- Email: [email protected]
Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthiaroretzi
Gene Expression Patterns 3 (2003) 71–75 www.elsevier.com/locate/modgep
Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi Yoriko Nakamuraa,*, Kazuhiro W. Makabeb, Hiroki Nishidaa a
Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuda, Yokohama 226-8501, Japan Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
b
Received 15 October 2002; received in revised form 12 November 2002; accepted 13 November 2002
Abstract The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Ascidian embryo; Halocynthia roretzi; Maternal mRNA; RNA localization; cDNA project; Type I postplasmic RNA; Posterior localization; Glucose transporter; g1-related protein; HrPEM-3; HrGLUT; HrPEN-1; HrPEN-2
1. Results and discussion It has been suggested that maternal factors localized to the posterior-vegetal cytoplasm (PVC) in Halocynthia roretzi eggs play important roles in control of cleavage pattern, determination of the anterior – posterior axis, autonomous specification of muscle fate, and generation of differences in responsiveness to inductive signals in mesenchyme and notochord precursor blastomeres. These functions were revealed in micromanipulative experiments in which PVC was removed and transplanted (Nishida, 1997, 2002). In ascidians, some maternal mRNAs are localized to the posterior pole of embryos (Yoshida et al., 1996; Satou and Satoh, 1997). These RNAs are called posterior end mark (PEM) or postplasmic RNAs. They are categorized into two groups depending on localization in eggs. Type I postplasmic RNAs are already localized to the PVC in fertilized eggs and are then concentrated in the *
Corresponding author. Tel.: þ 81-45-924-5722; fax: þ81-45-924-5722. E-mail address: [email protected] (Y. Nakamura).
centrosome-attracting body (CAB; Hibino et al., 1998; Nishikata et al., 1999) during cleavages. By contrast, type II postplasmic RNAs are not strongly localized in eggs, but are concentrated in the CAB after cleavage starts (Sasakura et al., 2000). The results from the removal and transplantation of the PVC of eggs suggest that type I postplasmic RNA would be more important than the type II. So far, five type I postplasmic mRNAs have been identified in Halocynthia roretzi: macho-1 (a maternal muscle determinant), HrPEM, HrWnt-5, HrPOPK-1, and HrZF-1 (Sasakura et al., 1998a,b, 2000; Nishida and Sawada, 2001). In a cDNA project of maternal mRNAs in Halocynthia eggs (MAGEST database, Kawashima et al., 2000; Makabe et al., 2001), RNA localization and expression of 2626 clones have been observed by whole-mount in situ hybridization. In the database, there are 56 postplasmic clones representing 29 mRNAs that accumulate in the CAB of 8-cell embryos. To distinguish type I and type II postplasmic RNAs, we examined the localization of maternal mRNAs of every postplasmic clone. Four novel type I postplasmic mRNAs were identified in addition to five that have been identified
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(02)00069-8
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Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
Table 1 Postplasmic RNAs in Halocynthia Genea HrPEM HrPEM-3 HrWnt-5 HrPOPK-1 HrGLUT HrPEN-1 HrZF-1 HrPEN-2 macho-1 Total in MAGEST a b c d
In situb
ESTc
13 4 3 3 2 2 1 1 0
39 29 10 13 9 2 2 2 8
2626
32660
Accession no.
In situ hoursd
Similarity
Ref.
AB045129 AB091123 AB006608 AB014885 AB091122 AB091124 AB029332 AB091125 AB045124
0.5 0.5 1.5 3.5 4.0 4.5 3.0 6.5 2.5
Ciona pem Ciona pem-3 Wnt-5 Kinase Glucose transporter g1-related protein Zn finger No similarity Zn finger
Nishida and Sawada, 2001 Present study Sasakura et al., 1998a Sasakura et al., 1998b Present study Present study Sasakura et al., 2000 Present study Nishida and Sawada, 2001
Genes identified in the present study are shown in boldface. Frequency of the clones among total clones for which whole-mount in situ hybridization was carried out. Frequency of the clones among total clones in MAGEST database. Hours of coloring reaction required to obtain enough staining in whole-mount in situ hybridization.
