A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra.

Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.

Subcellular localization of the epitheliopeptide, Hym-301, in Hydra.

Peptides, as signaling molecules, play a number of roles in cell activities. An epitheliopeptide, Hym-301, has been described as a peptide involved in morphogenesis in hydra. However, little is known about the intracellular location of the peptide or its specific functions. To investigate the mechanism of morphogenesis that involves peptidic molecules, we have examined the intracellular localization of Hym-301 in hydra by using immunohistochemical and immunogold electron-microscopic analyses. We have found that the pattern of distribution of mature peptide is slightly different from that of its mRNA, and that the peptide is stored in vesicles located adjacent to the cell membrane. We have also found that the peptide is released both extracellularly and internally to the cytoplasm of the cells. Based upon these observations, we have constructed a possible model mechanism of homeostatic regulation of the distribution of the Hym-301 peptide in a dynamic tissue context.

Neuropeptides trigger oocyte maturation and subsequent spawning in the hydrozoan jellyfish Cytaeis uchidae.

Oocyte maturation and subsequent spawning in hydrozoan jellyfish are generally triggered by light-dark cycles. To examine if the initiation of the maturation process after light stimulus is mediated by neurotransmitters, neuropeptides isolated originally from Hydra magnipapillata were applied to sexually mature female medusae of the hydrozoan jellyfish Cytaeis uchidae. Among the Hydra neuropeptides tested, Hym-53 (NPYPGLW-NH2 ), as well as a nonphysiological peptide, CGLWamide (CGLW-NH2 ), were most effective in inducing oocyte maturation and spawning. Hym-355 (FPQSFLPRG-NH2 ) also triggered these events, but the stimulatory effect was weaker. Since Hym-53-OH (NPYPGLW) and Hym-355-OH (FPQSFLPRG) had no effect, amidation at the C-terminus may be critical for the stimulatory activities of the peptides. Exposure to Hym-53 for 2 min was sufficient to trigger of oocyte maturation, and the spawned eggs were able to be fertilized and to develop normally. Transmission electron microscopy confirmed that bundles of axon-like structures that contain dense-core synaptic vesicles and microtubules are present in the ovarian ectodermal epithelium overlying the oocytes. In addition, immunohistological analyses revealed that some of the neurons in the ectodermal epithelium are GLWamide- and PRGamide-positive. These results suggest that a neuropeptide signal transduction pathway is involved in mediating the induction of oocyte maturation and spawning in this jellyfish.

Microtubules are involved in regulating body length in hydra.

Little is known about how the size of an adult animal is determined and regulated. To investigate this issue in hydra, we altered the body size by surgically removing a part of the body column and/or by axial grafting, and examined changes of column length with time. When the body column was shortened it elongated and resumed the original length within 24-48 h. This increase in the body column length was not accompanied by an increase in the number of epithelial cells in the body column. Instead, each of the epithelial cells elongated longitudinally, leading to elongation of the body column. When the body column surpassed the original length, the column shortened over time. This was not accompanied by a decrease in cell number but by the shortening and thickening of the epithelial cells. TEM analysis showed that formation of microtubule arrays takes place longitudinally along the body axis in elongated cells and perpendicular to the axis in shortened cells. Treatment with a drug that degrades microtubules completely blocked changes in body length. These observations suggest that microtubules are involved in regulating the length of the hydra body column by altering the shape of the epithelial cells. We propose from these observations that hydra has a mechanism for detecting the metrical distance between the two ends of the body column.

Important roles for epithelial cell peptides in hydra development.

It has been convincingly shown that peptides play important roles in the regulation and maintenance of a variety of tissues and organs in living animals. However, little is known concerning the potential role of peptides as signaling molecules in developmental processes. In Hydra, there is circumstantial evidence that small diffusible molecules act as morphogens in the regulation of patterning processes. In order to view the entire spectrum of peptide signaling molecules, we initiated a project aiming at the systematic identification of peptide signaling molecules in Hydra. In this review, we describe three peptide signaling molecules and one family of peptides that function as signaling molecules in the processes of axial pattern formation and neuron differentiation in Hydra. These peptides are produced by epithelial cells and are therefore termed "epitheliopeptides". We discuss the importance of epitheliopeptides in developmental processes within a subset of hydrozoans.

