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Molecular cloning and expression analysis of dystroglycan during Xenopus laevis embryogenesis
Mechanisms of Development 119S (2002) S49–S54 www.elsevier.com/locate/modo
Molecular cloning and expression analysis of dystroglycan during Xenopus laevis embryogenesis Andrea Lunardi, Luciana Dente* Dipartimento di Fisiologia e Biochimica, Sezione di Biologia Cellulare e dello Sviluppo, University of Pisa, Via G. Carducci 13, Ghezzano, Pisa 56010, Italy Received 26 July 2002; received in revised form 6 September 2002; accepted 11 September 2002
Abstract Dystroglycan is a transmembrane receptor protein that provides a structural linkage between extracellular matrix components and cytoskeletal proteins. It was originally characterized as a member of dystrophin associated protein complex in muscle but, unlike other proteins of this complex, mutations in the dystroglycan gene have not been implicated as a cause of muscular dystrophies. Indeed, dystroglycan is an essential gene, expressed early in development that, if removed in knockout mice, provokes lethal defects before the onset of myogenesis. Dystroglycan is synthesized as a precursor propeptide that is post-translationally cleaved and glycosylated to yield alpha and beta subunits. We have cloned and characterized a cDNA clone, containing the complete coding region of the dystroglycan precursor, from a Xenopus laevis cDNA library. We have performed a spatial and temporal analysis of its expression in X. laevis embryos, using whole-mount in situ hybridization and reverse transcription-polymerase chain reaction analysis. Early expression of dystroglycan in a variety of tissues of different embryological derivation suggests a crucial role in morphogenetic events, especially during central nervous system differentiation. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xenopus; Expression pattern; Dystroglycan; Muscular dystrophy; Dystrophin; Dystrophin associated proteins; DAP complex; CNS; Extracellular matrix; Basement membrane; Ectodermal layer; Eye; Ganglional cells
1. Results Search in NCBI-EST GenBank for dystroglycan homologs in Xenopus laevis provided two EST clones (acc. no. BE506803 and BG037666) showing considerable homology to 5 0 and 3 0 extremities of the human dystroglycan coding region (acc. no. L19711). We designed specific primers to isolate the complete coding region of dystroglycan from a stage 28/30 X. laevis head cDNA library, using polymerase chain reaction amplification. Sequence analysis of a 2661 bases cDNA fragment revealed a 886 amino acids long putative protein that shares high homology with human dystroglycan. In particular, the percentage of identity is 95% in the cytoplasmic and transmembrane regions of the beta subunits, 56% in the extracellular region of beta and 60% in the alpha subunits, respectively. A comparison between Xenopus dystroglycan sequence and those of human (L19711.1), mouse (AAH07150.1), bovine (BAA23650.1) and rabbit (X64393.1), is shown in Fig. 1. The highest homology among different species is always found in the beta-subunits, confirming the biological relevance of this * Corresponding author. E-mail address: [email protected] (L. Dente).
region. The cleavage site (Ser), the potential membrane targeting signals and the N and O-linked glycosylation sites are identical to those proposed in the human homolog (Deyst et al., 1995; Smalheiser and Kim, 1995; Holt et al., 2000) (Fig. 1). More recently the sequence of zebrafish dystroglycan has been cloned (Parsons et al., 2002). The percentage of identity is 96% in the cytoplasmic and transmembrane regions of the beta subunits, 36% in the extracellular region of the beta subunits and 56% in the alpha subunits, respectively. Temporal expression of X-dystroglycan mRNA (X-dg) during early X. laevis embryogenesis was revealed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. X-dg is present in embryos as a maternal transcript; zygotic expression persists in all the analyzed stages (Fig. 2). Spatial expression pattern was also analyzed using wholemount in situ hybridization and subsequent histological sections. The earliest expression of X-dg is detectable in early neurula stage embryos (st. 13) in the anterior half of the midline (Fig. 3A). At midneurula stage (st. 15) X-dg expression is present in the neural plate, where the neurogenic cells orient medio-laterally to participate in neural tube formation, and in the notochord, as revealed by sections (data not shown). A diffuse and punctate expression
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00091-1
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Fig. 1. Comparison of the amino acid sequences of dystroglycan proteins. Xenopus dystroglycan residues are in bold. Identities are in red colour, gaps are indicated by hyphens. Putative signal and transmembrane domains are boxed. The conserved serine, proposed as post-translational processing site is highlighted in yellow; N- and O-glycosilation sites are shaded in gray. Blue dots indicate two group of residues that have been suggested to be a targeting motif for basolateral surface of epithelial cells (Kachinsky et al., 1999).