so far (Table 1). In the other 20 mRNA, 13 were apparently type II, and we could not obtain the significant staining of the CAB in seven mRNAs. We cloned 50 termini of those clones by 50 -rapid amplification of cDNA ends (RACE) and determined their complete sequences and expression patterns. Table 1 summarizes the prevalence in the MAGEST database of all nine type I postplasmic mRNAs and other information. Six mRNAs appeared two or more
times in in situ hybridized clones (second column). Therefore, it is likely that these nine mRNAs cover most members of the major type I postplasmic mRNAs that show high prevalence. 1.1. HrPEM-3 (Figs. 1B and 2A) One mRNA showed similarity to pem-3 throughout its
Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
length (Fig. 2A). pem-3 is a postplasmic RNA of Ciona savignyi (Satou, 1999). In Halocynthia, transcripts of HrPEM-3 were detected in the cortical cytoplasm, except in the animal pole region, of unfertilized eggs. After fertilization, the mRNA was concentrated at the vegetal pole after the first phase of ooplasmic segregation and was then translocated to the posterior-vegetal region after the second phase. During three rounds of cleavage, the transcripts were further concentrated in the small posterior region that corresponds to the CAB in B4.1 blastomeres. From the 16to the 110-cell stage, maternal mRNAs always segregated into the posteriormost blastomeres that inherited the CAB. The B7.6 pair of the 110-cell embryo do not divide further during embryogenesis, and give rise to two cells in the endodermal strand at the caudal tip (Nishida, 1987). Accordingly, the signal for HrPEM-3 was still visible in the endodermal strand of tailbud embryos. The localized pattern of maternal HrPEM-3 RNA is typical of type I postplasmic RNA, represented by macho-1 (Fig. 1A). Zygotic expression of this gene started at the gastrula
73
stage (Fig.1B, arrowheads). In ascidians, the initiation of zygotic transcripts is usually detected as nuclear signals (Yasuo and Satoh, 1993). The signals were found in two rows of cells in the neural plate. At the tailbud-stage, the signals were found in the mesenchyme cells and weakly in adhesive organs. 1.2. HrGLUT (Figs. 1C and 2B) This clone had similarity to mammalian glucose transporter (Kayano et al., 1990) throughout its length (Fig. 2B). The localization pattern of the maternal mRNA was exactly the same as that of HrPEM-3. Zygotic expression was detected in the mesenchyme and the nervous system (nerve cord, brain, and adhesive organs) at the neurula and tailbud-stages (Fig. 1C, arrowheads). 1.3. HrPEN-1 (Figs. 1D,E and 2C) HrPEN-1 (after Posterior-ENd transcripts) exhibited
Fig. 1. Temporal and spatial localization and expression of type I postplasmic mRNA, as revealed by whole-mount in situ hybridization. (A) macho-1. (B) HrPEM-3. (C) HrGLUT. (D, E) HrPEN-1. Embryos were cleared in (D) but not in (E) to show faint zygotic expression in ectoderm blastomeres. (F) HrPEN-2. Zygotic expression of the genes is indicated by yellow (ectoderm), white (neural plate), red (adhesive organs), brain (orange), green (nerve cord), and blue (mesenchyme) arrowheads. In (B), zygotic expression of HrPEM-3 in the adhesive organs (red arrowhead) is faint and invisible in this lateral view of the tailbud embyo. Ani, animal pole; Veg, vegetal pole; A, anterior; P, posterior; UF, unfertilized egg; 1st phase, first phase of ooplasmic segregation; 2nd phase, second phase of ooplasmic segregation. Unfertilized eggs to second phase eggs are lateral views. Two-cell embryos to neurulae are shown in polar views, except for 8-cell embryos, which are lateral views. Tailbud embryos are lateral views. Scale bar: 100 mm.
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Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
Fig. 2. Comparison of the amino-acid sequences between newly identified type I postplasmic mRNA (upper) and known proteins (lower). Schematic diagrams of (A) HrPEM-3 and Ciona PEM-3 proteins, (B) HrGLUT and human glucose transporter (type 5) proteins, and (C) HrPEN-1 and mouse g1-related proteins. The KH domain (for K homology) is highlighted in red, and the RING finger domain in yellow in (A); the cytoplasmic domain in green, the transmembrane domain in purple, and the extracellular domain in pink in (B); and the zinc finger domain in light blue, and the serine/glutamic acid/proline-rich region in green in (C). Broken lines indicate the edges of the region in which a high degree of similarity was observed. Numbers indicate the number of amino acid residues.
