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RETRACTED: Cloning and expression of a novel armadillo motif containing gene in Xenopus
Gene Expression Patterns 2 (2002) 79–81 www.elsevier.com/locate/modgep
Cloning and expression of a novel armadillo motif containing gene in Xenopus Jae-Young Chang, Jin-Kwan Han* Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang, Kyungbuk, 790-784, South Korea Received 19 July 2002; accepted 25 July 2002
Abstract We report an isolation of a cDNA containing armadillo motif (Xamp: Xenopus armadillo motif protein) and its expression during Xenopus development. The open reading frame of Xamp encodes a predicted protein of 275 amino acids including an armadillo motif, and a bipartite nuclear localization signal. Xamp shares significant homology with a putative mouse protein (GeneBank AK009402) in the database. It is expressed both maternally and zygotically. Xamp is localized to the animal region of an egg and in the ectoderm of a gastrula stage embryo. At the neurula stages, Xamp is expressed in the dorsal region of neural tube from which presumptive sensory neurons arise. In addition to its neural tissue specific expression, Xamp transcripts are found to be localized in the developing gut tube. At the early tadpole stage, Xamp is expressed predominantly in the pharyngeal endoderm. As further development proceeds, its expression domain expands to include the entire foregut region but excludes the midgut and hindgut regions. This polarized pattern of expression persists until stage 46 after which, anterior specific expression of Xamp sharply decreases. These results suggest that Xamp may have a role in the neural tissue specification and gut endoderm patterning during the Xenopus development. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Xenopus laevis; Armadillo repeat motif; Pharynx; Endoderm; Gut; Embryogenesis
1. Results Members of the armadillo (arm) repeat protein family such as adenomatous polyposis coli (APC) and b-catenin encode the repeating 42 amino acid motifs, which was first reported in armadillo gene product of the Drosophila (Peifer et al., 1994). They are implicated in the Wnt signaling, intercellular junction, and nuclear transport using their arm motifs as the protein–protein interaction domain (Fleckenstein et al., 1998). To obtain genes expressed stage-specifically in the developing tissues of Xenopus embryo, we performed ordered differential display polymerase chain reaction (PCR) (Matz et al., 1997). Here, we report an isolation and expression pattern of a cDNA encoding a protein with an armadillo motif, which is named Xamp (Xenopus armadillo motif protein; GenBank accession No. AF466017). It encodes a predicted protein of 275 amino acids containing an armadillo motif and a bipartite nuclear localization signal (Fig. 1). BLAST search for similarity shows that the derived amino acid sequence of Xamp is 93% identical to that of a mouse putative protein (AK009402) and is highly similar to a human * Corresponding author. Tel.: 182-54-279-2126; fax: 182-54-279-2199. E-mail address: [email protected] (J.-K. Han).
hypothetical protein (FLJ10511) (Fig. 1). Among these homologues, amino acids containing armadillo motif and nuclear localization signal (NLS) are most conserved. In addition to these regions, 70 amino acids downstream of the NLS are quite similar, showing 96% identity among three proteins. Reverse transcriptase polymerase chain reaction (RTPCR) analysis of the temporal expression of Xamp reveals that Xamp transcripts are maternally expressed and are present throughout early development although the zygotic expression is reduced after gastrula stage (Fig. 2). Whole mount in situ hybridization shows that the maternal transcripts of Xamp are localized in the animal region of a fertilized egg (Fig. 3A). At the early gastrula (stage 10), Xamp staining is found in the entire ectoderm with higher intensity on the dorsal neuroectodermal region of the embryo (Fig. 3B). In the neurula embryo (stage 18), its expression in the neural tube shows a distinctive pattern of bilateral stripes along the anterior–posterior axis (Fig. 3C). Tissue sections demonstrate that this staining is localized in the head and dorsal most region of neural tube from which presumptive sensory neurons arise (Fig. 3D). Xamp expression is further examined in a developing gut. At the early tadpole stage (stage 36), Xamp transcripts are first detected in the developing pharynx (Fig. 3E). After 2.5 days of development (stages 38 and 39) at which the
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J.-Y. Chang, J.-K. Han / Gene Expression Patterns 2 (2002) 79–81
explants but not in the mid/hindgut region (Fig. 4B). In a fully differentiated gut (stage 49) explant, however, anterior specific expression of Xamp mostly disappears (Fig. 4B). These results suggest that Xamp may play a role in the processes of neural tissue specification and foregut differentiation during embryonic development of Xenopus laevis. 2. Material and methods 2.1. Isolation of Xamp
Fig. 1. A comparison of the predicted amino acid sequences of Xamp and its homologues. Identical amino acids are indicated by the shaded background. An armadillo motif is shown as underlined. Box represents the nuclear localization signal. These sequences are aligned using the CLUSTALW method.
