Dev Genes Evol (2001) 211:299–308 DOI 10.1007/s004270100156
O R I G I N A L A RT I C L E
Kazufumi Mochizuki · Chiemi Nishimiya-Fujisawa Toshitaka Fujisawa
Universal occurrence of the vasa-related genes among metazoans and their germline expression in Hydra Received: 24 November 2000 / Accepted: 19 January 2001 / Published online: 5 May 2001 © Springer-Verlag 2001
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 vasrelated 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. Keywords Vasa-related genes · Sponge · Hydra · Planaria · Germline expression
Introduction The Drosophila protein Vasa (Vas) is a putative ATPdependent RNA helicase which belongs to the DEAD box protein family (Hay et al. 1988; Lasko and Ashburner 1988). The DEAD box proteins share eight characterEdited by N. Satoh The accession numbers of the genes reported in this paper are: Cnvas1 (AB047382), Cnvas2 (AB047383), CnPL10 (AB047381), Povas1 (AB047385), PoPL10 (AB047384), Plvas1 (AB047388) K. Mochizuki · T. Fujisawa (✉) Department of Genetics, The Graduate University for Advanced Studies, Mishima 411–8540, Japan e-mail:
[email protected] K. Mochizuki · C. Nishimiya-Fujisawa · T. Fujisawa Department of Developmental Genetics, National Institute of Genetics, Mishima 411–8540, Japan
istic sequence motifs, including the DEAD (Asp-GluAla-Asp) box (Linder et al. 1989). The DEAD box proteins are present in a wide range of organisms and are involved in splicing processes, RNA editing, rRNA processing, translational initiation, nuclear mRNA export and mRNA degradation (Luking et al. 1998). Among the DEAD box proteins, the Vas- and PL10related proteins are very similar to each other. The PL10related proteins occur and their amino acid sequences are highly conserved in a diverse range of eukaryotes from yeast (Chuang et al. 1997; Kawamukai 1999) to plants (Lin et al. 1999) and animals (Leroy et al. 1989; Gururajan et al 1991; Gee and Conboy 1994; Sowden et al. 1995; Olsen et al. 1997; Fujimura and Takamura 2000). The DED1 gene, one of the PL10-related genes in yeast, may be required for translational initiation of almost all mRNA, and the mouse PL10 can rescue the phenotype of a yeast mutant with a chromosomal ded1 deletion (Chuang et al. 1997). This result indicates that DED1 and PL10 are functional homologues and the functions of the PL10-related genes may be conserved among eukaryotes. Although PL10 is expressed during spermatogenesis in mice (Leroy et al. 1989), other PL10-related genes, such as DEAD2 and DEAD3 (also called ERH) in mice, An3 in frogs and PL10a in zebrafish, are expressed in a variety of tissues (Gururajan et al. 1991; Gee and Conboy 1994; Sowden et al. 1995; Olsen et al. 1997). Thus, the PL10-related genes may have wider functions. In contrast, the vas-related genes have only been reported in metazoans such as vertebrates, insects, and a single prochordate and nematode (Hay et al. 1988; Lasko and Ashburner 1988; Roussell and Bennett 1993; Fujiwara et al. 1994; Komiya et al. 1994; Komiya and Tanigawa 1995; Gruidl et al. 1996; Olsen et al. 1997; Yoon et al. 1997; Nakao 1999; Castrillon et al. 2000; Fujimura and Takamura 2000; Kuznicki et al. 2000; Shinomiya et al. 2000; Tsunekawa et al. 2000; Yoshizaki et al. 2000). The mRNA or the products of the vas-related genes have been shown to be localized in germline cells in all animals examined to date. In some cases, for example, vasa in Drosophila, glh-1, glh-2 and glh-4 in
300 using the Dayhoff PAM matrix (Dayhoff 1979) and trees were produced by the neighbor joining (NJ) method (Saitou and Nei 1987). Gaps in the amino acid alignment were excluded from the calculations. For the bootstrap analysis, 1,000 data sets were produced by bootstrap resampling (Felsenstein 1985) from the alignments and the consensus tree was obtained by the majority-rule consensus method (Margush and McMorris 1981). These calculations were performed with the PHYLIP software version 3.5c (Felsenstein 1993).
