Selective inhibition of protein kinases blocks the formation of a new axis, the beginning of budding, in Hydra.

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.

Parameters of self-organization in Hydra aggregates.

Self-organization has been demonstrated in a variety of systems ranging from chemical-molecular to ecosystem levels, and evidence is accumulating that it is also fundamental for animal development. Yet, self-organization can be approached experimentally in only a few animal systems. Cells isolated from the simple metazoan Hydra can aggregate and form a complete animal by self-organization. By using this experimental system, we found that clusters of 5-15 epithelial cells are necessary and sufficient to form de novo head-organizing centers in an aggregate. Such organizers presumably arise by a community effect from a small number of cells that express the conserved HyBra1 and HyWnt genes. These local sources then act to pattern and instruct the surrounding cells as well as generate a field of lateral inhibition that ranges up to 1,000 microm. We propose that conserved patterning systems in higher animals originate from extremely robust and flexible molecular self-organizing systems that were selected for during early metazoan evolution.

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.

A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra.

During the course of a systematic screening of peptide signaling molecules in Hydra a novel peptide, Hym-355 (FPQSFLPRG-NH(2)), was identified. A cDNA encoding the peptide was isolated and characterized. Using both in situ hybridization and immunohistochemistry, Hym-355 was shown to be expressed in neurons and hence is a neuropeptide. The peptide was shown to specifically enhance neuron differentiation throughout the animal by inducing interstitial cells to enter the neuron pathway. Further, co-treatment with a PW peptide, which inhibits neuron differentiation, nullified the effects of both peptides, suggesting that they act in an antagonistic manner. This effect is discussed in terms of a feedback mechanism for maintaining the steady state neuron population in Hydra.

Cngsc, a homologue of goosecoid, participates in the patterning of the head, and is expressed in the organizer region of Hydra.

We have isolated Cngsc, a hydra homologue of goosecoid gene. The homeodomain of Cngsc is identical to the vertebrate (65-72%) and Drosophila (70%) orthologues. When injected into the ventral side of an early Xenopus embryo, Cngsc induces a partial secondary axis. During head formation, Cngsc expression appears prior to, and directly above, the zone where the tentacles will emerge, but is not observed nearby when the single apical tentacle is formed. This observation indicates that the expression of the gene is not necessary for the formation of a tentacle per se. Rather, it may be involved in defining the border between the hypostome and the tentacle zone. When Cngsc(+) tip of an early bud is grafted into the body column, it induces a secondary axis, while the adjacent Cngsc(-) region has much weaker inductive capacities. Thus, Cngsc is expressed in a tissue that acts as an organizer. Cngsc is also expressed in the sensory neurons of the tip of the hypostome and in the epithelial endodermal cells of the upper part of the body column. The plausible roles of Cngsc in organizer function, head formation and anterior neuron differentiation are similar to roles goosecoid plays in vertebrates and Drosophila. It suggests widespread evolutionary conservation of the function of the gene.

CnOtx, a member of the Otx gene family, has a role in cell movement in hydra.

Otx genes have been identified in a variety of organisms and are commonly associated with the patterning of anterior structures. In some vertebrates, Otx genes are also expressed in the prechordal mesoderm, where they may have a role in cell movement. Here we report the characterization of CnOtx, an Otx gene in hydra, thereby providing evidence that Otx genes appeared early in metazoan evolution. CnOtx is expressed at high levels in developing buds and aggregates, where it appears to have a role in the cell movements that are involved in the formation of new axes. Further, the gene is expressed at a low level throughout the body column of hydra. This latter pattern may reflect a role for CnOtx in specifying tissue as competent to be anterior, although the gene does not have a direct role in the formation of the head.

HyBra1, a Brachyury homologue, acts during head formation in Hydra.

