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Vg1-RBP is conserved between vertebrates and Drosophila
Mechanisms of Development 96 (2000) 129±132
www.elsevier.com/locate/modo
Gene expression pattern
The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila Jacob Nielsen a,*, Finn Cilius Nielsen b, Rasmus Kragh Jakobsen a, Jan Christiansen a a
Institute of Molecular Biology, University of Copenhagen, Sùlvgade 83 H, DK-1307 Copenhagen K, Denmark b Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark Received 2 May 2000; received in revised form 25 May 2000; accepted 30 May 2000
Abstract The human IGF-II mRNA-binding proteins (IMPs) 1±3, and their Xenopus homologue Vg1 RNA-binding protein (Vg1-RBP) are RNAbinding proteins implicated in mRNA localization and translational control in vertebrate development. We have sequenced the Drosophila homologue (dIMP) of these genes, and examined its expression pattern in Drosophila embryos by in situ hybridization. The study shows that dIMP exhibits a biphasic expression pattern. In the early stages of development, a maternal pool of dIMP mRNA is evenly distributed in the embryo and degraded by the end of stage 4. Expression reappears in the developing central nervous system, where dIMP is expressed throughout neurogenesis. In addition, dIMP is present in the pole cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: RNA localization; Central nervous system; RNA-binding proteins; K-homology domain; Biphasic expression; In situ hybridization
1. Results Localization of mRNA by RNA-binding proteins is emerging as an important feature in development. A number of mRNA transport systems have been described in Drosophila embryogenesis, and more recently, a few systems have been described in vertebrates (reviewed in Bashirullah et al., 1998). The closely related human IGF-II mRNA-binding proteins (IMP)/Vg1 RNA-binding protein (Vg1-RBP) family of RNA-binding proteins is one of these vertebrate systems. Independent work has led to the identi®cation of several members of this protein family, which are involved in apparently unrelated processes in various organisms. Xenopus Vg1 RNA-binding protein (Vg1-RBP) was found to be involved in localization of the Vg1 mRNA to the vegetal pole of the Xenopus egg during its maturation (Deshler et al., 1998; Havin et al., 1998), chicken zipcode binding protein 1 (ZBP-1) was suggested to mediate b-actin mRNA sorting in fetal ®broblasts (Ross et al., 1997), and IMP 1±3 was found to localize subcytoplasmically within cells and regulate IGF-II translation in late development (Nielsen et al., 1999). In spite of their different names, these proteins are closely related with 69±95% amino acid identity between each member and they seem to have arisen * Corresponding author. Tel.: 145-3532-2008; fax: 145-3532-2040. E-mail address: [email protected] (J. Nielsen).
from two gene duplications that occurred shortly before the segregation of vertebrates (Fig. 1A). Moreover, this protein family seems to be involved in mRNA localization in both early and late vertebrate development In order to identify homologues of this protein family in Drosophila, where mRNA localization is better understood, we examined the Drosophila expressed sequence tag (EST) database. Three ESTs (AI534274, AI543547 and AA142189) exhibited some similarity to IMP/Vg1-RBP, and sequencing of these ESTs revealed that they represent a Drosophila homologue of IMP/Vg1-RBP, which we name dIMP. Fig. 1B shows a comparison of dIMP with the vertebrate homologues. The vertebrate proteins all have six potential RNA-binding domains: two RNA recognition motifs (RRMs) and four K-homology (KH) domains (underlined in Fig. 1B). The most striking feature of dIMP is that it lacks the two N-terminal RRMs, but dIMP is about 47% identical with any individual family member within a 420 amino acid region encompassing the four KH domains. To gain insight into the generation of dIMP mRNA during Drosophila embryogenesis, a whole mount in situ hybridization analysis was carried out (Fig. 2). Maternally derived dIMP mRNA is found evenly distributed in the whole Drosophila embryo. This pool of maternal mRNA is present in decreasing amounts until the end of stage 4, where the staining disappears completely except in the pole cells. The dIMP pole cell staining is already apparent at stage 4, and staining
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00383-X
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J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
Fig. 1. Comparison of the Drosophila IMP homologue with vertebrate IMP family members. (A) Unrooted phylogenetic tree. Analysis was performed with the Phylip package (Felsenstein, 1989); distance matrix and parsimony programs gave identical trees. (B) Amino acid comparison, black and grey boxes indicate amino acid identity and similarity, respectively. The two predicted RRMs (not present in the Drosophila homologue) and the four maxi KH domains are underlined. hIMP-1±3: human IGF-II mRNA-binding protein 1±3 (AF117106, AF117107 and AF117108); dIMP: Drosophila melanogaster IMP homologue (AF241237); xVg1RBP: Xenopus Vg1 RNA-binding protein/Vera (AF064634); ZBP-1: chicken zipcode-binding protein 1 (AF026527). The zebra®sh orthologue of Vg1-RBP/IMP3 and the mouse orthologue of IMP-1 have also been cloned (Zhang et al., 1999; Doyle et al., 1998), but have been omitted from the alignment.
