Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.     

Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization

The freshwater polyp Hydra magnipapillata has a primitive nervous system that produces at least three distinct classes of neuropeptides: various peptides having the C-terminal sequence Arg-Phe-NH2 (the Hydra-RFamide family), Leu-Trp-NH2 (the Hydra-LWamide family), and a single peptide having the C-terminal sequence Lys-Val-NH2 (Hydra-KVamide). The various Hydra-RFamides are synthesized by three different preprohormones: preprohormone-A, -B, and -C. The various Hydra-LWamides are synthesized by a single preprohormone (prepro-Hydra-LWamide), as is Hydra-KVamide (prepro-Hydra-KVamide). Using a wholemount double-labeling two-color in situ hybridization technique and RNA probes specific for each of these five Hydra preprohormone mRNAs, we found that specific sets of neurons express each of the five preprohormones, except for the peduncle region of Hydra (an area just above the basal disk), where a population of neurons exists that expresses both preprohormones-A and preproHydra-KVamide mRNAs. The functional significance of this coexpression is unclear. This is the first report on the coexpression of two well-characterized preprohormones (yielding two well-characterized neurohormone families) in cnidarians. This report also shows that there are at least six neurochemically different populations of neurons in Hydra.     

Molecular Cloning of a Preprohormone from Hydra magnipapillata Containing Multiple Copies of Hydra-LWamide (Leu-Trp-NH2) Neuropeptides: Evidence for Processing at Ser and Asn Residues

Abstract: The simple, freshwater polyp Hydra is often used as a model to study development in cnidarians. Recently, a neuropeptide, 2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a polyp. Here, we have cloned a preprohormone from Hydra magnipapillata containing 11 (eight different) immature neuropeptide sequences that are structurally related to the metamorphosis-inducing neuropeptide from sea anemones. During the final phase of our cloning experiments, another research team independently isolated and sequenced five of the neuropeptides originally found on the preprohormone. Comparison of these mature neuropeptide structures with the immature neuropeptide sequences on the preprohormone shows that most immature neuropeptide sequences are preceded by Ser or Asn residues, indicating that these residues must be novel processing sites. Thus, the structure of the Hydra prepro-hormone confirms our earlier findings that cnidarian pre-prohormones contain unusual or novel processing sites. Nearly all neuropeptide copies located on the Hydra preprohormone will give rise to mature neuropeptides with a C-terminal Gly-Leu-Trp-NH₂ sequence (the most frequent one being Gly-Pro-Pro-Pro-Gly-Leu-Trp-NH₂; Hydra-LWamide I; three copies). Based on their structural similarities with the metamorphosis-inducing neuropeptide from sea anemones, the mature peptides derived from the Hydra-LWamide preprohormone are potential candidates for being developmentally active neurohormones in Hydra .     

Further characterization of the PW peptide family that inhibits neuron differentiation in <Emphasis Type="Italic">Hydra</Emphasis>

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Toshio Takahashi, Osamu Koizumi equally contributed to this study.     

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.     

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.     

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.     

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.     

Hym-301, a novel peptide, regulates the number of tentacles formed in hydra

Hym-301 is a peptide that was discovered as part of a project aimed at isolating novel peptides from hydra. We have isolated and characterized the gene Hym-301, which encodes this peptide. In an adult, the gene is expressed in the ectoderm of the tentacle zone and hypostome, but not in the tentacles. It is also expressed in the developing head during bud formation and head regeneration. Treatment of regenerating heads with the peptide resulted in an increase in the number of tentacles formed, while treatment with Hym-301 dsRNA resulted in a reduction of tentacles formed as the head developed during bud formation or head regeneration. The expression patterns plus these manipulations indicate the gene has a role in tentacle formation. Furthermore, treatment of epithelial animals indicates the gene directly affects the epithelial cells that form the tentacles. Raising the head activation gradient, a morphogenetic gradient that controls axial patterning in hydra, throughout the body column results in extending the range of Hym-301 expression down the body column. This indicates the range of expression of the gene appears to be controlled by this gradient. Thus, Hym-301 is involved in axial patterning in hydra, and specifically in the regulation of the number of tentacles formed.     

Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps

Summary Antisera to the sequence Arg-Phe-amide (RF-amide) have a high affinity to the nervous system of fixed hydroid polyps. Whole-mount incubations of several Hydra species with RFamide antisera visualize the three-dimensional structure of an ectodermal nervous system in the hypostome, tentacles, gastric region and peduncle. In the hypostome of Hydra attenuata a ganglion-like structure occurs, consisting of numerous sensory cells located in a region around the mouth opening and a dense plexus of processes which project mostly radially towards the bases of the tentacles. In Hydra oligactis an ectodermal nerve ring was observed lying at the border of hypostome and tentacle bases. This nerve ring consists of a few large ganglion cells with thick processes forming a circle around the hypostome. This is the first direct demonstration of a nerve ring in a hydroid polyp.Incubation of Hydractinia echinata gastrozooids with RFamide antisera visualizes an extremly dense plexus of neuronal processes in body and head regions. A ring of sensory cells around the mouth opening is the first group of neurons to show RFamide immunoreactivity during the development of a primary polyp. In gonozooids the oocytes and spermatophores are covered with strongly immunoreactive neurons.All examples of whole-mount incubations with RF-amide antisera clearly show that hydroid polyps have by no means a diffuse nerve net, as is often believed, and that neuronal centralization and plexus formation are common in these animals. The examples also show that treatment of intact fixed animals with RFamide antisera is a useful technique to study the anatomy or development of a principal portion of the hydroid nervous system.     

Enhancement of foot formation in Hydra by a novel epitheliopeptide, Hym-323

During the course of a systematic screening of peptide signaling molecules in Hydra magnipapillata, a novel peptide, Hym-323, which enhances foot regeneration was identified. The peptide is 16 amino acids long, and is encoded in the precursor protein as a single copy. Northern blot analysis, in situ hybridization analysis and immunohistochemistry showed that it was expressed in both ectodermal and endodermal epithelial cells throughout the body, except for the basal disk and the head region. The peptide enhanced foot regeneration by acting on epithelial cells. Lateral transplantation experiments indicated that the foot activation potential was increased in the peptide-treated tissue. These results suggest that Hym-323 is a peptide involved in a foot-patterning process in Hydra.     

The role of alpha-amidated neuropeptides in hydroid development--LWamides and metamorphosis in Hydractinia echinata

Peptides are increasingly attracting attention as primary signals in the control of development. Even though a large number of peptides have been characterized in cnidarians, little experimental evidence addresses their endogenous role. The life cycle of Hydractinia echinata includes metamorphosis from planula larva into the adult stage of the polyp. This process of stage conversion includes internal signalling, which controls cell cycle activity, cell differentiation, cell death and proportion-controlled morphogenesis. LWamide peptides are considered to be part of the control system. We implemented methods to silence gene activity by dsRNAi in Hydractinia and show a substantial knock-down of LWamide gene activity. In addition, LWamide function was knocked-out pharmacologically by targeting the biosynthesis of amidated peptides and thus preventing functional LWamides. Here we show that extinction of bioactive LWamides from planulae causes loss of metamorphosis competence, a deficiency which can be rescued by synthetic LWamide peptides. Thus, it is shown that LWamides are indispensable and act by conveying outer metamorphosis stimuli to target cells within the animal. Considering non-availability of genetic analysis and the so-far limited success in expressing transgenes in hydroids, gene functions are difficult to analyse in hydroids. The approach as outlined here is suitable for functional analysis of genes encoding amidated peptides in hydroids.     

The Drosophila DSP1 gene encoding an HMG 1-like protein: genomic organization,evolutionary conservation and expression

The gene that encodes the dorsal switch protein (DSP1) has been isolated from a Drosophila melanogaster cosmid library. It is organized into seven exons and six introns. The relative position of the introns within the region coding for the high mobility group (HMG) domains are identical to those of vertebrate HMG 1/2 genes. The close similarity between DSP1 and HMG 1/2 genes strongly suggests that these genes derived from a common ancestral gene. DSP1 encodes, at least, two distinct mRNAs that differ in the length of their 5′-untranslated region and coding sequence. Detailed sequence analysis shows that alternative splicing of precursor mRNA gives rise to the two isoform mRNAs found in Drosophila cells.     

p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas

To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.     

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.     

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 183–197, 1998