A homologue of the T-box gene, Brachyury, has been isolated from hydra. The gene, termed HyBra1, is expressed in the endoderm and is associated with the formation of the hypostome, the apical part of the head in four different developmental situations. In adults, which are continuously undergoing patterning, HyBra1 is continuously expressed in the hypostome. During budding, hydra's asexual form of reproduction, the gene is expressed in a small area that will eventually form the hypostome of the developing bud before any morphological sign of budding is apparent. The gene is also expressed very early during head regeneration and is confined to the region that will form the hypostome. During embryogenesis, HyBra1 is expressed shortly before hatching in the region that will form the apical end of the animal, the hypostome. The absence of expression at the apical end of decapitated animals of reg-16, a head formation-deficient mutant, provides additional evidence for a role of HyBra1 during head formation. Further, treatments that alter the head activation gradient have no effect on HyBra1 expression indicating the role of the gene is confined to head formation. Transplantation experiments indicate that the expression occurs before head determination has occurred, but expression does not irreversibly commit tissue to forming a head. A comparison of the function of the Brachyury homologues suggests an evolutionary conservation of a molecular mechanism that has been co-opted for a number of developmental processes throughout evolution.

The novel signal peptides, pedibin and Hym-346, lower positional value thereby enhancing foot formation in hydra.

Signaling molecules affecting patterning processes are usually proteins and rarely peptides. Two novel peptides, pedibin and Hym-346, that are closely related to one another have been isolated from Hydra vulgaris and Hydra magnipapillata. Several experiments indicate that both cause a reduction in the positional value gradient, the principle patterning process governing the maintenance of form in the adult hydra. The peptides cause an increase in the rate of foot regeneration following bisection of the body column. Treatment of animals with either peptide for an extended period of time resulted in an apical extension of the range of expression of CnNk-2 along the body column. Such an extension is correlated with a decrease in positional value. Transplantation of tissue treated with Hym-346 results in an increase in the fraction forming feet, and aggregates derived from Hym-346 tissue form more feet and fewer heads. The latter two experiments provide a direct measure of the lowering of positional value in the treated tissue. These results suggest that peptides play signaling roles in patterning processes in cnidaria and, plausibly, in more complex metazoans as well.

Embryogenesis in hydra.

Embryogenesis in hydra includes a variable period of dormancy; and this period, as well as subsequent stages through hatching, takes place within a thick cuticle that hinders observation. Thus, although the early stages of development have been well-characterized qualitatively, the middle and later stages are only poorly understood. Here, we provide a detailed description of the stages of embryogenesis, including the time required to traverse each of the stages, and the changes that occur in the type and number of cells throughout the stages. The events of cleavage and gastrulation occur within the first 48 h. Cleavage is holoblastic and unipolar and leads to a single-layered coeloblastula. Gastrulation occurs by ingression and is followed by the deposition of the thick cuticle. Thereafter, during the variable period of dormancy ranging from 2-24 weeks, little occurs; the important events are the conversion of the outer layer into an ectoderm and the appearance of the interstitial cell lineage. During the last 2 days before hatching, the endoderm and gastric cavity form, while stem cells of the interstitial cell lineage proliferate and differentiate into neurons, nematocytes, and secretory cells. Finally, the cuticle cracks, and the hatchling enlarges and emerges from the cuticle as a functional animal. The formation of the gastric cavity and the hatching of the embryo are both explicable in terms of the osmotic behavior of the animal and the hydrostatic forces generated by this behavior. Characteristics of development that are common to hydra and triploblastic phyla are presented.

Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families.

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of Hydra magnipapillata were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display-PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone, Anthopleura fuscoviridis, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species, Hydractinia serrata. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of Hydractinia serrata. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.

Budhead, a fork head/HNF-3 homologue, is expressed during axis formation and head specification in hydra.

Accumulating evidence indicates that a common set of genes and mechanisms regulates the developmental processes of a variety of triploblastic organisms despite large variation in their body plans. To what extent these same genes and mechanisms are also conserved among diploblasts, which arose earlier in metazoan evolution, is unclear. We have characterized a hydra homologue of the fork head/HNF-3 class of winged-helix proteins, termed budhead, whose expression patterns suggest a role(s) similar to that found in vertebrates. The vertebrate HNF-3 beta homologues are expressed early in embryogenesis in regions that have organizer properties, and later they have several roles, among them an important role in rostral head formation. In the adult hydra, where axial patterning processes are continuously active, budhead is expressed in the upper part of the head, which has organizer properties. It is also expressed during the formation of a new axis as part of the development of a bud, hydra's asexual form of reproduction. Expression during later stages of budding, during head regeneration and the formation of ectopic heads, indicates a role in head formation. It is likely that budhead plays a critical role in head as well as axis formation in hydra.