stomach begins to be bent to the left as the first sign of gut morphogenesis, Xamp is localized to the pharynx and liver bud of foregut (Fig. 3F). At 3 days of development (stage 41), the expression domain of Xamp expands to include the foregut containing liver, stomach, and pancreas but excludes the midgut and hindgut regions (Fig. 3G). This polarized pattern of expression persists until stage 46 (data not shown). Interestingly, however, Xamp expression sharply diminishes in the gut of stage 49 embryos in which the pharynx and foregut have fully differentiated cell types (Fig. 3H). The epithelium of gut tube including pharynx, esophagus, stomach, and intestine comes from the endoderm, whereas the outer smooth muscle and connective tissue surrounding the gut originate from the mesoderm. In situ hybridization on the sections of tadpole reveals that Xamp mRNAs are localized to the endodermal cells lining the lumen of the pharynx and foregut rather than mesodermal tissues (Fig. 3I). To further verify the dynamic expression of Xamp along the anterior–posterior axis of gut tube, RT-PCR was carried out with gut tissues dissected from different regions of the developing gut. Precisely, the whole gut of tadpole from various stages was divided into pharynx, foregut, and mid–hindgut regions (Fig. 4A). Consistent with the results from in situ hybridization, at stages 36 and 41/42, Xamp transcripts are enriched in the pharyngeal and foregut
Fig. 2. The temporal expression pattern of Xamp during Xenopus embryogenesis. RT-PCR analysis shows that Xamp transcript is expressed maternally. After gastrula stage, Xamp mRNA is reduced in a whole embryo. 2RT lane is a control of RT-PCR on stage 8 whole embryo RNA in the absence of reverse transcriptase. ODC serves as a loading control.
Approximately 10 6 plaques of a Xenopus head cDNA library were screened using cDNA probe prepared from the ordered differential display polymerase chain reaction (ODD-PCR) (Matz et al., 1997). Twelve positive clones were obtained and rescued as pBluescript phagemids by in vivo excision following the manufacturer’s protocol (Stratagene). Of these clones, one with the longest insert was sequenced. 2.2. RT-PCR analysis RT-PCR analysis was done as described in Wilson and Melton (1994). Total number of RNAs from embryos or endodermal explants were used for cDNA synthesis. The following primers give Xamp product of 418 bp: forward, 5 0 -GAACAAGATAAAGCTGTATCTCGCGT-3 0 , reverse,
Fig. 4. Spatiotemporal regulation of Xamp expression in developing gut endoderm. (A) Schematic diagram of gut isolation and dissection. (B) RTPCR analysis of Xamp from the isolated gut tube explants. Xamp is strongly expressed in the pharyngeal explants of stage 36 gut. At the stage 41/42, Xamp is expressed in both pharynx and foregut explants. At stage 49, Xamp expression disappears throughout the whole gut. -RT lane is a control of RT-PCR on pharynx RNA of stage 36 in the absence of reverse transcriptase. EF1-a served as a positive control for quantifying RNA levels in the different samples. Abbreviations: F, foregut; G, gut tube; M/H, mid–hindgut; P, pharynx.
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Fig. 3. Localization of Xamp transcripts in whole embryos and isolated guts. (A) The lateral view of a fertilized egg showing that maternal transcripts of Xamp are localized in the animal region of egg. (B) The dorsolateral view of an early gastrula embryo (stage 10). Xamp mRNA is restricted to the dorsal ectoderm. Arrowhead indicates the dorsal lip of blastopore. (C) The dorsal view of a neurula embryo (stage 18). Xamp is expressed in the prospective sensory neuron area of the neural tube (arrow). (D) A sagittal section of stage 18/19 embryo showing Xamp expression in the most dorsal region of neural tube (nt). (E–H) Isolated whole guts. (E) Stage 36 embryonic gut shows Xamp localization in the developing pharynx (ph). Anterior is to the left. (F) Left side view of Xamp expression in the stage 38/39 gut. Xamp is expressed in pharynx (ph) and liver bud (li) of foregut. (G) Ventrolateral view of stage 41 gut. Xamp is expressed in the pharynx (ph) and foregut including liver (li), pancreas (p), and stomach (st). The posterior expression boundary of Xamp expands to the transitional zone/intestine boundary (arrowhead). (H) Fully differentiated stage 49 gut. No positive signaling is detected at this stage. (I) A frontal section of the stage 42 tadpole shows that Xamp expression is detected in the endoderm of pharynx and foregut-derived organs. Abbreviations: int, intestine; li, liver; nt, neural tube; p, pancreas; pe, pharyngeal endoderm; st, stomach; si, small intestine.