in the pole cells is visible until stage 7 (see arrow in Fig. 2). At this point, the internalized pole cells become impossible to discern because of the high expression in the surrounding tissue. The dIMP expression outside the pole cells reappears at the end of stage 5; initially in four diffuse circumferential stripes. As gastrulation proceeds, this pattern is gradually overtaken by a broadening expression, that initiates around the neurogenic regions at the ventral and cephalic furrows and in an additional anterior stripe in the head, possibly the
procephalic neurogenic region ± see Fig. 2, stages 8 and 9. In the later part of neurogenesis, there is strong expression in both the ventral nerve cord and the embryonic brain. In addition, there is a broadening expression in the epidermis, which by stage 11 covers most of the embryo. After stage 11, the epidermal staining gradually fades leaving a distinct staining of the developing central nervous system. Following the more distinct staining of the embryo, the stained pole cells become distinguishable again ± see arrows in Fig. 2, stage 14.
J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
131
Fig. 2. Spatial expression of dIMP mRNA in Drosophila embryos as shown by in situ hybridization. The stage of embryos is indicated above each embryo. All embryos are shown with the anterior pointing to the left. For the stage 9, 12, 14 and 16 embryos, the same embryo is shown in lateral-, ventral- and dorsal views in upper, middle and lower panels, respectively. Hybridization with a sense probe revealed no staining except in the posterior spiracles in stage 16 embryos. The pole cells (pc) are marked by arrows in stage 7 and 14 embryos.
IMP/Vg1-RBP expression in mice, zebra®sh and Xenopus (Mueller-Pillasch et al., 1999; Nielsen et al., 1999; Zhang et al., 1999) exhibit some similarities to the expression of dIMP. Similar to Drosophila, the vertebrate IMP/ Vg1-RBP expression was shown to be biphasic with an initial maternal component and later zygotic transcription. Moreover, the early expression in the developing nervous system seems to be evolutionarily conserved. However, in late vertebrate embryogenesis IMP/Vg1-RBP becomes
expressed in a number of other tissues derived from all germ layers, which is not the case for dIMP that is restricted to the central nervous system and the pole cells. 2. Materials and methods 2.1. Sequencing The expressed sequence tags (ESTs) AI534274 and
132
J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
AI543547 containing the entire dIMP coding region, and EST AA142189 containing the ®rst 1389 bases of the coding region was obtained from Research Genetics. The sequence has been submitted to Genbank (AF241237). 2.2. In situ hybridization Antisense digoxigenin-labeled probe was generated from the EST AA142189. The plasmid was linearized with Apa1 and transcribed with a digoxigenin-labelling kit using T3 polymerase according to the manufacturers instructions (Boehringer±Mannheim). The resulting 1732-nucleotide transcript was subjected to alkaline digestion resulting in fragments with an average size of 250 bases. Drosophila melanogaster ¯ies of the Oregon strain and their embryos were raised at 258C using standard procedures. In situ hybridization followed by visualization with alkaline phosphatase-conjugated antibody to digoxigenin, X-phos and NBT (all from Boehringer±Manheim) were performed essentially as described by (Tautz and Pfei¯e, 1989).
