Toshitaka Fujisawa

Analysis of a piwi-Related Gene Implicates Small RNAs in Genome Rearrangement in Tetrahymena

During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation. These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression. We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.

Universal occurrence of the vasa-related genes among metazoans and their germline expression in Hydra

Abstract. The vasa (vas)-related genes are members of the DEAD box protein family and are involved in germ cell formation in higher metazoans. In the present study, we cloned the vas-related genes as well as the PL10-related genes, other members of the DEAD box protein family, from lower metazoans: sponge, Hydra and planaria. The phylogenetic analysis suggested that the vas-related genes arose by duplication of a PL10-related gene before the appearance of sponges but after the diversion of fungi and plants. The vas-related genes in Hydra, Cnvas1 and Cnvas2 were strongly expressed in germline cells and less strongly expressed in multipotent interstitial stem cells and ectodermal epithelial cells. These results suggest that the vas-related genes occur universally among metazoans and that their expression in germline cells was established at least before cnidarian evolution.

Expression and evolutionary conservation of nanos-related genes in Hydra

Abstractu2002The Drosophila gene nanos encodes two particular zinc finger motifs which are also found in germline-associated factors from nematodes to vertebrates. We cloned two nanos (nos)-related genes, Cnnos1 and Cnnos2 from Hydra magnipapillata. Using whole-mount in situ hybridization, the expression of Cnnos1 and Cnnos2 was examined. Cnnos1 was specifically expressed in multipotent stem cells and germline cells, but not in somatic cells. Cnnos2 was weakly expressed in germline cells and more specifically in the endoderm of the hypostome where it appears to be involved in head morphogenesis. In addition to structural conservation in the zinc finger domain of nanos-related genes, functional conservation of Cnnos1 was also demonstrated by the finding that a Cnnos1 transgene can partially rescue the nosRC phenotype that is defective in the egg production of Drosophila. Thus, the function of nanos-related genes in the germline appears to be well conserved from primitive to highly evolved metazoans.

A cnidarian neuropeptide of the GLWamide family induces metamorphosis of reef-building corals in the genus Acropora

Abstract. Coral larvae appear to sense appropriate environments for settlement and start metamorphosis by converting external cues into internal signals, although little is known about these molecular mechanisms. A family of neuropeptides, GLWamides, are thought to be such internal signals, acting hormonally to induce metamorphosis in some hydrozoan species. Here we report that one member of the GLWamide peptide family, Hym-248, can induce metamorphosis of planula larvae in the genus Acropora. The Acropora planulae responded to the peptide in a concentration-dependent manner. The GLWamide peptide would mimic endogenous molecules to start metamorphosis in Acropora as in case of hydrozoans. In addition, the peptide could be applied to produce coral seedlings with the aim of reef restoration.

Hydra regeneration and epitheliopeptides

Hydra has been well known for over 200 years for its remarkable regenerative capacity. In addition to small pieces excised from the body, reaggregates of dissociated single cells can also regenerate. Although the cellular events involved in the regeneration process have been well characterized, the underlying molecular mechanisms are yet to be uncovered. Recently, however, transcription factors and signaling molecules, both proteins and short peptides, have been identified and their role suggested in patterning and morphogenesis. In this article, a regeneration study at the tissue level is first described and then the importance of epithelial cells in regeneration is stressed. Finally, the recent study on morphogenetic peptides derived from epithelial cells is reviewed. Developmental Dynamics 226:182–189, 2003.

Identification of an astacin matrix metalloprotease as target gene for Hydra foot activator peptides

Abstractu2002Peptides serve as important signaling molecules in development and differentiation in Hydra. Two peptides, Hym-346 and pedibin, have recently been identified to act as morphogenetic signals for foot differentiation. In screening for target genes for Hym-346 we have isolated an astacin matrix metalloprotease, termed foot activator responsive matrix metalloprotease (Farm1). Farm1 is normally expressed in epithelial cells of the gastric region and absent in apical and basal tissue. Incubation of polyps in peptides Hym-346/pedibin causes immediate downregulation of Farm1 expression. A structurally unrelated peptide, Hym-323, which also enhances foot formation in Hydra, also downregulates Farm1 expression. Treatment of polyps with the ectopic feet-inducing agent LiCl also resulted in decreased level of Farm1 transcripts. Thus metalloproteinase Farm1 is a transcriptional target of positional signals specifying foot differentiation and appears to play a potent role in basal patterning processes.

Selective protein kinase inhibitors block head-specific differentiation in hydra

Several studies have suggested that morphogenesis and patterning in hydra are regulated through pathways involving protein kinase C (PKC). Nevertheless, the complete signal system for regeneration in hydra is still not completely understood. Using inhibitors of different signalling pathways we are dissecting this system. We found that sphingosine (2 microM), staurosporine (0.1 microM), PP1/AGL1872 (1 microM) and H7 (25 microM) were able to inhibit head but not foot regeneration. The inhibition was reversible. When the inhibitor was replaced with hydra medium the animals continue their regeneration in a normal way. The exception was PP1/AGL1872, in this case the animals regenerated only one or two tentacles. These results imply that head and foot regeneration are independent processes and they are not directly related as has been proposed. Sphingosine and PP1/AGL1872 inhibit the transcription of ks1, an early regeneration gene, at 24 and 48 h of treatment. Sphingosine 2 microM arrested the cells on the G1 phase of the cell cycle, but 1 microM of PP1/AGL1872 did not. The regeneration was not affected if the animals were exposed to inhibitors of human growth factor receptors. We propose that head regeneration in hydra may be regulated at least by two pathways, one going through PKC and the other through Src. The first pathway could be related to cellular proliferation and the second one to cellular differentiation.

Identification of a vasopressin-like immunoreactive substance in hydra

Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells.