- Email: [email protected]
Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development
Mechanisms of Development 94 (2000) 283±286
www.elsevier.com/locate/modo
Gene expression pattern
Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development Ingrid Fetka, Annalisa Radeghieri, Tewis Bouwmeester* Developmental Biology Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany Received 17 January 2000; received in revised form 7 February 2000; accepted 7 February 2000
Abstract RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Here we present the molecular cloning and spatio-temporal expression of Xseb-4, which codes for a putative RNA binding protein containing a single RNA recognition motif (RRM). XSEB-4 shares 60±65% identity with the mammalian SEB-4 proteins. Xseb-4 is strongly expressed maternally. Zygotic transcription is initiated in the early gastrula embryo in paraxial mesoderm that is fated to give rise to somites. During the course of gastrulation and neurulation Xseb-4 expression in somitic paraxial mesoderm is centered within the XmyoD expression domain. As development proceeds Xseb-4 expression is in addition initiated in the cardiac primordium and the lens vesicle. In the heart expression is con®ned to the myocardium. Thus, the RRM-containing putative RNA binding protein XSEB-4 is differentially expressed during embryonic development in Xenopus. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: RNA binding; RRM motif; Muscle; Heart; Spatio-temporal expression; Xenopus laevis
1. Results RNA binding proteins play diverse roles in the post-transcriptional regulation of RNA metabolism in vertebrates and invertebrates (reviewed in Burd and Dreyfuss, 1994). One of the largest families of RNA binding proteins is characterized by a RNA recognition motif (RRM), a domain composed of 80±100 amino acid residues that contains two RNP consensus sequences (RNP1 and RNP2). Besides general roles in the control of RNA metabolism RRMcontaining proteins have been implicated in different tissue-speci®c processes during embryonic development; e.g. Drosophila Elav is involved in neurogenesis (Good, 1997) and Drosophila Squid controls localization of Gurken and pair-rule transcripts during oogenesis and in blastoderm embryos, respectively (Lall et al., 1999; Norvell et al., 1999). In Xenopus several RRM proteins have been identi®ed though in most cases their function has remained elusive (Gerber et al., 1999; Perron et al., 1999). Here we present the molecular cloning and the spatiotemporal expression characteristics of the Xenopus orthologue of seb-4, which encodes a RRM-containing protein encompassing 227 amino acid residues. SMART search * Corresponding author. Tel.: 149-6221-387-603; fax: 149-6221-387166. E-mail address: [email protected] (T. Bouwmeester).
revealed the presence of a single RRM (amino acids 12± 84) and three segments of low compositional complexity (Schultz et al., 1998). XSEB-4 is 65% identical to mouse SEB-4 and 62% to its human counterpart (Fig. 1). In addition it shares 43% identity with C. elegans T22B2.4 suggesting that T22B2.4 is the C. elegans orthologue of SEB-4. To determine the subcellular localization of XSEB-4 we fused the coding region amino-terminally to eGFP and analyzed the distribution of this chimeric protein in ectodermal explants. Upon overexpression XSEB-4.GFP is found in the cytoplasm in a punctate pattern and in the nucleus (Fig. 2B). The spatio-temporal expression was determined by RTPCR analysis and whole-mount in situ hybridization. Xseb-4 is strongly expressed in the unfertilized egg and these maternal transcripts are prolonged till the blastula stage without apparent localization (Figs. 2A and 3A). Zygotic transcription commences at st. 10.5 in two dorso-lateral domains ¯anking the axial midline, which is similar to the activation domain of XmyoD (Hopwood et al., 1989) (Fig. 3B,G). However, in contrast to XMyoD expression, which extends ventral-laterally around the blastopore, Xseb-4 expression is con®ned to those dorso-lateral domains (compare Fig. 3B,C with 3G,H; red arrowheads). During gastrulation and neurulation Xseb-4 expression remains centered within the XmyoD expression territory, though again no staining is observed around the closing blastopore. These data indicate that XmyoD-positive
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00284-7
284
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
Fig. 1. Amino acid sequence alignment of Xenopus SEB-4, mouse SEB-4, human SEB-4 and C. elegans T22B2.4. The single RNA recognition motif (RRM) is underlined. Identical residues with XSEB-4 are shaded in black. The GenBank accession number of Xseb-4 is AF223427. Accession numbers of the other proteins are; mSEB-4 (X75316), hSEB-4 (X75316) and T22B2.4 (AF047662).