Further characterization of the PW peptide family that inhibits neuron differentiation in Hydra.

From an evolutionary point of view, Hydra has one of the most primitive nervous systems among metazoans. Two different groups of peptides that affect neuron differentiation were identified in a systematic screening of peptide signaling molecules in Hydra. Within the first group of peptides, a neuropeptide, Hym-355, was previously shown to positively regulate neuron differentiation. The second group of peptides encompasses the PW family of peptides that negatively regulate neuron differentiation. In this study, we identified the gene encoding PW peptide preprohormone. Moreover, we made the antibody that specifically recognizes LPW. In situ hybridization and immunohistochemical analyses showed that the PW peptides and the gene encoding them were expressed in ectodermal epithelial cells throughout the body except for the basal disk. The PW peptides are produced by epithelial cells and are therefore termed "epitheliopeptides." Together with Hym-355, the PW family peptides mediate communication between neurons and epithelial cells and thereby maintain a specific density of neurons in Hydra.

Cnidarians and the evolutionary origin of the nervous system.

Cnidarians are widely regarded as one of the first organisms in animal evolution possessing a nervous system. Conventional histological and electrophysiological studies have revealed a considerable degree of complexity of the cnidarian nervous system. Thanks to expressed sequence tags and genome projects and the availability of functional assay systems in cnidarians, this simple nervous system is now genetically accessible and becomes particularly valuable for understanding the origin and evolution of the genetic control mechanisms underlying its development. In the present review, the anatomical and physiological features of the cnidarian nervous system and the interesting parallels in neurodevelopmental mechanisms between Cnidaria and Bilateria are discussed.

Regionalized nervous system in Hydra and the mechanism of its development.

The last common ancestor of Bilateria and Cnidaria is considered to develop a nervous system over 500 million years ago. Despite the long course of evolution, many of the neuron-related genes, which are active in Bilateria, are also found in the cnidarian Hydra. Thus, Hydra is a good model to study the putative primitive nervous system in the last common ancestor that had the great potential to evolve to a more advanced one. Regionalization of the nervous system is one of the advanced features of bilaterian nervous system. Although a regionalized nervous system is already known to be present in Hydra, its developmental mechanisms are poorly understood. In this study we show how it is formed and maintained, focusing on the neuropeptide Hym-176 gene and its paralogs. First, we demonstrate that four axially localized neuron subsets that express different combination of the neuropeptide Hym-176 gene and its paralogs cover almost an entire body, forming a regionalized nervous system in Hydra. Second, we show that positional information governed by the Wnt signaling pathway plays a key role in determining the regional specificity of the neuron subsets as is the case in bilaterians. Finally, we demonstrated two basic mechanisms, regionally restricted new differentiation and phenotypic conversion, both of which are in part conserved in bilaterians, are involved in maintaining boundaries between the neuron subsets. Therefore, this study is the first comprehensive analysis of the anatomy and developmental regulation of the divergently evolved and axially regionalized peptidergic nervous system in Hydra, implicating an ancestral origin of neural regionalization.

Hydra peptide project 1993-2007.

A systematic screening of peptide signaling molecules (<5000 da) in Hydra magnipapillata (the Hydra Peptide Project) was launched in 1993 and at least the first phase of the project ended in 2007. From the project a number of interesting suggestions and results have been obtained. First, a simple metazoan-like Hydra appears to contain a few hundred peptide signaling molecules: half of them neuropeptides and the rest epitheliopeptides that are produced by epithelial cells. Second, epitheliopeptides were identified for the first time in Hydra. Some exhibit morphogen-like activities, which accord with the notion that epithelial cells are primarily responsible for patterning in Hydra. A family of epitheliopeptides was involved in regulating neuron differentiation possibly through neuron-epithelial cell interaction. Third, many novel neuropeptides were identified. Most of them act directly on muscle cells inducing contraction or relaxation. Some were involved in cell differentiation and morphogenesis. During the course of this study, a number of important technical innovations (e.g. genetic manipulations in transgenic Hydra, high-throughput purification techniques, etc.) and expressed sequence tag (EST) and genome databases were introduced in Hydra research. They have already helped to identify and characterize novel peptides and will contribute even more to the Hydra Peptide Project in the near future.