Acknowledgements We would like to thank Robert De Lotto and Daniel St Johnston for advice. The work was supported by the Danish Medical Research Council and the Novo Nordisk Foundation.
References Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D., 1998. RNA localization in development. Annu. Rev. Biochem. 67, 335±394. Deshler, J.O., Highett, M.I., Abramson, T., Schnapp, B.J., 1998. A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates. Curr. Biol. 8, 489±496. Doyle, G.A., Betz, N.A., Leeds, P.F., Fleisig, A.J., Prokipcak, R.D., Ross, J., 1998. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 26, 5036±5044. Felsenstein, J., 1989. PHYLIP ± Phylogeny Inference Package (Version 3.2). Cladistics 5, 164±166. Havin, L., Git, A., Elisha, Z., Oberman, F., Yaniv, K., Schwartz, S.P., Standart, N., Yisraeli, J.K., 1998. RNA-binding protein conserved in both microtubule- and micro®lament-based RNA localization. Genes Dev. 12, 1593±1598. Mueller-Pillasch, F., Pohl, B., Wilda, M., Lacher, U., Beil, M., Wallrapp, C., Hameister, H., Knochel, W., Adler, G., Gress, T.M., 1999. Expression of the highly conserved RNA binding protein KOC in embryogenesis. Mech. Dev. 88, 95±99. Nielsen, J., Christiansen, J., Lykke-Andersen, J., Johnsen, A.H., Wewer, U.M., Nielsen, F.C., 1999. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol. Cell Biol. 19, 1262±1270. Ross, A.F., Oleynikov, Y., Kislauskis, E.H., Taneja, K.L., Singer, R.H., 1997. Characterization of a beta-actin mRNA zipcode-binding protein. Mol. Cell Biol. 17, 2158±2165. Tautz, D., Pfei¯e, C., 1989. A non-radioactive in situ hybridization method for the localization of speci®c RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81±85. Zhang, Q., Yaniv, K., Oberman, F., Wolke, U., Git, A., Fromer, M., Taylor, W.L., Meyer, D., Standart, N., Raz, E., Yisraeli, J.K., 1999. Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development. Mech. Dev. 88, 101±106.
www.elsevier.com/locate/modo
Gene expression pattern
The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila Jacob Nielsen a,*, Finn Cilius Nielsen b, Rasmus Kragh Jakobsen a, Jan Christiansen a a
Institute of Molecular Biology, University of Copenhagen, Sùlvgade 83 H, DK-1307 Copenhagen K, Denmark b Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark Received 2 May 2000; received in revised form 25 May 2000; accepted 30 May 2000
Abstract The human IGF-II mRNA-binding proteins (IMPs) 1±3, and their Xenopus homologue Vg1 RNA-binding protein (Vg1-RBP) are RNAbinding proteins implicated in mRNA localization and translational control in vertebrate development. We have sequenced the Drosophila homologue (dIMP) of these genes, and examined its expression pattern in Drosophila embryos by in situ hybridization. The study shows that dIMP exhibits a biphasic expression pattern. In the early stages of development, a maternal pool of dIMP mRNA is evenly distributed in the embryo and degraded by the end of stage 4. Expression reappears in the developing central nervous system, where dIMP is expressed throughout neurogenesis. In addition, dIMP is present in the pole cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: RNA localization; Central nervous system; RNA-binding proteins; K-homology domain; Biphasic expression; In situ hybridization
1. Results Localization of mRNA by RNA-binding proteins is emerging as an important feature in development. A number of mRNA transport systems have been described in Drosophila embryogenesis, and more recently, a few systems have been described in vertebrates (reviewed in Bashirullah et al., 1998). The closely related human IGF-II mRNA-binding proteins (IMP)/Vg1 RNA-binding protein (Vg1-RBP) family of RNA-binding proteins is one of these vertebrate systems. Independent work has led to the identi®cation of several members of this protein family, which are involved in apparently unrelated processes in various organisms. Xenopus Vg1 RNA-binding protein (Vg1-RBP) was found to be involved in localization of the Vg1 mRNA to the vegetal pole of the Xenopus egg during its maturation (Deshler et al., 1998; Havin et al., 1998), chicken zipcode binding protein 1 (ZBP-1) was suggested to mediate b-actin mRNA sorting in fetal ®broblasts (Ross et al., 1997), and IMP 1±3 was found to localize subcytoplasmically within cells and regulate IGF-II translation in late development (Nielsen et al., 1999). In spite of their different names, these proteins are closely related with 69±95% amino acid identity between each member and they seem to have arisen * Corresponding author. Tel.: 145-3532-2008; fax: 145-3532-2040. E-mail address: [email protected] (J. Nielsen).