Fig. 2. Temporal expression and subcellular localization of XSEB-4. (A) RT-PCR analysis of Xseb-4 expression during embryonic development. Note the strong maternal contribution and a gradual increase in zygotic expression. Numbers indicate the developmental stages according to Nieuwkoop and Faber (1967). (B) Subcellular localization of XSEB-4.GFP fusion protein in ectodermal explants. Note a punctate cytoplasmic and nuclear distribution. Rhodamin phalloidin stains the cell cortex.
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
285
Fig. 3. Whole-mount in situ hybridization showing the spatio-temporal expression pattern of Xseb-4. (A±J) Comparative expression analysis of Xseb-4 (A±E) and XmyoD (F±J) during gastrulation and neurulation. Note that Xseb-4 expression is embedded in the expression domain of XmyoD, but does not extend around the closing blastopore (red arrowheads). (K±M) Comparative expression of Xseb-4 (K), XmyoD (L) and Xnkx-2.5 (M) at stage 27. Additional expression commences in the lens vesicle and the heart. (O±Q) Transverse sections of a st. 32 embryo (N) from anterior to posterior direction through the lens (O), heart (P) and tailbud (Q). Expression in the heart is restricted to the myocardium. Abbreviations: le, lens; hm, head mesenchyme; ov, otic vesicle; m, myocardium; so, somites.
paraxial mesoderm precursors engaging in convergence and extension do not yet activate Xseb-4 (compare Fig. 3D,E with 3I,J; red arrowheads). As development proceeds expression is additionally detected in the cardiac primordium (Fig. 3K; compared with XmyoD (L) and Xnkx-2.5 (M)). Activation starts around stage 19, well after induction of the most upstream component for cardiac development, Xnkx-2.5 (Tonissen et al., 1994). Xseb-4 expression in the heart is restricted to the myocardium (Fig. 3P). Furthermore, expression commences in the lens vesicle, a spot close to the mouth, the ventral aspect of the otic vesicle as well as presumably head mesenchyme (Fig. 3K,N±Q). In conclusion, Xseb-4, encoding a single RRM-containing putative RNA binding protein, is differentially expressed during embryogenesis.
2. Experimental procedures 2.1. Isolation of Xenopus seb-4 Xseb-4 was identi®ed as a false-positive in a two-hybrid
screening aimed at identifying interaction partners of XFD12 0 (Fetka et al., 2000). pCS2-Xseb-4.GFP was generated by in frame fusion of the coding region, which was ampli®ed with T3 and (R) 5 0 -CTGCGGATCCTGCATGCGGTCGGCTTGCAG, with the enhanced version of GFP. For microinjection pCS2-Xseb-4.GFP was linearized with sacII and transcribed with Sp6. Chimeric XSEB-4.GFP was detected by confocal microscopy. 2.2. Embryo manipulations and in situ hybridizations RT-PCR analysis and whole-mount in situ hybridization were done as described (Bouwmeester et al., 1996). Xseb-4 was ampli®ed by the following primer pair; (F) 5'-GACTCCAGCCTCAGGAAGTAC; (R) 5'-AGGCCTTTGTACAAGTGCTGG. Digoxygenin labeled antisense RNA was transcribed from; SK-Xseb-4 (EcoRI, Sp6), KS-XmyoD (HindIII, Sp6) and pGEM3Z-Xnkx-2.5 (HindIII, T7). Acknowledgements We would like to thank Andre Brandli for the tailbud
286
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
cDNA library, Paul Krieg for the Xnkx-2.5 construct, Jun Wu for assistance with confocal microscopy, Henry Jasper and Ralph Rupp for sharing information prior to publication and Jochen Wittbrodt for comments on the manuscript. References Bouwmeester, T., Kim, S.-H., Sasai, Y., Lu, B., De Robertis, E.M., 1996. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382, 595±601. Burd, C.G., Dreyfuss, G., 1994. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615±621. Fetka, I., Doederlein, G., Bouwmeester, T., 2000. Neuroectodermal speci®cation and regionalization of the Spemann organizer in Xenopus. Mech. Dev. 93, 49±58. Gerber, W.V., Yatshievych, T.A., Antin, P.B., Correia, K.M., Conlon, R.A., Krieg, P.A., 1999. The RNA-binding protein gene. hermes, is expressed at high levels in the developing heart. Mech. Dev. 80, 77±86. Good, P.J., 1997. The role of elav-like genes, a conserved family encoding RNA-binding proteins in growth and development. Semin. Cell. Dev. Biol. 8, 577±584.
Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 8, 3409±3417. Lall, S., Francis-Lang, H., Flament, A., Norvell, A., Schupbach, T., IshHorowicz, D., 1999. Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts. Cell 98, 171±180. Nieuwkoop, P., Faber, J., 1967. Normal Table of Xenopus laevis, North Holland, (Daudin), Amsterdam. Norvell, A., Kelley, R.L., Wehr, K., Schupbach, T., 1999. Speci®c isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis. Genes Dev. 13, 864±876. Perron, M., Furrer, M.-P., Wegnez, M., Theodore, L., 1999. Xenopus elavlike genes are differentially expressed during neurogenesis. Mech. Dev. 84, 139±142. Schultz, J., Milpetz, F., Bork, P., Ponting, C.P., 1998. SMART, a simple modular architecture research tool: identi®cation of signalling domains. Proc. Natl. Acad. Sci. USA 95, 5857±5864. Tonissen, K.F., Drysdale, T.A., Lints, T.J., Harvey, R.P., Krieg, P.A., 1994. XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development. Dev. Biol. 162, 325± 328.
www.elsevier.com/locate/modo
Gene expression pattern
Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development Ingrid Fetka, Annalisa Radeghieri, Tewis Bouwmeester* Developmental Biology Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany Received 17 January 2000; received in revised form 7 February 2000; accepted 7 February 2000
Abstract RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Here we present the molecular cloning and spatio-temporal expression of Xseb-4, which codes for a putative RNA binding protein containing a single RNA recognition motif (RRM). XSEB-4 shares 60±65% identity with the mammalian SEB-4 proteins. Xseb-4 is strongly expressed maternally. Zygotic transcription is initiated in the early gastrula embryo in paraxial mesoderm that is fated to give rise to somites. During the course of gastrulation and neurulation Xseb-4 expression in somitic paraxial mesoderm is centered within the XmyoD expression domain. As development proceeds Xseb-4 expression is in addition initiated in the cardiac primordium and the lens vesicle. In the heart expression is con®ned to the myocardium. Thus, the RRM-containing putative RNA binding protein XSEB-4 is differentially expressed during embryonic development in Xenopus. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: RNA binding; RRM motif; Muscle; Heart; Spatio-temporal expression; Xenopus laevis
1. Results RNA binding proteins play diverse roles in the post-transcriptional regulation of RNA metabolism in vertebrates and invertebrates (reviewed in Burd and Dreyfuss, 1994). One of the largest families of RNA binding proteins is characterized by a RNA recognition motif (RRM), a domain composed of 80±100 amino acid residues that contains two RNP consensus sequences (RNP1 and RNP2). Besides general roles in the control of RNA metabolism RRMcontaining proteins have been implicated in different tissue-speci®c processes during embryonic development; e.g. Drosophila Elav is involved in neurogenesis (Good, 1997) and Drosophila Squid controls localization of Gurken and pair-rule transcripts during oogenesis and in blastoderm embryos, respectively (Lall et al., 1999; Norvell et al., 1999). In Xenopus several RRM proteins have been identi®ed though in most cases their function has remained elusive (Gerber et al., 1999; Perron et al., 1999). Here we present the molecular cloning and the spatiotemporal expression characteristics of the Xenopus orthologue of seb-4, which encodes a RRM-containing protein encompassing 227 amino acid residues. SMART search * Corresponding author. Tel.: 149-6221-387-603; fax: 149-6221-387166. E-mail address: [email protected] (T. Bouwmeester).