Caenorhabditis elegans, and XVLG-1 protein in Xenopus have been shown to be involved in the formation and/or maintenance of the germline cells (Lasko and Ashburner 1988; Ikenishi and Tanaka 1997; Kuznicki et al. 2000). The sequence similarity of the Vas- and PL10-related proteins and the restricted existence of the vas-related genes to metazoans appear to suggest that an ancestor of the vas-related genes was derived from an ancient PL10related gene and thereafter acquired the specificity in germline cells. However, it is not known whether the vas-related genes occur and are involved in germline formation in lower metazoans. In order to gain insight into early events in the evolution of the vas-related genes, we examined whether the vas-related genes are present in lower metazoans. As a result, we obtained both vas- and PL10-related genes from sponge, Hydra and planaria. The evolutionary relationship of the vas- and PL10-related genes and the detailed analysis of their expression in Hydra are reported.
In situ hybridization (ISH) on whole mounts of Hydra was carried out as described previously (Grens et al. 1995; Mochizuki et al. 2000). Northern blot analysis was carried out by a standard method (Sambrook et al. 1989). The cRNA probes for ISH and cDNA probes for northern blot analysis corresponded to the following sequence of each cDNA: Cnvas1 (2,248b–2,582b), Cnvas2 (1b–842b), CnPL10 (1b–826b), Cnnos1 (a cnidarian nanos homologue 1; Mochizuki et al. 2000; 1b–696b), CnASH (an achaetescute homologue; Grens et al. 1995; 50b–815b), CnDmc1 (a meiosis-specific recombinase homologue; Bishop et al. 1992; 1b–631b; accession no.: AB047582) and GAPDH (1b–245b; accession no.: AB044096).
Materials and methods
Axial grafting
Animals All Hydra used in the present study belong to the species H. magnipapillata (Cnidaria, Hydrozoa). Five types of animals derived from strain nem-1 were used: normal male, normal female, pseudo-epithelial male, pseudo-epithelial female and epithelial Hydra. Pseudo-epithelial Hydra consists of epithelial cells and germline cells, while epithelial Hydra consists of only epithelial cells (Sugiyama and Fujisawa 1978; Nishimiya-Fujisawa and Sugiyama 1993, 1995). For double in situ hybridization, normal polyps of strain 105 were used. Occasional sex conversion occurs in this strain. Culturing of Hydra and induction of sexual differentiation were performed as described previously (Sugiyama and Fujisawa 1977; Nishimiya-Fujisawa and Sugiyama 1993). A freshwater planarian, Dugesia dorotocephala (Platyhelminthes, Turbellaria), was cultured in Hydra culture solution at 18°C and fed with Artemia daily. The animals were starved for 1 week before RNA extraction. Isolation of vasa /PL10 class genes RT-PCR was performed as described previously (Mochizuki et al. 2000). PCR was carried out with 37 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 60 s. For PCR, the following degenerated primers were designed from the amino acid sequences (in brackets) of two different regions within the well-conserved domains of the Vas/PL10 class proteins: vasFW(MACAQTG), CCGGATCCATGGC(ACGT)TG(CT)GC(ACGT)CA(AG)AC(ACGT)G; vasRV(DRMLDMGF), CCAAGCTTAA(ACGT)CCCAT(AG)TC(ACGT)A(AG)CAT(ACGT)C(GT)(AG)TC, where restriction sites for BamHI and HindIII are underlined. Sequences of the 5′and 3′-regions of the cDNAs were obtained by a modified RACE method (Mochizuki et al. 2000). Total RNA from a freshwater sponge, Ephydatia fluviatilis (Porifera, Demospongiae) was a kind gift of Drs. T. Iwabe and T. Miyata (Kyoto University). Phylogenetic analyses Multiple amino acid alignments were performed with the CLUSTALW software package version 1.6 (Thompson et al. 1994) and edited manually. Genetic distance measurements were obtained
Expression analyses of mRNA
An apical half of the epithelial Hydra and a basal half of the normal male Hydra were grafted as described previously (Sugiyama and Fujisawa 1978). Twenty-four hours after grafting, the animals were fixed and used for whole-mount ISH.