similarity to mammalian g1-related protein, although it lacks the C-terminal half of mammalian g1-related protein (Fig. 2C). Two independent clones from MAGEST were sequenced. Both had a poly-A stretch at the 30 end, but did not have the C-terminal region of mammalian g1-related protein. g1-related protein in mice has similarity to Drosophila g1, which is expressed in mesoderm. g1-related protein is suggested to play a role in regulating apoptosis of myeloid precursor cells induced by withdrawal of growth factor (Bouchard and Cote, 1993; Baker and Reddy, 2000). The maternal mRNA showed the typical localization pattern of type I postplasmic RNA, although the signals were weaker than those of HrPEM-3. Zygotic expression was transiently detected at the 16- to 64-cell stages in the animal hemisphere (Fig. 1E, arrowheads). 1.4. HrPEN-2 (Fig. 1F) This clone showed similarity only to a human protein
with unknown function. The localization of the maternal mRNA was typical of type I postplasmic RNA. The signal was weaker than that of HrPEM-3. No zygotic expression was detected. 1.5. Localization of type I postplasmic RNAs Nine kinds of type I postplasmic RNAs are now known in Halocynthia, and their distributions of maternal mRNAs in embryos are the same. Therefore, these RNAs would be anchored and translocated by the same mechanisms utilizing 30 -untranslated region as proposed for several postplasmic RNAs by Sasakura and Makabe, 2002. Although CAB was discovered as a structure that is involved in unequal cleavages (Hibino et al., 1998; Nishikata et al., 1999), it serves as a multifunctional complex that is also involved in the localization of mRNAs. By regulating both cleavage planes and RNA localization, the CAB may ensure that postplasmic RNAs are infallibly
Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
partitioned into one of the daughter cells after division. The present work will provide a basis for analyzing the roles of localized factors in ascidian eggs and embryos.
2. Experimental procedures 2.1. In situ hybridization Whole-mount in situ hybridization was performed as described Miya et al. (1994, 1997). The HrGLUT and HrPEN-1 antisense RNA probes were synthesized using a full-length cDNA as template. The HrPEM-3 probe was transcribed from 1083 –3690 bp fragment of HrPEM-3 cDNA. The HrPEN-2 probe was generated from 1522 – 4015 bp fragment of HrPEN-2 cDNA. Most embryos were then dehydrated in a graded series of ethanol and rendered transparent with a 1:2 mixture (v/v) of benzyl alcohol and benzyl benzoate.
Acknowledgements We thank T. Kawashima (Kyoto University) and Y. Kohara (National Institute of Genetics) for providing MAGEST plasmids. We also thank members of the Asamushi Marine Biological Station and the Otsuchi Marine Research Center for help in collecting live ascidian adults, and members of the Misaki Marine Biological Laboratory for help in maintaining them. This work was supported by the Research for the Future Program of the Japanese Society for the Promotion of Science, and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References Baker, S.J., Reddy, E.P., 2000. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1. Gene 248, 33 –40. Bouchard, M.L., Cote, S., 1993. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein. Gene 125, 205–209. Hibino, T., Nishikata, T., Nishida, H., 1998. Centrosome-attracting body: a novel structure closely related to unequal cleavages in the ascidian embryo. Dev. Growth Differ. 40, 85–95. Kawashima, T., Kawashima, S., Kanehisa, M., Nishida, H., Makabe, K.W., 2000. MAGEST: Maboya gene expression patterns and sequence tags. Nucleic Acids Res. 28, 133 –135. Kayano, T., Burant, C.F., Fukumoto, H., Gould, G.W., Fan, Y., Eddy, R.L., Byers, M.G., Shows, T.B., Seino, S., Bell, G.I., 1990. Human facilitative glucose transporters. Isolation, functional characterization,
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and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J. Biol. Chem. 265, 13276–13282. Makabe, K.W., Kawashima, T., Kawashima, S., Minokawa, T., Adachi, A., Kawamura, H., Ishikawa, H., Yasuda, R., Yamamoto, H., Kondoh, K., Arioka, S., Sasakura, Y., Kobayashi, A., Yagi, K., Shojima, K., Kondoh, Y., Kido, S., Tsujinami, M., Nishimura, N., Takahashi, M., Nakamura, T., Kanehisa, M., Ogasawara, M., Nishikata, T., Nishida, H., 2001. Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development. Development 128, 2555–2567. Miya, T., Makabe, K.W., Satoh, N., 1994. Expression of a gene for major mitochondrial protein, ADP/ATP translocase, during embryogenesis in the ascidian Halocynthia roretzi. Dev. Growth Differ. 36, 39–48. Miya, T., Morita, K., Suzuki, A., Ueno, N., Satoh, N., 1997. Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate specification. Development 124, 5149–5159. Nishida, H., 1987. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev. Biol. 121, 526 –541. Nishida, H., 1997. Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int. Rev. Cytol. 176, 245 –306. Nishida, H., 2002. Specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and signaling molecules. Int. Rev. Cytol. 217, 227–276. Nishida, H., Sawada, K., 2001. macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409, 724–729. Nishikata, T., Hibino, T., Nishida, H., 1999. The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. Dev. Biol. 209, 72– 85. Sasakura, Y., Makabe, K.W., 2002. Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos. Dev. Biol. 250, 128 –144. Sasakura, Y., Ogasawara, M., Makabe, K.W., 1998a. HrWnt-5: a maternally expressed ascidian Wnt gene with posterior localization in early embryos. Int. J. Dev. Biol. 42, 573 –579. Sasakura, Y., Ogasawara, M., Makabe, K.W., 1998b. Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi. Mech. Dev. 76, 161–163. Sasakura, Y., Ogasawara, M., Makabe, K.W., 2000. Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos. Dev. Biol. 220, 365 –378. Satou, Y., 1999. posterior end mark 3 ( pem-3), an ascidian maternally expressed gene with localized mRNA encodes a protein with Caenorhabditis elegans MEX-3-like KH domains. Dev. Biol. 212, 337– 350. Satou, Y., Satoh, N., 1997. posterior end mark 2 ( pem-2), pem-4, pem-5, and pem-6: maternal genes with localized mRNA in the ascidian embryo. Dev. Biol. 192, 467–481. Yasuo, H., Satoh, N., 1993. Function of vertebrate T gene. Nature 364, 582– 583. Yoshida, S., Marikawa, Y., Satoh, N., 1996. posterior end mark, a novel maternal gene encoding a localized factor in the ascidian embryo. Development 122, 2005–2012.
Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi Yoriko Nakamuraa,*, Kazuhiro W. Makabeb, Hiroki Nishidaa a
Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuda, Yokohama 226-8501, Japan Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
b
Received 15 October 2002; received in revised form 12 November 2002; accepted 13 November 2002
Abstract The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Ascidian embryo; Halocynthia roretzi; Maternal mRNA; RNA localization; cDNA project; Type I postplasmic RNA; Posterior localization; Glucose transporter; g1-related protein; HrPEM-3; HrGLUT; HrPEN-1; HrPEN-2
1. Results and discussion It has been suggested that maternal factors localized to the posterior-vegetal cytoplasm (PVC) in Halocynthia roretzi eggs play important roles in control of cleavage pattern, determination of the anterior – posterior axis, autonomous specification of muscle fate, and generation of differences in responsiveness to inductive signals in mesenchyme and notochord precursor blastomeres. These functions were revealed in micromanipulative experiments in which PVC was removed and transplanted (Nishida, 1997, 2002). In ascidians, some maternal mRNAs are localized to the posterior pole of embryos (Yoshida et al., 1996; Satou and Satoh, 1997). These RNAs are called posterior end mark (PEM) or postplasmic RNAs. They are categorized into two groups depending on localization in eggs. Type I postplasmic RNAs are already localized to the PVC in fertilized eggs and are then concentrated in the *
Corresponding author. Tel.: þ 81-45-924-5722; fax: þ81-45-924-5722. E-mail address: [email protected] (Y. Nakamura).
centrosome-attracting body (CAB; Hibino et al., 1998; Nishikata et al., 1999) during cleavages. By contrast, type II postplasmic RNAs are not strongly localized in eggs, but are concentrated in the CAB after cleavage starts (Sasakura et al., 2000). The results from the removal and transplantation of the PVC of eggs suggest that type I postplasmic RNA would be more important than the type II. So far, five type I postplasmic mRNAs have been identified in Halocynthia roretzi: macho-1 (a maternal muscle determinant), HrPEM, HrWnt-5, HrPOPK-1, and HrZF-1 (Sasakura et al., 1998a,b, 2000; Nishida and Sawada, 2001). In a cDNA project of maternal mRNAs in Halocynthia eggs (MAGEST database, Kawashima et al., 2000; Makabe et al., 2001), RNA localization and expression of 2626 clones have been observed by whole-mount in situ hybridization. In the database, there are 56 postplasmic clones representing 29 mRNAs that accumulate in the CAB of 8-cell embryos. To distinguish type I and type II postplasmic RNAs, we examined the localization of maternal mRNAs of every postplasmic clone. Four novel type I postplasmic mRNAs were identified in addition to five that have been identified
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(02)00069-8
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Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
Table 1 Postplasmic RNAs in Halocynthia Genea HrPEM HrPEM-3 HrWnt-5 HrPOPK-1 HrGLUT HrPEN-1 HrZF-1 HrPEN-2 macho-1 Total in MAGEST a b c d
In situb
ESTc
13 4 3 3 2 2 1 1 0
39 29 10 13 9 2 2 2 8
2626
32660
Accession no.
In situ hoursd
Similarity
Ref.
AB045129 AB091123 AB006608 AB014885 AB091122 AB091124 AB029332 AB091125 AB045124
0.5 0.5 1.5 3.5 4.0 4.5 3.0 6.5 2.5
Ciona pem Ciona pem-3 Wnt-5 Kinase Glucose transporter g1-related protein Zn finger No similarity Zn finger
Nishida and Sawada, 2001 Present study Sasakura et al., 1998a Sasakura et al., 1998b Present study Present study Sasakura et al., 2000 Present study Nishida and Sawada, 2001
Genes identified in the present study are shown in boldface. Frequency of the clones among total clones for which whole-mount in situ hybridization was carried out. Frequency of the clones among total clones in MAGEST database. Hours of coloring reaction required to obtain enough staining in whole-mount in situ hybridization.
so far (Table 1). In the other 20 mRNA, 13 were apparently type II, and we could not obtain the significant staining of the CAB in seven mRNAs. We cloned 50 termini of those clones by 50 -rapid amplification of cDNA ends (RACE) and determined their complete sequences and expression patterns. Table 1 summarizes the prevalence in the MAGEST database of all nine type I postplasmic mRNAs and other information. Six mRNAs appeared two or more
times in in situ hybridized clones (second column). Therefore, it is likely that these nine mRNAs cover most members of the major type I postplasmic mRNAs that show high prevalence. 1.1. HrPEM-3 (Figs. 1B and 2A) One mRNA showed similarity to pem-3 throughout its
Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
length (Fig. 2A). pem-3 is a postplasmic RNA of Ciona savignyi (Satou, 1999). In Halocynthia, transcripts of HrPEM-3 were detected in the cortical cytoplasm, except in the animal pole region, of unfertilized eggs. After fertilization, the mRNA was concentrated at the vegetal pole after the first phase of ooplasmic segregation and was then translocated to the posterior-vegetal region after the second phase. During three rounds of cleavage, the transcripts were further concentrated in the small posterior region that corresponds to the CAB in B4.1 blastomeres. From the 16to the 110-cell stage, maternal mRNAs always segregated into the posteriormost blastomeres that inherited the CAB. The B7.6 pair of the 110-cell embryo do not divide further during embryogenesis, and give rise to two cells in the endodermal strand at the caudal tip (Nishida, 1987). Accordingly, the signal for HrPEM-3 was still visible in the endodermal strand of tailbud embryos. The localized pattern of maternal HrPEM-3 RNA is typical of type I postplasmic RNA, represented by macho-1 (Fig. 1A). Zygotic expression of this gene started at the gastrula
73
stage (Fig.1B, arrowheads). In ascidians, the initiation of zygotic transcripts is usually detected as nuclear signals (Yasuo and Satoh, 1993). The signals were found in two rows of cells in the neural plate. At the tailbud-stage, the signals were found in the mesenchyme cells and weakly in adhesive organs. 1.2. HrGLUT (Figs. 1C and 2B) This clone had similarity to mammalian glucose transporter (Kayano et al., 1990) throughout its length (Fig. 2B). The localization pattern of the maternal mRNA was exactly the same as that of HrPEM-3. Zygotic expression was detected in the mesenchyme and the nervous system (nerve cord, brain, and adhesive organs) at the neurula and tailbud-stages (Fig. 1C, arrowheads). 1.3. HrPEN-1 (Figs. 1D,E and 2C) HrPEN-1 (after Posterior-ENd transcripts) exhibited
Fig. 1. Temporal and spatial localization and expression of type I postplasmic mRNA, as revealed by whole-mount in situ hybridization. (A) macho-1. (B) HrPEM-3. (C) HrGLUT. (D, E) HrPEN-1. Embryos were cleared in (D) but not in (E) to show faint zygotic expression in ectoderm blastomeres. (F) HrPEN-2. Zygotic expression of the genes is indicated by yellow (ectoderm), white (neural plate), red (adhesive organs), brain (orange), green (nerve cord), and blue (mesenchyme) arrowheads. In (B), zygotic expression of HrPEM-3 in the adhesive organs (red arrowhead) is faint and invisible in this lateral view of the tailbud embyo. Ani, animal pole; Veg, vegetal pole; A, anterior; P, posterior; UF, unfertilized egg; 1st phase, first phase of ooplasmic segregation; 2nd phase, second phase of ooplasmic segregation. Unfertilized eggs to second phase eggs are lateral views. Two-cell embryos to neurulae are shown in polar views, except for 8-cell embryos, which are lateral views. Tailbud embryos are lateral views. Scale bar: 100 mm.
