strains, when deprived of interstitial cells, lose their distinguishing size differences. Thus both epithelial and interstitial cells (or interstitial cell derivatives) mutually participate in hydra's development.

Animals↗

Temporal relationships of host cell and algal mitosis in the green hydra symbiosis.

In fed hydra or excised regenerating peduncles there are increases in the mitotic indices of both digestive cells and the symbiotic algae that reside within them. Conversely, algal and digestive cell mitotic indices decrease in starved hydra. The temporal relationships of algal and host cell division differ in fed hydra and regenerating peduncles. After feeding, algal and digestive cell mitotic indices both reach a peak at about the same time; during regeneration, first the algae and then the digestive cells divide. Thus, mitotic digestive cells in regenerating peduncles contain more algae than those in gastric regions of fed hydra. However, in both cases mitotic digestive cells contain more algae than non-mitotic cells. The algae appear to be partitioned at random between daughter digestive cells at teleophase. It is suggested that the division of the symbiotic algae is closely related to that of the digestive cells in which they maintained. Mitosis of algae is stimulated by host cell mitosis, but in non-dividing cells algal mitosis is restricted. Possible mechanisms by which the host digestive cells could restrict algal division are discussed.

Animals↗

Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis.

An aposymbiotic strain of Hydra viridis became infected with free-living Chlorella sp. A stable symbiosis formed that differed in its characteristics from other known Chlorella/Hydra symbioses. The algae reproduced and formed clusters in host endodermal cells, inside large vacuoles filled with an electron-dense substance. A few algae were found to be digested by the hydra, but the apparently uncontrolled reproduction rate of the algae more than compensated for this loss. Surplus algae were expelled into the coelenteron and eventually into the surrounding medium. The expelled algae were repeatedly re-engulfed by the hydra during its feeding, forming a process of continuous reinfection. We suggest that such repeated reinfection of the hydra by the expelled algae provides the host with an endless number of Chlorella from which it might in time select a suitable adapted, controllable symbiont. The present newly formed symbiosis might serve as a model for the study of evolution of algal endosymbioses.

Animals↗

4-nonylphenol-induced toxicity and apoptosis in Hydra attenuata.

Effects of 4-nonylphenol (4-NP) on the morphology and survival of the cnidarian Hydra attenuata were studied under acute exposure conditions. The lethal concentration value inducing 50% mortality after 96 h was 97.5 +/- 20 microg/L, whereas the lethal concentration value inducing 10% mortality after 96 h was 64 +/- 25.5 microg/L. The no-observed-effect concentration based on morphological criteria was less than 25 microg/L. Hydra was one of the most sensitive freshwater invertebrate species behind the amphipod Hyalella azteca. Toxicity effects appeared rapidly and did not evolve substantially between 24 and 96 h of exposure. Induction of apoptosis was registered during the first hour of exposure to 4-NP at lethal concentrations, indicating rapid effects of the chemical. Abnormal increase of apoptosis may explain the acute toxicity of 4-NP in hydra. Results show that hydra viability is affected in the short term at 4-NP concentrations normally found in contaminated sites, but not at those concentrations reflecting lower levels of environmental contamination.

Animals↗

Immunocytochemically defined populations of neurons progressively increase in size through embryogenesis of Hydra vulgaris.

The hydra nervous system shares many features with nervous systems of more complex organisms but serves as a unique model system due to its simplicity and constant regeneration. Development of neuron populations during and after hydra embryogenesis is not well understood. In this study, neurons were identified at prehatching and posthatching stages with RFamide or JD1 antisera. These populations were further subdivided into ganglion, sensory, or unclassifiable neurons, and all identified populations were statistically analyzed over developmental time. RFamide-positive neurons appeared 20 days after the cuticle formed around the embryo. The JD1-positive neuron population appeared just after hatching, but by adulthood it had surpassed the size of the RFamide-positive population. All neuron populations progressively increased through their adult levels. Density of most of the populations, however, did not. For instance, during the 5-fold increase in size that the hydra experienced between 5 days posthatching and adulthood, the number of RFamide-positive neurons rose approximately 2-fold and the number of JD1-positive neurons 4-fold. However, the density of neurons in each of these populations fell. These data do not support the hypothesis that large-scale culling of neurons during development, frequently found in other animals, occurs in hydra.

Animals↗

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.