Results Lower metazoans also have vas-related genes By RT-PCR and RACE methods, we obtained two vas/PL10-related genes from the sponge, E. fluviatilis (Povas1 and PoPL10) and three from H. magnipapillata (Cnvas1, Cnvas2 and CnPL10). We also obtained by RTPCR three cDNA fragments from the planaria, D. dorotocephala. The deduced amino acid sequence of one of them showed 72% identity with that of the vas-like gene of D. japonica, DjvlgA, and the second one showed 94% identity with DjvlgB (Shibata et al. 1999). Thus, they were respectively considered to be the homologues of DjvlgA and DjvlgB, and were omitted from further analysis. The third cDNA fragment, Plvas1, was a novel gene. Figure 1A shows the amino acid alignment of the conserved RNA helicase domain (Fig. 1B) of these and other Vas/PL10-related proteins. All of them share eight characteristic sequence motifs (Fig. 1A, underlined). The Vas/PL10-related proteins are schematically shown in Fig. 1B. In the helicase domain (shown in green), the identities of PoPL10, CnPL10, DjVLGA and DjVLGB are significantly higher when compared with mouse PL10 than with Vas (Fig. 1B). This strongly suggests that they are the PL10-related proteins. The phylogenetic tree constructed by using amino acid sequences of the helicase domain of various Vas/PL10-related proteins (Fig. 1C) also supported this view. On the other hand, CnVAS1, CnVAS2, PoVAS1 and PlVAS1 were classified
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Fig. 1A–C Comparison of the deduced amino acid sequences of the Vas- and PL10-related proteins. A Alignment of the RNA helicase domains of the Vas- and PL10-related proteins. Eight conserved motifs are underlined. The sequences used to design the PCR primers are marked with arrows. B Schematic comparison of the Vasand PL10-related proteins. C Phylogenetic analysis of the Vas- and PL10-related proteins. The phylogenetic trees were constructed by the Neighbor Joining method using the sequences presented in A.
The Bootstrap probabilities (%) are shown on each branch. Protein IDs in DDBJ/EMBL/GenBank are in brackets. At Arabidopsis thaliana, Bm Bombyx mori, Ce Caenorhabditis elegans, Ci Ciona intestinalis, Dd, Dugesia dorotocephala, Dj Dugesia japonica, Dm Drosophila melanogaster, Dr Danio rerio, Ef Ephydatia fluviatilis, Gg Gallus gallus, Hm Hydra magnipapillata, Mm Mus musculus, Om Oncorhynchus mykiss, Rn Rattus norvegicus, Sc Saccharomyces cerevisiae, Sp Schizosaccharomyces pombe, Xl Xenopus laevis
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as Vas-related proteins by the phylogenetic analysis with a bootstrap probability of 81% (Fig. 1C), although their identities to Vas and PL10 were similar (Fig. 1B). Interestingly, CnVAS1 and CnVAS2, respectively, had seven and one retrovirus type zinc finger motifs (CCHC motif) in their N-terminal regions (Fig. 1B). Vas-related proteins containing the CCHC motif are known only in a nematode, C. elegans (GLH-1, GLH-2, GLH-3 and GLH-4; Roussell and Bennett 1993; Gruidl et al. 1996; Kuznicki et al. 2000), and an ascidian, Ciona intestinalis (Ci-DEAD1; Fujimura and Takamura 2000). The CCHC motif found in the gag proteins of retroviruses (Rein 1994) and CNBP in vertebrates (Rajavashisth et al. 1989; Yasuda et al. 1995) are considered to bind to nucleic acids. By contrast, PoVAS1 had RGG motifs, which are frequently found in the N-terminal regions of Vas-related and PL10-related proteins (Fig. 1B). The RGG motif in some proteins binds to RNA (Kiledjian and Dreyfuss 1992). No characteristic motifs have yet been found in the N-terminal region of PlVAS1. Despite the high identity values among all PL10-related proteins in animals, fungi and Arabidopsis, the animal PL10-related proteins clustered themselves and exhibited a close association with the Vas-related proteins with a bootstrap probability of 62% (Fig. 1C). This suggests that the vas-related genes arose by duplication of an ancestral PL10-related gene before the appearance of sponges but after the diversion of fungi and plants. CnVAS1 and CnVAS2 from Hydra show greater homology to each other than to any other vas-related protein. In addition, we also obtained the cDNA fragments of Cnvas1 and Cnvas2 from a jellyfish, Aurelia aurita (data not shown). These data suggest that Cnvas1 and Cnvas2 were probably duplicated during cnidarian evolution. The branching order of the Vas-related proteins of sponge, Hydra and planaria does not reflect the evolutionary order. Furthermore, the occurrence of the CCHC or RGG motifs in the Vas-related proteins does not correlate with the branching order. These discrepancies may be attributable either to the difference in the rate of amino acid changes in different organisms or to the possibility that some of these genes may be paralogues. Hydra Cnvas1 and Cnvas2 are expressed in multipotent stem cells and germline cells, but not in somatic cells of the interstitial cell lineage All the vas-related genes so far reported are expressed in germline cells (Hay et al. 1988; Lasko and Ashburner 1988; Roussell and Bennett 1993; Fujiwara et al. 1994; Komiya et al. 1994; Komiya and Tanigawa 1995; Gruidl et al. 1996; Olsen et al. 1997; Yoon et al. 1997; Nakao 1999; Castrillon et al. 2000; Fujimura and Takamura 2000; Kuznicki et al. 2000; Shinomiya et al. 2000; Tsunekawa et al. 2000; Yoshizaki et al. 2000), while the PL10-related genes are expressed in a variety of tissues (Leroy et al. 1989; Gururajan et al. 1991; Gee and
Conboy 1994; Sowden et al. 1995; Olsen et al. 1997). To examine whether the vas-related genes in lower metazoans are also expressed in germline cells, the expression patterns of Cnvas1, Cnvas2 and CnPL10 in Hydra were analyzed with whole-mount ISH. Hydra tissue is composed of epithelial cell lineages and the interstitial(i-) cell lineage (Bode 1996). The icell lineage consists of multipotent stem cells and their differentiation products. The multipotent stem cells undergo self-renewal and produce germline stem cells as well as three types of somatic cells, nematocytes, neurons and gland cells (David and Gierer 1974; David and Murphy 1977; Bode et al. 1987; Bosch and David 1987). Both multipotent and germline stem cells are relatively large in cell size (10–12 µm in diameter under the present ISH conditions) with a large conspicuous nucleus. They are referred to as large i-cells (David 1973; Bode 1996). In contrast, differentiating somatic cells are generally small in size (5–7 µm in diameter) and are called small i-cells (David 1973; Heimfeld and Bode 1984; Bode et al. 1990; Bode 1996). In normal female polyps, Cnvas1 and Cnvas2 were strongly expressed in clumps of large i-cells, which are typical for female germline cells (Littlefield 1991; Nishimiya-Fujisawa and Sugiyama 1995), and weakly expressed in scattered large i-cells in singles or in pairs, which are typical for multipotent stem cells (David