from two gene duplications that occurred shortly before the segregation of vertebrates (Fig. 1A). Moreover, this protein family seems to be involved in mRNA localization in both early and late vertebrate development In order to identify homologues of this protein family in Drosophila, where mRNA localization is better understood, we examined the Drosophila expressed sequence tag (EST) database. Three ESTs (AI534274, AI543547 and AA142189) exhibited some similarity to IMP/Vg1-RBP, and sequencing of these ESTs revealed that they represent a Drosophila homologue of IMP/Vg1-RBP, which we name dIMP. Fig. 1B shows a comparison of dIMP with the vertebrate homologues. The vertebrate proteins all have six potential RNA-binding domains: two RNA recognition motifs (RRMs) and four K-homology (KH) domains (underlined in Fig. 1B). The most striking feature of dIMP is that it lacks the two N-terminal RRMs, but dIMP is about 47% identical with any individual family member within a 420 amino acid region encompassing the four KH domains. To gain insight into the generation of dIMP mRNA during Drosophila embryogenesis, a whole mount in situ hybridization analysis was carried out (Fig. 2). Maternally derived dIMP mRNA is found evenly distributed in the whole Drosophila embryo. This pool of maternal mRNA is present in decreasing amounts until the end of stage 4, where the staining disappears completely except in the pole cells. The dIMP pole cell staining is already apparent at stage 4, and staining
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00383-X
130
J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
Fig. 1. Comparison of the Drosophila IMP homologue with vertebrate IMP family members. (A) Unrooted phylogenetic tree. Analysis was performed with the Phylip package (Felsenstein, 1989); distance matrix and parsimony programs gave identical trees. (B) Amino acid comparison, black and grey boxes indicate amino acid identity and similarity, respectively. The two predicted RRMs (not present in the Drosophila homologue) and the four maxi KH domains are underlined. hIMP-1±3: human IGF-II mRNA-binding protein 1±3 (AF117106, AF117107 and AF117108); dIMP: Drosophila melanogaster IMP homologue (AF241237); xVg1RBP: Xenopus Vg1 RNA-binding protein/Vera (AF064634); ZBP-1: chicken zipcode-binding protein 1 (AF026527). The zebra®sh orthologue of Vg1-RBP/IMP3 and the mouse orthologue of IMP-1 have also been cloned (Zhang et al., 1999; Doyle et al., 1998), but have been omitted from the alignment.
in the pole cells is visible until stage 7 (see arrow in Fig. 2). At this point, the internalized pole cells become impossible to discern because of the high expression in the surrounding tissue. The dIMP expression outside the pole cells reappears at the end of stage 5; initially in four diffuse circumferential stripes. As gastrulation proceeds, this pattern is gradually overtaken by a broadening expression, that initiates around the neurogenic regions at the ventral and cephalic furrows and in an additional anterior stripe in the head, possibly the
procephalic neurogenic region ± see Fig. 2, stages 8 and 9. In the later part of neurogenesis, there is strong expression in both the ventral nerve cord and the embryonic brain. In addition, there is a broadening expression in the epidermis, which by stage 11 covers most of the embryo. After stage 11, the epidermal staining gradually fades leaving a distinct staining of the developing central nervous system. Following the more distinct staining of the embryo, the stained pole cells become distinguishable again ± see arrows in Fig. 2, stage 14.
J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
131
Fig. 2. Spatial expression of dIMP mRNA in Drosophila embryos as shown by in situ hybridization. The stage of embryos is indicated above each embryo. All embryos are shown with the anterior pointing to the left. For the stage 9, 12, 14 and 16 embryos, the same embryo is shown in lateral-, ventral- and dorsal views in upper, middle and lower panels, respectively. Hybridization with a sense probe revealed no staining except in the posterior spiracles in stage 16 embryos. The pole cells (pc) are marked by arrows in stage 7 and 14 embryos.