A novel neuropeptide (FRamide) family identified by a peptidomic approach in Hydra magnipapillata.

In the course of systematic identification of peptide signaling molecules combined with the expressed sequence tag database from Hydra, we have identified a novel neuropeptide family that consists of two members with FRamide at the C-terminus; FRamide-1 (IPTGTLIFRamide) and FRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained a single copy each of FRamide-1 and FRamide-2 precursor sequences. Expression analysis by whole-mount in situ hybridization showed that the gene was expressed in a subpopulation of neurons that were distributed throughout the body from tentacles to basal disk. Double in situ hybridization analysis showed that the expressing cell population was further subdivided into one population consisting of neurons expressing both the FRamide and Hym176 (neuropeptide) genes and the other consisting of neurons expressing only the FRamide gene. FRamide-1 evoked elongation of the body column of 'epithelial' Hydra that was composed of epithelial cells and gland cells but lacked all the cells in the interstitial stem cell lineage, including neurons. In contrast, FRamide-2 evoked body column contraction. These results suggest that both of the neuropeptides directly act on epithelial cells as neurotransmitters and regulate body movement in an axial direction.

Degeneration after sexual differentiation in hydra and its relevance to the evolution of aging.

Aging occurs in most multicellular animals, yet some primitive animals do not show any sign of aging. This raises the following question: How have metazoans acquired the trait of aging in the course of evolution? Comparative studies of various species have provided a clue to this question by showing that sexually reproducing organisms predominantly undergo aging. The evolutionary theory "pleiotropy" also postulates aging as a price for facilitating the reproduction in the early life stage of an organism. For investigating the association between sexual reproduction and aging, a sexual phase-inducible organism in a laboratory would be suitable. One of such organisms is hydra, a genus of Cnidaria. Asexual hydra has been considered to be immortal, but there is the possibility that hydra undergoes aging after sexual reproduction. To search for signs of aging in hydra, we studied sexually differentiated Hydra oligactis at the individual and cellular levels. As a result, we found a significant decline in the capacities for food capture, contractile movements, and reproduction. More importantly, we discovered an exponential increase in the mortality rate of the population. These observations suggest that the degenerative process in H. oligactis represents the aging process. Furthermore, we found that the number of germ cells increased, whereas the number of somatic cells concomitantly decreased. The observed change of the cell composition is thus consistent with the "pleiotropy" theory of aging.

Motility of endodermal epithelial cells plays a major role in reorganizing the two epithelial layers in Hydra.

Cell-cell interactions and cell rearrangements play important roles during development. Aggregates of Hydra cells reorganize into the two epithelial layers and subsequently form a normal animal. Examination of the formation of the two layers under various situations, indicates that the motility of endodermal epithelial cells, but not the differential adhesive forces of the two types of epithelial cells, plays the critical role in setting up the two epithelial layers. (1) When aggregates of ectodermal cells and of endodermal cells were placed in direct contact, the endodermal cells migrated into the interior of the ectodermal aggregate. This process was completely inhibited by cytochalasin B although initial firm attachment between the two aggregates was not blocked. (2) A single endodermal epithelial cell placed in contact with an ectodermal aggregate, actively extended pseudopod-like structures and migrated toward the center of the ectodermal aggregate. In contrast, an ectodermal epithelial cell remained in contact with an endodermal aggregate and never exhibited migratory behavior. Cytochalasin treatment of only endodermal epithelial cells abolished the migration. (3) One to 4 endodermal epithelial cells and/or ectodermal epithelial cells were placed in contact with one another forming up to 4-cell aggregates. Endodermal epithelial cells exhibited high motility that can be attributed to the migratory movement described above. Finally, formation of actin bundles, as visualized with rhodamine-phalloidin, was always correlated with pseudopod formation in endodermal epithelial cells during early and mid stages of aggregate formation.

GENETIC ANALYSIS OF DEVELOPMENTAL MECHANISMS IN HYDRA I. SEXUAL REPRODUCTION OF HYDRA MAGNIPAPILLATA AND ISOLATION OF MUTANTS.