CnNK-2, an NK-2 homeobox gene, has a role in patterning the basal end of the axis in hydra.

NK-2-class homeobox genes have been identified in a variety of metazoans, from sponges to arthropods and vertebrates, and have been shown to play roles in a variety of cell and tissue specifications. Here we describe the characterization of the NK-2 homolog CnNK-2 from Hydra vulgaris, a freshwater cnidarian. CnNK-2 expression is restricted to the endodermal epithelial cells of hydra and is primarily in the peduncle, the lower end of the body column. In some species it is graded along the apical-basal axis with a maximum in the basal tissue of the lower peduncle, adjacent to the foot. CnNK-2 expression invariably precedes foot formation as part of the normal tissue dynamics of the adult as well as during asexual reproduction by budding, foot regeneration, or ectopic foot formation. Manipulations which alter the gradient of positional value along this axis affect CnNK-2 expression in a manner which indicates that expression of this gene is closely linked to the gradient. The normal and altered patterns of expression of this gene extend the understanding of the regulation of foot formation in hydra.

Patterns of oriented cell division during the steady-state morphogenesis of the body column in hydra.

In an adult hydra, the tissue of the body column is in a dynamic state. The epithelial cells of both layers are constantly in the mitotic cycle. As the tissue expands, it is continuously displaced along the body axis in either an apical or basal direction, but not in a circumferential direction. Using a modified whole mount method we examined the orientation of mitotic spindles to determine what role the direction of cell division plays in axial displacement. Surprisingly, the direction of cell division was found to differ in the two epithelial layers. In the ectoderm it was somewhat biased in an axial direction. In the endoderm it was strongly biased in a circumferential direction. For both layers, the directional biases occurred throughout the length of the body column, with some regional variation in its extent. As buds developed into adults, the bias in each layer increased from an almost random distribution to the distinctly different orientations of the adult. Thus, to maintain the observed axial direction of tissue displacement, rearrangement of the epithelial cells of both layers must occur continuously in the adult as well as in developing animals. How the locomotory and contractile behavior of the muscle processes of the epithelial cells may effect changes in cell shape, and thereby influence the direction of cell division in each layer, is discussed.

Evolutionary conservation of a cell fate specification gene: the Hydra achaete-scute homolog has proneural activity in Drosophila.

Members of the Achaete-scute family of basic helix-loop-helix transcription factors are involved in cell fate specification in vertebrates and invertebrates. We have isolated and characterized a cnidarian achaete-scute homolog, CnASH, from Hydra vulgaris, a representative of an evolutionarily ancient branch of metazoans. There is a single achaete-scute gene in Hydra, and the bHLH domain of the predicted gene product shares a high degree of amino acid sequence similarity with those of vertebrate and Drosophila Achaete-scute proteins. In Hydra, CnASH is expressed in a subset of the interstitial cells as well as differentiation intermediates of the nematocyte pathways. In vitro translated CnASH protein can form heterodimers with the Drosophila bHLH protein Daughterless, and these dimers bind to consensus Achaete-scute DNA binding sites in a sequence-specific manner. Ectopic expression of CnASH in wild-type late third instar Drosophila larvae and early pupae leads to the formation of ectopic sensory organs, mimicking the effect of ectopic expression of the endogenous achaete-scute genes. Expression of CnASH in flies that are achaete and scute double mutants gives partial rescue of the mutant phenotype, comparable to the degree of rescue obtained by ectopic expression of the Drosophila genes. These results indicate that the achaete-scute type of bHLH genes for cell fate specification, as well as their mode of action, arose early and have been conserved during metazoan evolution.

Migrating interstitial cells differentiate into neurons in hydra.

Interstitial cell migration has been observed numerous times in grafted hydra, but the extent to which graft injuries might stimulate the migration of otherwise nonmotile cells was unknown. The present study describes the migration and differentiation of vital dye-labeled interstitial cells in intact, normal hydra. Interstitial cells, stained with a fluorescent vital dye, migrated away from a labeled patch of ectodermal cells and subsequently were found throughout the body column. Shortly thereafter, labeled neurons began to appear among the migrating cells. The number of migrating interstitial cells remained constant over 5 days, whereas the number of labeled neurons increased. Labeled interstitial cells and neurons accumulated primarily in the head and peduncle, as observed in previous studies of short-term migration patterns in grafted hydra. The present study shows that migration of interstitial cells occurs in normal nongrafted hydra and that the accumulation patterns parallel results from graft experiments. The population of migrating cells appears to be limited to neuron precursors.