IMP/Vg1-RBP expression in mice, zebra®sh and Xenopus (Mueller-Pillasch et al., 1999; Nielsen et al., 1999; Zhang et al., 1999) exhibit some similarities to the expression of dIMP. Similar to Drosophila, the vertebrate IMP/ Vg1-RBP expression was shown to be biphasic with an initial maternal component and later zygotic transcription. Moreover, the early expression in the developing nervous system seems to be evolutionarily conserved. However, in late vertebrate embryogenesis IMP/Vg1-RBP becomes
expressed in a number of other tissues derived from all germ layers, which is not the case for dIMP that is restricted to the central nervous system and the pole cells. 2. Materials and methods 2.1. Sequencing The expressed sequence tags (ESTs) AI534274 and
132
J. Nielsen et al. / Mechanisms of Development 96 (2000) 129±132
AI543547 containing the entire dIMP coding region, and EST AA142189 containing the ®rst 1389 bases of the coding region was obtained from Research Genetics. The sequence has been submitted to Genbank (AF241237). 2.2. In situ hybridization Antisense digoxigenin-labeled probe was generated from the EST AA142189. The plasmid was linearized with Apa1 and transcribed with a digoxigenin-labelling kit using T3 polymerase according to the manufacturers instructions (Boehringer±Mannheim). The resulting 1732-nucleotide transcript was subjected to alkaline digestion resulting in fragments with an average size of 250 bases. Drosophila melanogaster ¯ies of the Oregon strain and their embryos were raised at 258C using standard procedures. In situ hybridization followed by visualization with alkaline phosphatase-conjugated antibody to digoxigenin, X-phos and NBT (all from Boehringer±Manheim) were performed essentially as described by (Tautz and Pfei¯e, 1989).
Acknowledgements We would like to thank Robert De Lotto and Daniel St Johnston for advice. The work was supported by the Danish Medical Research Council and the Novo Nordisk Foundation.
References Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D., 1998. RNA localization in development. Annu. Rev. Biochem. 67, 335±394. Deshler, J.O., Highett, M.I., Abramson, T., Schnapp, B.J., 1998. A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates. Curr. Biol. 8, 489±496. Doyle, G.A., Betz, N.A., Leeds, P.F., Fleisig, A.J., Prokipcak, R.D., Ross, J., 1998. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 26, 5036±5044. Felsenstein, J., 1989. PHYLIP ± Phylogeny Inference Package (Version 3.2). Cladistics 5, 164±166. Havin, L., Git, A., Elisha, Z., Oberman, F., Yaniv, K., Schwartz, S.P., Standart, N., Yisraeli, J.K., 1998. RNA-binding protein conserved in both microtubule- and micro®lament-based RNA localization. Genes Dev. 12, 1593±1598. Mueller-Pillasch, F., Pohl, B., Wilda, M., Lacher, U., Beil, M., Wallrapp, C., Hameister, H., Knochel, W., Adler, G., Gress, T.M., 1999. Expression of the highly conserved RNA binding protein KOC in embryogenesis. Mech. Dev. 88, 95±99. Nielsen, J., Christiansen, J., Lykke-Andersen, J., Johnsen, A.H., Wewer, U.M., Nielsen, F.C., 1999. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol. Cell Biol. 19, 1262±1270. Ross, A.F., Oleynikov, Y., Kislauskis, E.H., Taneja, K.L., Singer, R.H., 1997. Characterization of a beta-actin mRNA zipcode-binding protein. Mol. Cell Biol. 17, 2158±2165. Tautz, D., Pfei¯e, C., 1989. A non-radioactive in situ hybridization method for the localization of speci®c RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81±85. Zhang, Q., Yaniv, K., Oberman, F., Wolke, U., Git, A., Fromer, M., Taylor, W.L., Meyer, D., Standart, N., Raz, E., Yisraeli, J.K., 1999. Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development. Mech. Dev. 88, 101±106.