Amino Acid Sequence↗

Colchicine, nocodazole and trifluralin: different effects of microtubule polymerization inhibitors on the uptake and migration of endosymbiotic algae in Hydra viridis.

Hydra viridis (= Chlorohydra viridissima) the freshwater coelenterate, is symbiotic. Each individual animal harbours Chlorella sp. in its endodermal cells. The symbiosis may be disestablished, the partners grown independently, and then re-established experimentally. The most effective method to produce alga-free hydra was developed by Pardy (1976). In this study algae from homogenized H. viridis, English (= European) strain, were either used directly or first grown in pure culture and then injected to re-establish the symbiosis. The uptake of algae grown in culture was compared with that of algae released directly from fresh hydra. Fewer cultured algae were taken up. Four strains were tested: English, Carolina, Frome, and Jubilee. Each takes up a characteristic number of Chlorella when injected in excess. Colchicine, nocodazole and trifluralin, were tested for their effects on either uptake or migration. Colchicine had no effect. Nocodazole and trifluralin reduced both the number of algae taken up and the rate at which they were transported to the distal region of the hydra digestive cell. The effects of these drugs tended to be proportional to the concentration between 10(-8) and 10(-5) M.

Animals↗

Commitment of stem cells to nerve cells and migration of nerve cells precursors in preparatory bud development in Hydra.

Budding in Hydra starts as an evagination of the double-layered tissue in the parent animal's gastric region. Five hours later the density of nerve cells in the bud's tissue doubles, representing the first detectable difference from the cellular composition of the surrounding tissue. These new nerve cells derive from multipotent stem cells which are in S-phase one day before evagination starts. Some of the bud's new nerve cells derive from stem cells which have migrated into the future bud's tissue after their commitment, apparently attracted by the bud anlage. The bud anlage recruits precursors of nerve cells even during starvation, during which nerve cell production ceases in other parts of the body. Furthermore, the bud anlage controls the duration of the development from commitment to final differentiation of the resulting nerve cells. Experiments with an inhibitor purified from hydra tissue indicate a tight correlation between stages of preparatory bud development and stages of recruitment of nerve cells for the bud. Whether or not precursors of nerve cells are involved in the control of bud formation in normal hydra, as compared to epithelial hydra which still bud though consisting of epithelial cells only, will be discussed.

Animals↗

Neuronal control of development in hydra.

Hydra is an excellent model system for developmental biology, because pattern formation processes can be easily studied in regeneration, transplantation, and reaggregation experiments. At the cellular level hydra has the advantage that it contains only a few basic cell types and that differentiation pathways are short. Two types of signals, produced and released by nerve cells, control the spatial and temporal patterns of differentiation. Positive signals induce specific local differentiation events, and negative signals inhibit the spread of such inductions to larger areas. Head-specific growth and differentiation are controlled by head activator and head inhibitor, food-specific processes are regulated by foot activator and foot inhibitor. The activators are peptides, the inhibitors are low-molecular-weight substances. The sequence of the head activator is known, and it is conserved throughout the animal kingdom. At the cellular level head activator exerts three types of effects in hydra. It stimulates cells to divide, and it is responsible for the determination and the final differentiation of nerve cells and head-specific epithelial cells. For nerve-cell differentiation the cAMP pathway is used as second messenger system. Components of this pathway were identified in hydra. In mammals head activator is produced by nerve and neuro-endocrine cells, and it acts as mitogen on cells of neural origin. It is present early in neural development and in abnormal neural development, such as brain and neuroendocrine tumours.

Amino Acid Sequence↗

Genome sizes and chromosomes in the basal metazoan Hydra.

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.

Journal Article↗

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts.

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.

Journal Article↗

HYDRA: a knowledge acquisition tool for expert systems that critique medical workup.

HYDRA is a computer-based knowledge acquisition tool under development to assist in the creation of expert systems which critique medical workup. To use HYDRA, a domain expert first outlines the recommended approaches to the workup of a chosen medical problem, using the Augmented Transition Network formalism. From this model, HYDRA produces a list of the various conditions for which critiquing comments may be required to react to all possible approaches that might be proposed by the user of the critiquing system. Domain-specific constraints can be used to restrict the number of conditions suggested. In this way, HYDRA assists the domain expert by providing a model for structuring the problem, and by breaking down the domain expert's work into a set of small, easily understood tasks.

Algorithms↗