74
Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
Fig. 2. Comparison of the amino-acid sequences between newly identified type I postplasmic mRNA (upper) and known proteins (lower). Schematic diagrams of (A) HrPEM-3 and Ciona PEM-3 proteins, (B) HrGLUT and human glucose transporter (type 5) proteins, and (C) HrPEN-1 and mouse g1-related proteins. The KH domain (for K homology) is highlighted in red, and the RING finger domain in yellow in (A); the cytoplasmic domain in green, the transmembrane domain in purple, and the extracellular domain in pink in (B); and the zinc finger domain in light blue, and the serine/glutamic acid/proline-rich region in green in (C). Broken lines indicate the edges of the region in which a high degree of similarity was observed. Numbers indicate the number of amino acid residues.
similarity to mammalian g1-related protein, although it lacks the C-terminal half of mammalian g1-related protein (Fig. 2C). Two independent clones from MAGEST were sequenced. Both had a poly-A stretch at the 30 end, but did not have the C-terminal region of mammalian g1-related protein. g1-related protein in mice has similarity to Drosophila g1, which is expressed in mesoderm. g1-related protein is suggested to play a role in regulating apoptosis of myeloid precursor cells induced by withdrawal of growth factor (Bouchard and Cote, 1993; Baker and Reddy, 2000). The maternal mRNA showed the typical localization pattern of type I postplasmic RNA, although the signals were weaker than those of HrPEM-3. Zygotic expression was transiently detected at the 16- to 64-cell stages in the animal hemisphere (Fig. 1E, arrowheads). 1.4. HrPEN-2 (Fig. 1F) This clone showed similarity only to a human protein
with unknown function. The localization of the maternal mRNA was typical of type I postplasmic RNA. The signal was weaker than that of HrPEM-3. No zygotic expression was detected. 1.5. Localization of type I postplasmic RNAs Nine kinds of type I postplasmic RNAs are now known in Halocynthia, and their distributions of maternal mRNAs in embryos are the same. Therefore, these RNAs would be anchored and translocated by the same mechanisms utilizing 30 -untranslated region as proposed for several postplasmic RNAs by Sasakura and Makabe, 2002. Although CAB was discovered as a structure that is involved in unequal cleavages (Hibino et al., 1998; Nishikata et al., 1999), it serves as a multifunctional complex that is also involved in the localization of mRNAs. By regulating both cleavage planes and RNA localization, the CAB may ensure that postplasmic RNAs are infallibly
Y. Nakamura et al. / Gene Expression Patterns 3 (2003) 71–75
partitioned into one of the daughter cells after division. The present work will provide a basis for analyzing the roles of localized factors in ascidian eggs and embryos.
2. Experimental procedures 2.1. In situ hybridization Whole-mount in situ hybridization was performed as described Miya et al. (1994, 1997). The HrGLUT and HrPEN-1 antisense RNA probes were synthesized using a full-length cDNA as template. The HrPEM-3 probe was transcribed from 1083 –3690 bp fragment of HrPEM-3 cDNA. The HrPEN-2 probe was generated from 1522 – 4015 bp fragment of HrPEN-2 cDNA. Most embryos were then dehydrated in a graded series of ethanol and rendered transparent with a 1:2 mixture (v/v) of benzyl alcohol and benzyl benzoate.
Acknowledgements We thank T. Kawashima (Kyoto University) and Y. Kohara (National Institute of Genetics) for providing MAGEST plasmids. We also thank members of the Asamushi Marine Biological Station and the Otsuchi Marine Research Center for help in collecting live ascidian adults, and members of the Misaki Marine Biological Laboratory for help in maintaining them. This work was supported by the Research for the Future Program of the Japanese Society for the Promotion of Science, and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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