5 0 -AGGCTGTCACTGAGTAACGCGT-3 0 . EF1-a and ODC (Ornithine decarboxylase) were used as a loading control. Embryos were staged according to Nieuwkoop and Faber (1967).
Health and Welfare (01-PJ1-PG3-20700-0024), and Brain Korea 21 project.
2.3. In situ hybridization
Chalmers, A.D., Slack, J.M.W., 1998. Development of the gut in Xenopus laevis. Dev. Dyn. 212, 509–521. Fleckenstein, D., Rohde, M., Klionsky, D.J., Ru¨ diger, M., 1998. Ye1013 p (Vac8 p), an armadillo repeat protein related to plakoglobin and importin a , is associated with the yeast vacuole membrane. J. Cell. Sci. 111, 3109–3118. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell. Biol. 36, 685–695. Matz, M., Usman, N., Shagin, D., Bogdanova, E., Lukyanov, S., 1997. Ordered differential display: a simple method for systematic comparison of gene expression profiles. Nucleic Acids Res. 25 (12), 2541– 2542. Nieuwkoop, P.D., Faber, J., 1967. Normal table of Xenopus laevis (Daudin), North-Holland, Amsterdam (Reprinted Garland, 1994). Peifer, M., Berg, S., Reynolds, A.B., 1994. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 76, 789–791. Wilson, P.A., Melton, D.A., 1994. Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol. 4(8), 676–686.
Digoxigenin-labelled Xamp antisense RNA was synthesized from NotI-cut pBS(SK)Xamp construct using T7 RNA polymerase. In situ hybridization was performed as previously described (Harland, 1991) with minor modification. Whole gut dissections were performed as in Chalmers and Slack (1998).
Acknowledgements We would like to thank Dr. Harland for Xenopus head cDNA library. We also thank other members in our laboratory for their helpful comments. This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of
References
Cloning and expression of a novel armadillo motif containing gene in Xenopus Jae-Young Chang, Jin-Kwan Han* Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang, Kyungbuk, 790-784, South Korea Received 19 July 2002; accepted 25 July 2002
Abstract We report an isolation of a cDNA containing armadillo motif (Xamp: Xenopus armadillo motif protein) and its expression during Xenopus development. The open reading frame of Xamp encodes a predicted protein of 275 amino acids including an armadillo motif, and a bipartite nuclear localization signal. Xamp shares significant homology with a putative mouse protein (GeneBank AK009402) in the database. It is expressed both maternally and zygotically. Xamp is localized to the animal region of an egg and in the ectoderm of a gastrula stage embryo. At the neurula stages, Xamp is expressed in the dorsal region of neural tube from which presumptive sensory neurons arise. In addition to its neural tissue specific expression, Xamp transcripts are found to be localized in the developing gut tube. At the early tadpole stage, Xamp is expressed predominantly in the pharyngeal endoderm. As further development proceeds, its expression domain expands to include the entire foregut region but excludes the midgut and hindgut regions. This polarized pattern of expression persists until stage 46 after which, anterior specific expression of Xamp sharply decreases. These results suggest that Xamp may have a role in the neural tissue specification and gut endoderm patterning during the Xenopus development. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Xenopus laevis; Armadillo repeat motif; Pharynx; Endoderm; Gut; Embryogenesis
1. Results Members of the armadillo (arm) repeat protein family such as adenomatous polyposis coli (APC) and b-catenin encode the repeating 42 amino acid motifs, which was first reported in armadillo gene product of the Drosophila (Peifer et al., 1994). They are implicated in the Wnt signaling, intercellular junction, and nuclear transport using their arm motifs as the protein–protein interaction domain (Fleckenstein et al., 1998). To obtain genes expressed stage-specifically in the developing tissues of Xenopus embryo, we performed ordered differential display polymerase chain reaction (PCR) (Matz et al., 1997). Here, we report an isolation and expression pattern of a cDNA encoding a protein with an armadillo motif, which is named Xamp (Xenopus armadillo motif protein; GenBank accession No. AF466017). It encodes a predicted protein of 275 amino acids containing an armadillo motif and a bipartite nuclear localization signal (Fig. 1). BLAST search for similarity shows that the derived amino acid sequence of Xamp is 93% identical to that of a mouse putative protein (AK009402) and is highly similar to a human * Corresponding author. Tel.: 182-54-279-2126; fax: 182-54-279-2199. E-mail address: [email protected] (J.-K. Han).