and Gierer 1974; David and Plotnik 1980; Fig. 2A, B, E, F). In normal male polyps, they were expressed strongly in strings of large i-cells, which are typical for male germline cells (Littlefield 1985; Littlefield et al. 1985; Nishimiya-Fujisawa and Sugiyama 1993), and weakly in scattered large i-cells (Fig. 2C, D, G, H). These expression patterns were very similar to those of Cnnos1 (Mochizuki et al. 2000). Since Cnnos1 is expressed strongly in germline stem cells in both female and male polyps and weakly in multipotent stem cells, the co-expression of Cnvas1 or Cnvas2 with Cnnos1 was analyzed by double ISH. Figure 2I and L, respectively, shows the first staining with the Cnvas1 and Cnvas2 probes, and Fig. 2J and M shows the second staining with the Cnnos1 probe. The expression of Cnvas1 and Cnvas2 overlapped completely with that of Cnnos1 (Fig. 2J, M). Unfortunately, the staining in the scattered cells was too weak to detect in double ISH. Therefore, it is not certain if the scattered cells stained with the Cnvas1 and Cnvas2 probes were identical to the multipotent stem cells expressing the Cnnos1 mRNA. Nematoblasts at an early differentiation stage are relatively large in cell size (David 1973; Grens et al. 1995) and therefore, some of the Cnvas1 or Cnvas2 positive cells could be nematoblasts. To examine this possibility, we performed double ISH using the CnASH plus Cnvas1 or Cnvas2 cRNA probes. CnASH is expressed in the nematoblasts just after commitment to mature stages in Hydra (Grens et al. 1995). The red and purple stains in Fig. 2K represent the CnASH and Cnvas1 mRNAs respectively. No double-stained cells were detected. Similarly, no double-stained cells were detected with the
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Fig. 3A–F Expression of Cnvas1 and Cnvas2 in whole mounts of sexually induced polyps. Expression of Cnvas1 (A) and Cnvas2 (D) in male polyps showing testes at early stages of formation. Expression of Cnvas1 (B) or Cnvas2 (E; purple) with CnDmc1 (red) in testes at late stages analyzed by double ISH. Expression of Cnvas1 (C) and Cnvas2 (F) in female polyps showing massive aggregation of germline cells and unfertilized eggs (insets). A and D, B and E, C and F and insets of C and F, respectively, share the same scale bars
thermore, the head region in which neuron precursors are always detected (David and Gierer 1974; Heimfeld and Bode 1981) was free of signals (Fig. 2A, C, E, G). Thus, the Cnvas1 and Cnvas2 expression appears to be confined to multipotent stem cells and germline stem cells. The expression probably ceases immediately after multipotent stem cells are committed to the nematocyte pathway and possibly to the nerve cell pathway as well. These results indicate that the expression of the vasrelated genes in germline cells was established before the appearance of cnidarians. Cnvas1 and Cnvas2 expression during gametogenesis
Fig. 2A–N Expression of Cnvas1 and Cnvas2 in whole mounts of normal polyps. A, B Cnvas1 in a female polyp; C, D Cnvas1 in a male polyp; E, F Cnvas2 in a female polyp; G, H Cnvas2 in a male polyp. I–N Double in situ hybridization (ISH). Expression of Cnvas1 (I) or Cnvas2 (L) visualized with red staining, followed by purple staining with Cnnos1 probe (J, M). Expression of Cnvas1 (K) or Cnvas2 (N; purple) and CnASH (red) detected with double ISH. A, C, E and G, B, D, F, H, K and N and I, J, L and M share common scale bars, respectively.