revealed the presence of a single RRM (amino acids 12± 84) and three segments of low compositional complexity (Schultz et al., 1998). XSEB-4 is 65% identical to mouse SEB-4 and 62% to its human counterpart (Fig. 1). In addition it shares 43% identity with C. elegans T22B2.4 suggesting that T22B2.4 is the C. elegans orthologue of SEB-4. To determine the subcellular localization of XSEB-4 we fused the coding region amino-terminally to eGFP and analyzed the distribution of this chimeric protein in ectodermal explants. Upon overexpression XSEB-4.GFP is found in the cytoplasm in a punctate pattern and in the nucleus (Fig. 2B). The spatio-temporal expression was determined by RTPCR analysis and whole-mount in situ hybridization. Xseb-4 is strongly expressed in the unfertilized egg and these maternal transcripts are prolonged till the blastula stage without apparent localization (Figs. 2A and 3A). Zygotic transcription commences at st. 10.5 in two dorso-lateral domains ¯anking the axial midline, which is similar to the activation domain of XmyoD (Hopwood et al., 1989) (Fig. 3B,G). However, in contrast to XMyoD expression, which extends ventral-laterally around the blastopore, Xseb-4 expression is con®ned to those dorso-lateral domains (compare Fig. 3B,C with 3G,H; red arrowheads). During gastrulation and neurulation Xseb-4 expression remains centered within the XmyoD expression territory, though again no staining is observed around the closing blastopore. These data indicate that XmyoD-positive
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00284-7
284
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
Fig. 1. Amino acid sequence alignment of Xenopus SEB-4, mouse SEB-4, human SEB-4 and C. elegans T22B2.4. The single RNA recognition motif (RRM) is underlined. Identical residues with XSEB-4 are shaded in black. The GenBank accession number of Xseb-4 is AF223427. Accession numbers of the other proteins are; mSEB-4 (X75316), hSEB-4 (X75316) and T22B2.4 (AF047662).
Fig. 2. Temporal expression and subcellular localization of XSEB-4. (A) RT-PCR analysis of Xseb-4 expression during embryonic development. Note the strong maternal contribution and a gradual increase in zygotic expression. Numbers indicate the developmental stages according to Nieuwkoop and Faber (1967). (B) Subcellular localization of XSEB-4.GFP fusion protein in ectodermal explants. Note a punctate cytoplasmic and nuclear distribution. Rhodamin phalloidin stains the cell cortex.
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
285
Fig. 3. Whole-mount in situ hybridization showing the spatio-temporal expression pattern of Xseb-4. (A±J) Comparative expression analysis of Xseb-4 (A±E) and XmyoD (F±J) during gastrulation and neurulation. Note that Xseb-4 expression is embedded in the expression domain of XmyoD, but does not extend around the closing blastopore (red arrowheads). (K±M) Comparative expression of Xseb-4 (K), XmyoD (L) and Xnkx-2.5 (M) at stage 27. Additional expression commences in the lens vesicle and the heart. (O±Q) Transverse sections of a st. 32 embryo (N) from anterior to posterior direction through the lens (O), heart (P) and tailbud (Q). Expression in the heart is restricted to the myocardium. Abbreviations: le, lens; hm, head mesenchyme; ov, otic vesicle; m, myocardium; so, somites.
paraxial mesoderm precursors engaging in convergence and extension do not yet activate Xseb-4 (compare Fig. 3D,E with 3I,J; red arrowheads). As development proceeds expression is additionally detected in the cardiac primordium (Fig. 3K; compared with XmyoD (L) and Xnkx-2.5 (M)). Activation starts around stage 19, well after induction of the most upstream component for cardiac development, Xnkx-2.5 (Tonissen et al., 1994). Xseb-4 expression in the heart is restricted to the myocardium (Fig. 3P). Furthermore, expression commences in the lens vesicle, a spot close to the mouth, the ventral aspect of the otic vesicle as well as presumably head mesenchyme (Fig. 3K,N±Q). In conclusion, Xseb-4, encoding a single RRM-containing putative RNA binding protein, is differentially expressed during embryogenesis.