A. Lunardi, L. Dente / Mechanisms of Development 119S (2002) S49–S54
Fig. 2. RT-PCR analysis to determine temporal expression of X-dg in whole embryos. X-dg is present in all the analyzed stages (top panel). Ornithine decarboxilase (ODC) amplification was used as control of the reaction (bottom panel). The line marked 2RT indicates all the ingredients of Xdg except for reverse transcriptase.
is also detectable in the epidermal ectoderm (Figs. 3B,C). At late neurula stage (st. 19) a strong expression site is localized in presumptive prosencephalic/mesencephalic region (Fig. 3D, arrowhead). Note the absence of expression in the region from which neural crest cells derive (Fig. 3E, dorsal view). During neural tube closure (st. 22), X-dg transcripts are clearly detectable in prosencephalon evaginations of the eye anlagen, in rhomboencephalon (Fig. 3F, arrowhead) and in the otic vescicle primordium (Fig. 3G, arrowhead). X-dg epidermal ectoderm expression is not present in the most anterior part of the embryo (Fig. 3G) and is limited to the sensorial layer of this tissue, as revealed by sections of this
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stage embryos (Figs. 3J,K). At early tail bud stage (st. 26) Xdg epidermal ectoderm signal disappears, while the expression remains concentrated in prosencephalon, mesencephalon, in a restricted anterior area of the rhomboencephalon and in the dorsomedian portion of eye vesicles, representing the prospective pigmented layer of the retina (Fig. 3H, arrowhead). Finally, at stage 33, X-dg transcripts are present in several tissues derived from different germ layers (Fig. 3I). Enlarged view of the dorsal trunk musculature shows dystroglycan expression at level of myotomes (Fig. 3L). On the other hand, using a cross-reacting antibody raised against human dystroglycan, we have detected immunoreactivity only at level of the transverse myosepta at this stage (Fig. 3M, arrowheads). A similar phenotype has been recently observed also in zebrafish embryos (Parsons et al., 2002). In the adult skeletal muscle the protein has specific sarcolemma localization, as already reported in human and mouse muscles (Fig. 3N). Serial transverse sections of stage 33 embryos from anterior to posterior revealed high expression in the olfactory placode (op), in the telencephalic ventricular zone (Fig. 4A), in the diencephalic ventricular region and in the stomodeum (Fig. 4B, arrowhead). In contrast, in the most posterior portion of the diencephalon X-dg expression is limited to
Fig. 3. Expression of X-dg during Xenopus development (A–M). Whole-mount in situ hybridization was carried out using digoxigenin-labeled cRNA probe. Nieuwkoop-Faber stages of embryogenesis are indicated in higher left corners. Dorsal views, anterior down (A, B, E); postero-lateral view (C); anterior views (D, F, H); lateral views, anterior on the right (G, I). Transverse section of stage 22 (J): arrow head indicates sensorial layer of the ectoderm; parasagittal section of stage 22 (K) shows strong expression in the eye anlage and in the diencephalon/mesencephalon regions. X-dg expression sites at stage 33 (arrow heads in I): central nervous system (black), heart (orange), lens epithelium (turquoise), mandibular, hyoid and branchial arches (yellow and green respectively), myotomes (purple), otic vesicle (red), pronephron (blue). Parasagittal and longitudinal sections of stage 33 showing X-dg transcripts (L) and protein localization (M) in the myotomes. Transverse section of adult skeletal muscle reveals protein localization in the sarcolemma (N).