CnASH and Cnvas2 probes (Fig. 2M). Thus, Cnvas1 and Cnvas2 are not expressed in cells in the nematocyte pathway. The neuron precursors are considered to be single or a pair of small i-cells (Heimfeld and Bode 1984; Bode et al. 1990). These small i-cells, however, were not observed among the Cnvas1- or Cnvas2-positive cells. Fur-
The results presented above indicate that the expression of both Cnvas1 and Cnvas2 was low in multipotent stem cells but upregulated in germline stem cells. Here, we examined their expression during gametogenesis. In sexually induced male polyps, proliferating germline cells migrate toward several sites on the body and form testes (Littlefield et al. 1985). Cells around the testis undergo spermatogenesis and mature sperms accumulate in the center of the testis. As shown in Fig. 3A and D respectively, Cnvas1 and Cnvas2 were expressed intensively in germline cells, which migrated massively toward developing testes, and the number of cells expressing the genes increased. However, as spermatogenesis proceeded, the expression declined. To examine at which stage this decline occurs, the double ISH was carried out using cRNA probes of Cnvas1 or Cnvas2 and CnDmc1. CnDmc1, a Hydra homologue of Dmc1 that is involved in meiotic recombination in yeast (Bishop et al. 1992), is
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expressed specifically in early spermatocytes and early oocytes/nurse cells (Mochizuki and Fujisawa, unpublished data). Since most of the cells expressing Cnvas1 or Cnvas2 mRNA did not overlap with those expressing CnDmc1 (Fig. 3B, E), the expression of Cnvas1 or Cnvas2 halted before or early in spermatocyte differentiation. During oogenesis, germline cells increase in number and form massive aggregates between the ectoderm and mesoglea thereby lifting up the ectoderm (Honneger et al. 1985). During this stage a single oocyte is produced within an aggregate and the rest of the germline cells become nurse cells which are eventually phagocytosed by the oocyte. The mature oocyte comes out of the ectodermal cell layer to become an egg on the body column. During oogenesis in normal polyps, the level of Cnvas1 and Cnvas2 expression and the number of cells expressing the genes continued to increase (Fig. 3C, F) and the messages accumulated in unfertilized eggs (Fig. 3E, F, insets). This suggests that Cnvas1 and Cnvas2 are expressed both in oocyte and nurse cells and presumably bear some maternal activity during embryogenesis. CnPL10 is expressed in multipotent stem cells, germline cells and somatic cells in the interstitial cell lineage CnPL10 was expressed in a larger number of i-cells in both female (Fig. 4A, B) and male (Fig. 4C, D) normal polyps compared to Cnvas1 (Fig. 2A–D) and Cnvas2 (Fig. 2E–G). The germline cells appeared to be stained, since clumps of large i-cells in the female (Fig. 4A, arrowheads), and large i-cells around the developing testis in the male (Fig. 4C, double arrowheads) were observed. This view was confirmed by the specific expression of CnPL10 in the germline cells in female (Fig. 4E, F) and male pseudo-epithelial Hydra (Fig. 4G, H). Thus, CnPL10 is expressed in germline cells. In addition, clusters of small i-cells, which are invariably nematoblasts (David 1973; David and Challoner 1974; Grens et al. 1995), were stained in large numbers. Therefore, the majority of the nematoblasts appeared to express CnPL10. However, the expression was probably not in mature nematocytes or neurons because no staining was observed in the tentacles, hypostome or peduncle where both or one of these cell types are abundant (Fig. 4A, C, arrows; Bode 1996). Finally, we examined CnPL10 expression in multipotent stem cells. Since large i-cells in singles or in pairs were stained both in male and female normal polyps, multipotent stem cells appeared to express CnPL10. This was confirmed by an independent experiment. When epithelial tissue lacking all cells in the i-cell lineage was axially grafted onto normal tissue, multipotent stem cells were shown to immigrate quickly into the epithelial tissue from the normal tissue (Sugiyama and Fujisawa 1978). The immigrated large i-cells in the epithelial tissue expressed CnPL10 (Fig. 4I, J, arrowheads). These results indicate that CnPL10 was also expressed in multipotent stem cells.