hypothetical protein (FLJ10511) (Fig. 1). Among these homologues, amino acids containing armadillo motif and nuclear localization signal (NLS) are most conserved. In addition to these regions, 70 amino acids downstream of the NLS are quite similar, showing 96% identity among three proteins. Reverse transcriptase polymerase chain reaction (RTPCR) analysis of the temporal expression of Xamp reveals that Xamp transcripts are maternally expressed and are present throughout early development although the zygotic expression is reduced after gastrula stage (Fig. 2). Whole mount in situ hybridization shows that the maternal transcripts of Xamp are localized in the animal region of a fertilized egg (Fig. 3A). At the early gastrula (stage 10), Xamp staining is found in the entire ectoderm with higher intensity on the dorsal neuroectodermal region of the embryo (Fig. 3B). In the neurula embryo (stage 18), its expression in the neural tube shows a distinctive pattern of bilateral stripes along the anterior–posterior axis (Fig. 3C). Tissue sections demonstrate that this staining is localized in the head and dorsal most region of neural tube from which presumptive sensory neurons arise (Fig. 3D). Xamp expression is further examined in a developing gut. At the early tadpole stage (stage 36), Xamp transcripts are first detected in the developing pharynx (Fig. 3E). After 2.5 days of development (stages 38 and 39) at which the
1567-133X/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0925-477 3(02)00294-0
80
J.-Y. Chang, J.-K. Han / Gene Expression Patterns 2 (2002) 79–81
explants but not in the mid/hindgut region (Fig. 4B). In a fully differentiated gut (stage 49) explant, however, anterior specific expression of Xamp mostly disappears (Fig. 4B). These results suggest that Xamp may play a role in the processes of neural tissue specification and foregut differentiation during embryonic development of Xenopus laevis. 2. Material and methods 2.1. Isolation of Xamp
Fig. 1. A comparison of the predicted amino acid sequences of Xamp and its homologues. Identical amino acids are indicated by the shaded background. An armadillo motif is shown as underlined. Box represents the nuclear localization signal. These sequences are aligned using the CLUSTALW method.
stomach begins to be bent to the left as the first sign of gut morphogenesis, Xamp is localized to the pharynx and liver bud of foregut (Fig. 3F). At 3 days of development (stage 41), the expression domain of Xamp expands to include the foregut containing liver, stomach, and pancreas but excludes the midgut and hindgut regions (Fig. 3G). This polarized pattern of expression persists until stage 46 (data not shown). Interestingly, however, Xamp expression sharply diminishes in the gut of stage 49 embryos in which the pharynx and foregut have fully differentiated cell types (Fig. 3H). The epithelium of gut tube including pharynx, esophagus, stomach, and intestine comes from the endoderm, whereas the outer smooth muscle and connective tissue surrounding the gut originate from the mesoderm. In situ hybridization on the sections of tadpole reveals that Xamp mRNAs are localized to the endodermal cells lining the lumen of the pharynx and foregut rather than mesodermal tissues (Fig. 3I). To further verify the dynamic expression of Xamp along the anterior–posterior axis of gut tube, RT-PCR was carried out with gut tissues dissected from different regions of the developing gut. Precisely, the whole gut of tadpole from various stages was divided into pharynx, foregut, and mid–hindgut regions (Fig. 4A). Consistent with the results from in situ hybridization, at stages 36 and 41/42, Xamp transcripts are enriched in the pharyngeal and foregut
Fig. 2. The temporal expression pattern of Xamp during Xenopus embryogenesis. RT-PCR analysis shows that Xamp transcript is expressed maternally. After gastrula stage, Xamp mRNA is reduced in a whole embryo. 2RT lane is a control of RT-PCR on stage 8 whole embryo RNA in the absence of reverse transcriptase. ODC serves as a loading control.