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Fig. 4. (A–F) Antero-posterior transverse sections of central nervous system at stage 33. Eye expression at level of ganglion cells layer in the neural retina (G); and in the optic stalk (H). Abbreviations: die, diencephalon; ep, epiphysis; gcl, ganglion cells layer; h, hypothalamus; mes, mesencephalon; os, optic stalk; rho, rhombencephalon; st, stomodeum.
the dorsal aspect (Fig. 4C). Furthermore, high signal level is detectable in the epiphysis (ep), hypotalamus (hyp), optic lobe of the mesencephalon (Fig. 4E) and in the dorsal rhomboencephalon (Fig. 4F). In the eye, X-dg is expressed in the ganglion cell layer (gcl) of the neural retina (Fig. 4G) and at level of the optic stalk (os) (Fig. 4H). Neural X-dg expression was further analyzed during early metamorphosis stage (st. 48) by whole-mount in situ hybridization of dissected brains (Fig. 5). This procedure allows to distinguish with more details X-dg specific transcripts, in particular in the ventral territories, where expression is limited to the optic chiasma, hypothalamus, pituitary gland and olfactory bulb. Cloning of the Xenopus dystroglycan coding region allowed a detailed analysis of its expression during embryogenesis. In particular, our analysis revealed for the first time a dynamic pattern of X-dg expression at level of central nervous system that suggests a direct involvement in differentiation of specific structures. A crucial role of dystroglycan in branching epithelial morphogenesis (Durbeej and Ekblom, 1997; Henry and Campbell, 1998) and in muscle differentiation (Brown et al., 1999; Leschziner et al., 2000) has been previously demonstrated. On the other hand, owing to the inability to generate viable dystroglycan-null mice, because of lethal defects in early basal membrane formation, the role of dystroglycan in neuronal cell differentiation has remained elusive (Williamson et al., 1997). Recently Moore et al. made ‘conditional’ knockout mice, where dystroglycan expression was inactivated in glia and brain
Fig. 5. (A–C) Expression of X-dg as detected in dissected brains of stage 48 embryos by whole-mount in situ hybridization. Lateral view, anterior right (A); ventral (B); and dorsal (C) view, anterior up.
A. Lunardi, L. Dente / Mechanisms of Development 119S (2002) S49–S54
neurons only. These mutants have brain malformations, such as disarray of cerebral cortical layering, aberrant migration of granule cells that resemble specific human congenital brain dystrophies (Moore et al., 2002) and give new insight into the role of dystroglycan in neuronal development (Ross, 2002). In contrast, complete removal of dystroglycan in zebrafish embryos causes loss of muscle integrity and necrosis, but not embryo lethality (Parsons et al., 2002). The characterization of the coding region and the expression pattern of Xenopus dystroglycan, presented in this paper, opens the possibility to design specific tools to perturb dystroglycan function and to test its role during morphogenetic events. 2. Methods 2.1. Xenopus dystroglycan cloning Two EST cDNA clones were obtained from IMAGE Consortium and sequenced to confirm and to complete the sequences derived from the NCBI-EST data. The clone BE50680 contains 50 bp of 5 0 untranslated region followed by the first 318 nucleotides of the coding region. The complete sequence of clone BG037666 revealed that it contains the last 600 bp of the coding region and 1168 bp of 3 0 untranslated region, followed by a poly(A) tail 16 bp long. To clone the complete Xenopus dystroglycan coding region, forward primer 5 0 -CTGTAACAGAACGCCAAAATGG-3 0 including the start codon and reverse primer 5 0 AGGCGAATTCTTAAGGAGGTACATAAGGGGGT-3 0 including the stop codon were synthesized and used to amplify the cDNA fragment from st. 28/30 head Xenopus cDNA library. The complete sequence of clone XDAG has been submitted with accession number AJ496325.