2. Experimental procedures 2.1. Isolation of Xenopus seb-4 Xseb-4 was identi®ed as a false-positive in a two-hybrid
screening aimed at identifying interaction partners of XFD12 0 (Fetka et al., 2000). pCS2-Xseb-4.GFP was generated by in frame fusion of the coding region, which was ampli®ed with T3 and (R) 5 0 -CTGCGGATCCTGCATGCGGTCGGCTTGCAG, with the enhanced version of GFP. For microinjection pCS2-Xseb-4.GFP was linearized with sacII and transcribed with Sp6. Chimeric XSEB-4.GFP was detected by confocal microscopy. 2.2. Embryo manipulations and in situ hybridizations RT-PCR analysis and whole-mount in situ hybridization were done as described (Bouwmeester et al., 1996). Xseb-4 was ampli®ed by the following primer pair; (F) 5'-GACTCCAGCCTCAGGAAGTAC; (R) 5'-AGGCCTTTGTACAAGTGCTGG. Digoxygenin labeled antisense RNA was transcribed from; SK-Xseb-4 (EcoRI, Sp6), KS-XmyoD (HindIII, Sp6) and pGEM3Z-Xnkx-2.5 (HindIII, T7). Acknowledgements We would like to thank Andre Brandli for the tailbud
286
I. Fetka et al. / Mechanisms of Development 94 (2000) 283±286
cDNA library, Paul Krieg for the Xnkx-2.5 construct, Jun Wu for assistance with confocal microscopy, Henry Jasper and Ralph Rupp for sharing information prior to publication and Jochen Wittbrodt for comments on the manuscript. References Bouwmeester, T., Kim, S.-H., Sasai, Y., Lu, B., De Robertis, E.M., 1996. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382, 595±601. Burd, C.G., Dreyfuss, G., 1994. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615±621. Fetka, I., Doederlein, G., Bouwmeester, T., 2000. Neuroectodermal speci®cation and regionalization of the Spemann organizer in Xenopus. Mech. Dev. 93, 49±58. Gerber, W.V., Yatshievych, T.A., Antin, P.B., Correia, K.M., Conlon, R.A., Krieg, P.A., 1999. The RNA-binding protein gene. hermes, is expressed at high levels in the developing heart. Mech. Dev. 80, 77±86. Good, P.J., 1997. The role of elav-like genes, a conserved family encoding RNA-binding proteins in growth and development. Semin. Cell. Dev. Biol. 8, 577±584.
Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 8, 3409±3417. Lall, S., Francis-Lang, H., Flament, A., Norvell, A., Schupbach, T., IshHorowicz, D., 1999. Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts. Cell 98, 171±180. Nieuwkoop, P., Faber, J., 1967. Normal Table of Xenopus laevis, North Holland, (Daudin), Amsterdam. Norvell, A., Kelley, R.L., Wehr, K., Schupbach, T., 1999. Speci®c isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis. Genes Dev. 13, 864±876. Perron, M., Furrer, M.-P., Wegnez, M., Theodore, L., 1999. Xenopus elavlike genes are differentially expressed during neurogenesis. Mech. Dev. 84, 139±142. Schultz, J., Milpetz, F., Bork, P., Ponting, C.P., 1998. SMART, a simple modular architecture research tool: identi®cation of signalling domains. Proc. Natl. Acad. Sci. USA 95, 5857±5864. Tonissen, K.F., Drysdale, T.A., Lints, T.J., Harvey, R.P., Krieg, P.A., 1994. XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development. Dev. Biol. 162, 325± 328.