Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones.

Oocyte meiotic maturation is crucial for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. By contrast, the few known maturation-inducing hormones (MIHs) that act on oocytes to initiate this process are highly variable in their molecular nature. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions, respectively, we deduced amidated tetrapeptide sequences from gonad transcriptome data and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We show that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light/light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIHs and the neuropeptide hormones that regulate reproduction upstream of MIHs in bilaterian species.

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.

The nerve ring in cnidarians: its presence and structure in hydrozoan medusae.

In our previous studies of the Hydra nerve ring, we proposed the following hypothesis: "The nerve ring in the hypostome of Hydra is a central nervous system (CNS)-like neuronal structure." Related to this hypothesis, we have started to survey the nerve ring immunocytochemically using antibodies against neuropeptides throughout the whole phylum of cnidarians. In the present study, we describe nerve rings in hydrozoan medusae. We examined the medusae of five hydrozoan species belonging to three orders: Eirene sp. (order Leptomedusae), Craspedacusta sowerbyi (order Limnomedusae), Sarsia tubulosa, Turritopsis nutricula, and Cladonema radiatum (order Anthomedusae). We observed a well-developed nerve ring in all species. The nerve ring runs circumferentially around the margin of the bell. In all cases, the nerve ring was visualized by plural antibodies, suggesting that it contains different neural subpopulations. In C. radiatum, antibodies against four different neuropeptides labeled the nerve ring. We established clear (without undesirable cross-reactions) double-staining procedures with two rabbit primary antibodies. Using the double-staining method, three neural subsets visualized by three antibodies revealed completely separate neural populations. The results show that the nerve ring is a common feature in hydrozoan medusae and has a complex heterogeneous structure composed of different neural subsets.

Inferences of evolutionary history of a widely distributed mangrove species, Bruguiera gymnorrhiza, in the Indo-West Pacific region.

Inference of genetic structure and demographic history is fundamental issue in evolutionary biology. We examined the levels and patterns of genetic variation of a widespread mangrove species in the Indo-West Pacific region, Bruguiera gymnorrhiza, using ten nuclear gene regions. Genetic variation of individual populations covering its distribution range was low, but as the entire species it was comparable to other plant species. Genetic differentiation among the investigated populations was high. They could be divided into two genetic clusters: the West and East clusters of the Malay Peninsula. Our results indicated that these two genetic clusters derived from their ancestral population whose effective size of which was much larger compared to the two extant clusters. The point estimate of speciation time between B. gymnorrhiza and Bruguiera sexangula was two times older than that of divergence time between the two clusters. Migration from the West cluster to the East cluster was much higher than the opposite direction but both estimated migration rates were low. The past Sundaland and/or the present Malay Peninsula are likely to prevent gene flow between the West and East clusters and function as a geographical or land barrier.

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.

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family.

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH(2). To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons.

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.

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber.

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.

Nerve ring of the hypostome in hydra: is it an origin of the central nervous system of bilaterian animals?

A hypothesis, 'the nerve ring in hydra shares a common origin with the central nervous system in bilaterian animals', is discussed in this review. The nerve ring of hydra is a ring of neurons whose neurites make a bundle running circumferentially around the hypostome just above the tentacle zone. This nervous structure has unique features in the hydra nervous system. It shows a tight association of neurons in contrast to the diffuse nerve net seen in other regions. It shows static developmental characters in contrast to the dynamic features of hydra nerve net present in other regions. Moreover, its structure and location are similar to the central nervous system (CNS) of other animals without a complex CNS such as nematodes and starfishes. Functions of the hydra nerve ring are also studied to test the hypothesis. The identified function is a crumpling of the tentacles, corresponding to the function of the inner nerve ring of hydrozoan jellyfish. The jellyfish nerve ring is considered to be a primitive central nervous system of radiates. Considering all the information available, the hypothesis is highly possible.

Three digestive movements in Hydra regulated by the diffuse nerve net in the body column.

The mammalian digestive tract undergoes various digestive movements such as peristalsis and segmentation movement. How those digestive movements and the underlying mechanisms appeared in evolution remains unraveled. A widely accepted view has been that, early in evolution, the digestive process was static based upon diffusion, and later it became dynamic involving digestive movements. Here, we report digestive movements which occur in Hydra, a member of the phylum Cnidaria. We find that the body column of Hydra undergoes a series of movements when fed with Artemia. Comparison of the movements to those in mammals showed similarities in appearance to esophageal reflex, segmentation movement, and defecation reflex. When nerve cells were eliminated, polyps showed only a weak segmentation movement, demonstrating that the diffuse nerve net in the body column of Hydra primarily regulates the movements just as the netlike enteric nervous system does in mammals. Elimination of both secretory gland cells and nerve cells resulted in the complete loss of movement, suggesting that the gland cells are involved in the weak movement. Overall, these observations suggest that the digestive process in Hydra is dynamic and that the diffuse nerve net regulates the digestive movements as a primitive form of enteric nervous system.

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.

Morphogenesis of the atrichous isorhiza, a type of nematocyst, in Hydra observed with a monoclonal antibody.

We produced a monoclonal antibody, AE03, which recognized mucous granules in the basal disk gland cells in Hydra and the secreted mucus with which they stick onto substrate. AE03 also recognized atrichous isorhizas, one of the four types of nematocyst present in tentacles, and their nematoblasts present in the body column. With this monoclonal antibody, we could observe the detailed morphogenesis of the atrichous isorhiza from the beginning of its formation. The elongation and invagination processes of external tubes and correspondence between the external tubes and the thread of discharged nematocysts were confirmed.