Fig. 4A–J Expression of CnPL10 in whole mounts. Low and high magnifications of a normal female (A, B), a normal male (C, D), a pseudo-epithelial female (E, F) and a pseudo-epithelial male (G, H) polyp. Arrows show tentacles, peduncle or hypostome, where no signal is seen. Arrowheads show clumps of female cells and double arrowheads indicate developing testes. I, J Low and high magnifications of CnPL10 expression in the epithelial tissue grafted onto normal tissue (see text for detail). Asterisks indicate the grafting border. Black arrowheads indicate large interstitial cells expressing CnPL10. A, C, E and G and B, D, F and H, respectively, share common scale bars
In summary, CnPL10 mRNA was shown to be expressed in the germline cells; multipotent stem cells and differentiating somatic cells of the interstitial cell lineage in Hydra. This expression pattern is reminiscent of DjvlgA, one of the PL10-related genes occurring in the planaria, D. japonica, which is expressed in germline cells, pluripotent stem cells (neoblasts) and differentiating somatic cells (Shibata et al. 1999).
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Fig. 5A–E Expression of Cnvas1, Cnvas2 and CnPL10 in epithelial cells. A Expression of Cnvas1 (1), Cnvas2 (2), CnPL10 (3), Cnnos1 (4) and GAPDH (5) analyzed by northern blot. e Epithelial Hydra, f normal female Hydra, m normal male Hydra. B–E Whole-mount ISH on epithelial polyps. B, C Cnvas2 expression. D, E CnPL10 expression. Arrowheads Developing bud, asterisks mesoglea, h head, p peduncle, t tentacle. B, D and C, E, respectively, share common scale bars. The staining around the basal disk in B and D is outside of the cells and thus non-specific. The sticky mucous substances secreted from basal disk cells probably trapped the antibody and developed the color after the prolonged reaction
The vas- and PL10-related genes are expressed in ectodermal epithelial cells in Hydra The expression of Cnvas1, Cnvas2 and CnPL10 mRNA was also observed in epithelial cells by both northern blot analysis (Fig. 5A) and whole-mount ISH (Fig. 5B–E). As shown in Fig. 5A (rows 1–3), these genes were expressed in both normal and epithelial Hydra indicating that they are also expressed in epithelial cells. This is in sharp contrast to Cnnos1, which is known to be specifically expressed in germline cells and multipotent stem cells (Mochizuki et al. 2000) but was not expressed in epithelial Hydra (Fig. 5A, row 4). Furthermore, the prolonged color reaction in ISH revealed the expression of Cnvas1 and Cnvas2 mRNA in the ectodermal epithelial cells of the body column, but neither in the head or foot regions nor the developing head of the bud. No expression in the endodermal epithelial cells was detected. Figure 5B,C shows only the result of Cnvas2 because the Cnvas1 expression was essentially the same as that of Cnvas2, though less strong. A similar expression pattern of CnPL10 mRNA was observed except that the gene was additionally expressed in the hypostome, excluding its tip (Fig. 5D, E). It is known that the ectodermal epithelial cells in the body column are undifferentiated cells and when they are displaced into two extremities, they differentiate into the head- or foot-specific epithelial cells (Bode 1996). Thus, Cnvas2 appears to be expressed in all kinds of undifferentiated cells; multipotent stem cells, gameterestricted cells and the ectodermal epithelial cells in the body column. In contrast, CnPL10 was expressed not only in undifferentiated cells but also differentiating cells.
However, none of the vas/PL10 genes were expressed in fully differentiated somatic cells in Hydra.