Nematocyte differentiation in hydra: commitment to nematocyte type occurs at the beginning of the pathway.

In hydra the four types of nematocytes arise by differentiation from the multipotent stem cells among the interstitial cells. It has been unclear where along the nematocyte pathway commitment to type occurs. Some evidence suggests that this commitment occurs at the beginning of the pathway, while other data suggest that it occurs at the terminal cell cycle midway through the pathway. Upon reduction of cell population sizes of the interstitial cell lineage by treatment with hydroxyurea, interstitial cells entering nematocyte pathways frequently undergo an amplification division. A nearest-neighbor analysis of pairs of nematoblast nests in such depleted animals has shown that the fraction of the nearest-neighbor pairs that are matched pairs, in which both nests are of the same type, is higher than predicted. A very high fraction of the matched pairs were identical pairs in which the number of cells in each nest was the same. Also, in a large majority of the identical pairs the nests were shown to be in the same stage of development. The simplest interpretation of these results is that the two daughters of the amplification division giving rise to the matched pair were committed to nematocyte type before the division occurred. In another experiment we show that the length of the G2 phase of the next-to-terminal cell cycle differs between desmonemes and stenoteles. This indicates that differences in the differentiation pathways of these two types exist before the terminal cell cycle. This result also supports the idea that commitment to type occurs at the beginning of the pathway. A means of reconciling the view that commitment occurs early with the view that it occurs late in teh differentiation pathway is discussed.

Expression of Cnox-2, a HOM/HOX gene, is suppressed during head formation in hydra.

Using PCR, six genes of the HOM/HOX class have been identified in hydra. One of them, Cnox-2, has been sequenced and resembles the Deformed gene of Drosophila. As shown previously, the expression pattern of Cnox-2 suggested that it may be involved in axial patterning as it was strongly expressed in the epithelial cells of the body column and foot, but only weakly expressed in the head. In addition, expression of Cnox-2 decreased sharply in body column tissue as it was converted into head tissue, thereby providing further support for a role in hydra pattern formation. To examine this possibility further, additional manipulations were carried out which convert body column tissue into either head or foot tissue. When body column tissue was converted into head tissue by regeneration, transplantation, or during budding, Cnox-2 expression was sharply reduced, indicating that expression of this gene is suppressed during head formation. When body column tissue was converted into foot tissue by regeneration or Li+ treatment, the changes in Cnox-2 expression patterns indicated that Cnox-2 had no direct role in foot formation. Surprisingly, however, the observed changes in Cnox-2 expression patterns indicate that following bisection, both head and foot regeneration processes are initiated at the injured edge of the bisected animal.

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.

Nerve ring of the hypostome in hydra. I. Its structure, development, and maintenance.

The anatomy and developmental dynamics of the nerve ring in the hypostome of Hydra oligactis were examined immunocytochemically with an antiserum against a neuropeptide and with neuron-specific monoclonal antibodies. The nerve ring is unique in the mesh-like nerve net of hydra. It is a distinct neuronal complex consisting of a thick nerve bundle running circumferentially at the border between the hypostome and tentacle zone. Immunostaining showed that the nerve ring was heterogeneous and contained at least four different subsets of neurons. During head regeneration and budding, the nerve ring appeared only after the nerve net of ganglion and sensory cells had formed. Every epithelial cell is continuously displaced with neurons toward either head or foot in an adult hydra. However, the ectoderm in the immediate vicinity of, and including, the nerve ring constitutes a stationary zone that is not displaced. Tissue immediately above this zone is displaced toward the tip of the hypostome, while tissue below is displaced along the tentacles. Correspondingly, the production of new neurons in the ring as measured by their differentiation kinetics is much slower than in surrounding areas. Thus, the nerve ring is static and stable in contrast to the dynamic features of the nerve net of hydra.