Hydra magnipapillata strains collected from various localities in Japan were induced to reproduce sexually. From the survival data of the progeny, it was calculated that H. magnipapillata contained an average of between 3.5 and 4.0 lethal equivalent units of recessive deleterious genes per gamate (between 7.0 and 8.0 per animal). Various types of developmental mutants were found among the offspring of crosses made between strains isolated from the same ponds. The mutant types isolated included mini strains, maxi strains, multi-headed strains, nematocyst-deficient strains, regeneration-deficient strains and male sterile strains. The characters of these strains were stably transmissible to the successive progeny produced by budding. These strains therefore were propagated by budding and maintained as clonal lines to be used later for developmental studies.

GENETIC ANALYSIS OF DEVELOPMENTAL MECHANISMS IN HYDRA VII. STATISTICAL ANALYSES OF DEVELOPMENTAL-MORPHOLOGICAL CHARACTERS AND CELLULAR COMPOSITIONS.

Various strains belonging to Hydra magnipapillata are examined for their developmental-morphological characters and relative abundance of the six basic types of cells, and the results are statistically analysed. Significant correlations are found between various (including seeming unrelated) characters. For example, budding rate, bud developmental rate and polyp size, which in theory can be all regulated by independent mechanisms, show strong correlations with each other. This suggests that the underlying mechanisms regulating these characters must be closely related to each other. Similar significant coerelations are also found between the relative abundance of various cell types, but not between the developmental-morphological characters and the cellular composition. The significance of these findings are discussed.

Genetic Analysis of Developmental Mechanisms in Hydra. XV. Nematocyte Differentiation in Strains with Altered Developmental Gradients*: (Hydra/nematocyst/nematocyte/interstitial cell/chimera).

Nematocyte differentiation from the interstitial stem cells in hydra occurs non-uniformly along the body column. The relative ratios of the 4 nematocyte types produced vary gradually from head to foot along the body axis (Bode and Smith, 1977). To find out whether this regional variation in nematocyte differentiation along the body column is related to the gradients of the head-activation and head-inhibition potentials, nematocyte differentiation patterns were examined in strains which have significantly different developmental gradients along their body columns. Five strains of hydra, including a wild-type, two mutant strains and two chimeric (mutnt/wild-type) strains, were investigated. It was found that the regional variations in the nematocyte differentiation were similar in all the strains examined, and that no significant differences of the variation existed that could be attributed to the differences of the developmental gradients in these strains. This suggests that nematocyte differentiation is strongly affected by the axial position along the body column, but that the gradients of the morphogenetic potentials involved in head formation are not involved in this effect. Instead, some other parameter(s) of axial position not directly associated with these gradients must be responsible for the positional effect on nematocyte differentiation.

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps.

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.

Sequential actions of ß-catenin and Bmp pattern the oral nerve net in Nematostella vectensis.

Animal evolution is closely linked to the emergence of the nervous system. At present it is unknown how the basic mechanisms of neural induction and formation of central nervous systems evolved. We addressed this question in Nematostella vectensis, a member of cnidarians, the ancient sister group of bilaterians. We found that ß-catenin signalling is crucial for the early induction of the embryonic nervous system. ß-Catenin activity at the blastopore induces specific neurogenic genes required for development of the oral nervous system. ß-Catenin signalling induces also Bmp signalling, which, at later larval stages, becomes indispensible for the maintenance and asymmetric patterning of the oral nervous system along the primary and secondary (directive) axes. We hypothesize that the consecutive and functionally linked involvement of ß-catenin and Bmp signalling in the formation of the cnidarian oral nervous system reflects an ancestral mechanism that evolved before the cnidarian/bilaterian split.

Peptide signaling in Hydra.

Peptides play a number of crucial roles as signaling molecules in metazoans. In order to elaborate a more complete picture of the roles played by peptides in a single organism, we launched the "Hydra Peptide Project". For this project, we used Hydra magnipapillata, a species belonging to Cnidaria, one of the most basal metazoan phyla, and using a peptidomic approach, we systematically identified a number of peptide signaling molecules, their encoding genes and their functions. In this article, we report the peptides isolated from Hydra and other cnidarians, as well as their synthesis, processing and release from the cells to the target. Possible peptide signaling pathways are overviewed and finally we discuss the evolution of the peptide signaling system.