Discussion In the present study, we have shown for the first time that lower metazoans also have at least one vas-related gene: Povas1 (sponge), Cnvas1 and Cnvas2 (Hydra) and Plvas1 (planaria). Our findings indicated that the vas-related genes occur ubiquitously among metazoans and strongly suggest that a vas-related gene was already present in a common ancestor of the metazoans. The vas-related genes were derived from a PL10-related gene 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 (Fig. 1C), possibly in a protozoa or some other primitive multicellular animal which was a common ancestor of all metazoans. In support of this view, no vas-related gene has been identified either in the whole genome of the yeast, S. cerevisiae, or in that portion of the genome of the plant, A. thaliana, thus far examined. However, no information is available in other plants, fungi, or protozoans. In order to understand the origin of the vas-related genes, it would be of particular importance to determine whether vas-related genes occur in protozoans. Functional relationship between the vas-related and PL10-related genes It is also of importance to determine the functional relationship between the vas- and PL10-related genes. Based on the present phylogenetic analysis (Fig. 1C), one can assume that animal PL10s inherited the functions of an ancestral PL10, and that the vas-related genes might have obtained new functions or lost some of them after duplication. DED1, a PL10-related gene in S. cerevisiae, appears to be essential for translational initiation of al-
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most all transcripts (Chuang et al. 1997), whereas the Vas protein in Drosophila binds to dIF2, a homologue of the yeast translational initiation factor 2, yIF2 (Carrera et al. 2000). Vas, therefore, probably works in translational initiation of some germline-specific genes, such as oskar, nanos and gurken (Gavis et al. 1996; Markussen et al. 1997; Tomancak et al. 1998). Although fragmentary, these observations suggest that the Vas- and PL10-related proteins are involved in translational initiation, though the difference appears to lie in their target transcripts. Another line of evidence based on the present findings also supports the latter view: i.e. CnPL10 was expressed in a wider range of tissues (Fig. 4) than Cnvas1 and Cnvas2 (Fig. 2). Both types of genes were expressed in germline cells, multipotent stem cells and ectodermal epithelial cells. CnPL10 was also expressed in nematoblasts. The ectodermal expression of both genes was restricted in the body column where the proliferation predominates and was not detected in the head (+ presumptive head in the bud) and foot regions where differentiation takes place or is completed (Fig. 5). These expression patterns appear to suggest that the genes are required for maintenance of the undifferentiated state or cell cycle progression. Our knowledge of the PL10-related genes in metazoans is currently limited, and further investigations of the PL10-related genes will be required, particularly in genetically accessible animals such as Drosophila. The recent discovery of a PL10-related gene in the Drosophila genome, CG9748 (Lasko 2000) should be of great help. Expression of the vas-related genes in germline is conserved at least from Cnidaria The expression of Cnvas1 and Cnvas2 of Hydra was detected not only in germline cells but also in multipotent stem cells and epithelial cells (Figs. 2, 3, 5). Since multipotent stem cells produce germline cells, they might be considered part of the germline. However, the ectodermal epithelial cells are somatic cells. Recently, it has been reported that Xvlg1, the vas-related gene in Xenopus, is expressed in somatic cells of the embryo (Ikenishi and Tanaka 2000). This observation, together with those of the present study raises the possibility that the vasrelated genes are expressed in a wider range of tissues and that the inability to detect the expression might be due to the detection sensitivity and/or the spatiotemporal variation of their expression. Nevertheless, the present results indicate that the expression of the vas-related genes in the germline is conserved in Cnidaria. Due to technical difficulties, we were unable to determine the expression of Povas1 in the sponge. The Vas-related proteins in fly, chicken, frog and nematode as well as the vas-mRNA in zebrafish have been localized in the germ plasm or related structures (Hay et al. 1988; Komiya et al. 1994; Roussell and Bennett 1993; Gruidl et al. 1996; Knaut et al. 2000; Kuznicki et al. 2000; Tsunekawa et al. 2000). Although
the sub-cellular localization of the mRNA or proteins of Cnvas1 and Cnvas2 are not known, they may be components of the germ plasm in Hydra. In Hydra, both multipotent stem cells and germline cells contain germinal granules (Noda and Kanai 1977). Their mass increases during egg formation but decreases as somatic cell differentiation proceeds. These features closely correlate with the expression of Cnvas1 and Cnvas2. In a protozoan, Tetrahymena, a conjusome, which is an electrondense structure similar to the germ plasm in metazoans, was observed only during conjugation (Janetopoulos et al. 1999). It would thus appear that a part of the system or machinery of germ-soma differentiation might be conserved from Protozoa. In this context as well, it would be of great value to investigate the vas-related genes in Protozoa. Acknowledgements This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (to T.F.).
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