Approximately 10 6 plaques of a Xenopus head cDNA library were screened using cDNA probe prepared from the ordered differential display polymerase chain reaction (ODD-PCR) (Matz et al., 1997). Twelve positive clones were obtained and rescued as pBluescript phagemids by in vivo excision following the manufacturer’s protocol (Stratagene). Of these clones, one with the longest insert was sequenced. 2.2. RT-PCR analysis RT-PCR analysis was done as described in Wilson and Melton (1994). Total number of RNAs from embryos or endodermal explants were used for cDNA synthesis. The following primers give Xamp product of 418 bp: forward, 5 0 -GAACAAGATAAAGCTGTATCTCGCGT-3 0 , reverse,
Fig. 4. Spatiotemporal regulation of Xamp expression in developing gut endoderm. (A) Schematic diagram of gut isolation and dissection. (B) RTPCR analysis of Xamp from the isolated gut tube explants. Xamp is strongly expressed in the pharyngeal explants of stage 36 gut. At the stage 41/42, Xamp is expressed in both pharynx and foregut explants. At stage 49, Xamp expression disappears throughout the whole gut. -RT lane is a control of RT-PCR on pharynx RNA of stage 36 in the absence of reverse transcriptase. EF1-a served as a positive control for quantifying RNA levels in the different samples. Abbreviations: F, foregut; G, gut tube; M/H, mid–hindgut; P, pharynx.
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Fig. 3. Localization of Xamp transcripts in whole embryos and isolated guts. (A) The lateral view of a fertilized egg showing that maternal transcripts of Xamp are localized in the animal region of egg. (B) The dorsolateral view of an early gastrula embryo (stage 10). Xamp mRNA is restricted to the dorsal ectoderm. Arrowhead indicates the dorsal lip of blastopore. (C) The dorsal view of a neurula embryo (stage 18). Xamp is expressed in the prospective sensory neuron area of the neural tube (arrow). (D) A sagittal section of stage 18/19 embryo showing Xamp expression in the most dorsal region of neural tube (nt). (E–H) Isolated whole guts. (E) Stage 36 embryonic gut shows Xamp localization in the developing pharynx (ph). Anterior is to the left. (F) Left side view of Xamp expression in the stage 38/39 gut. Xamp is expressed in pharynx (ph) and liver bud (li) of foregut. (G) Ventrolateral view of stage 41 gut. Xamp is expressed in the pharynx (ph) and foregut including liver (li), pancreas (p), and stomach (st). The posterior expression boundary of Xamp expands to the transitional zone/intestine boundary (arrowhead). (H) Fully differentiated stage 49 gut. No positive signaling is detected at this stage. (I) A frontal section of the stage 42 tadpole shows that Xamp expression is detected in the endoderm of pharynx and foregut-derived organs. Abbreviations: int, intestine; li, liver; nt, neural tube; p, pancreas; pe, pharyngeal endoderm; st, stomach; si, small intestine.
5 0 -AGGCTGTCACTGAGTAACGCGT-3 0 . EF1-a and ODC (Ornithine decarboxylase) were used as a loading control. Embryos were staged according to Nieuwkoop and Faber (1967).
Health and Welfare (01-PJ1-PG3-20700-0024), and Brain Korea 21 project.
2.3. In situ hybridization
Chalmers, A.D., Slack, J.M.W., 1998. Development of the gut in Xenopus laevis. Dev. Dyn. 212, 509–521. Fleckenstein, D., Rohde, M., Klionsky, D.J., Ru¨ diger, M., 1998. Ye1013 p (Vac8 p), an armadillo repeat protein related to plakoglobin and importin a , is associated with the yeast vacuole membrane. J. Cell. Sci. 111, 3109–3118. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell. Biol. 36, 685–695. Matz, M., Usman, N., Shagin, D., Bogdanova, E., Lukyanov, S., 1997. Ordered differential display: a simple method for systematic comparison of gene expression profiles. Nucleic Acids Res. 25 (12), 2541– 2542. Nieuwkoop, P.D., Faber, J., 1967. Normal table of Xenopus laevis (Daudin), North-Holland, Amsterdam (Reprinted Garland, 1994). Peifer, M., Berg, S., Reynolds, A.B., 1994. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 76, 789–791. Wilson, P.A., Melton, D.A., 1994. Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol. 4(8), 676–686.
Digoxigenin-labelled Xamp antisense RNA was synthesized from NotI-cut pBS(SK)Xamp construct using T7 RNA polymerase. In situ hybridization was performed as previously described (Harland, 1991) with minor modification. Whole gut dissections were performed as in Chalmers and Slack (1998).
Acknowledgements We would like to thank Dr. Harland for Xenopus head cDNA library. We also thank other members in our laboratory for their helpful comments. This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of
References