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with the corresponding X-dg sense probe. Histological examinations were performed as described in Pannese et al. (1998). Bleaching of pigmented embryos was done after colour reaction as described (Mayor et al., 1995). 2.4. Immunohistochemistry Dissected muscle and embryos were immersion-fixed for 1 h at 4 8C in 4% paraformaldehyde in 100 mM phosphate buffer pH 7.4, then cryoprotected by immersion in 25% sucrose in PBS overnight at 4 8C. Tissues were frozen in freezing medium (OCT compound; Sakura Finetek Europe B.V.), sectioned vertically at 12 mm thickness in a cryostat, collected on slides (SuperFrost Plus; Menzel-Glaser, Germany), air dried for 30 min, and stored at 280 8C until use. Immunohistochemistry labeling: blocking in PBS pH 7.4 containing 10% normal goat serum, 1% bovine serum albumine and 0.05% Triton X-100, sections were incubated with primary monoclonal antibody (43DAG/ 8D5, Novocastra) in PBS supplemented with 3% NGS and 0.05% Triton X-100 for 60 min at room temperature and then with secondary goat anti-mouse antibody coupled to rodamine-isothiocyanate (TRITC; Sigma T7657) or to peroxidase (HRP; Sigma A2304), using 3,3 0 diaminobenzidine (DAB; Sigma D4293) substrate. Acknowledgements We are grateful to Richard Harland for the gift of the st. 28/30 head library. We thank Massimiliano Andreazzoli, Federico Cremisi and Robert Vignali for a critical reading of the manuscript, Gaia Gestri, Michela Ori, Simona Casarosa and Giuseppe Lupo for help and suggestions in the experiments; Marzia Fabbri and Donatella De Matienzo for technical assistance and Salvatore De Maria for frog care. This work was supported by a grant from M.U.R.S.T to L.D.
2.2. RT-PCR analysis RT-PCR analysis was performed using total RNA isolated from embryos at the indicated stages and the SuperScript eII RNase H 2 Reverse Transcriptase kit (Life Technologies). X-dg primers (forward: 5 0 -TATCGGAAGAAGAGGAAAGGCA-3 0 ; reverse: 5 0 -GTGACCCTTTACCTTCCATAGG-3 0 ) and ODC primers (forward: 5 0 -GTCAATGATGGAGTGTATGGATC-3 0 ; reverse: 5 0 -TCCATTCCGCTCTCCTGAGCAC-3 0 ) were annealed at 54 8C for 26 cycles. 2.3. In situ hybridization Whole-mount in situ hybridization on X. leavis embryos was performed as described (Harland, 1991), except that BM purple (Roche) was used as a substrate for the alkaline phosphatase, using a X-dg antisense cRNA 600 bases long. Non-specific hybridization of X-dg riboprobe was evaluated
References Brown, S.C., Fassati, A., Popplewell, L., Page, A.M., Henry, M.D., Campbell, K.P., Dickson, G., 1999. Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction. J. Cell Sci. 112, 209–216. Deyst, K., Deyst, K.A, Bowe, M.A., Leszyk, J.D., Fallon, J.R., 1995. The alpha-dystroglycan-beta-dystroglycan complex. Membrane organization and relationship to an agrin receptor. J. Biol. Chem. 270, 25956– 25959. Durbeej, M., Ekblom, P., 1997. Dystroglycan and laminins: glycoconjugates involved in branching epithelial morphogenesis. Exp. Lung Res. 23, 109–118. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685–695. Henry, M.D., Campbell, K.P., 1998. A role for dystroglycan in basement membrane assembly. Cell 95, 859–870. Holt, K.H., Crosbie, R.H., Venzke, D.P., Campbell, K.P., 2000. Biosynthesis of dystroglycan: processing of a precursor propeptide. FEBS Lett. 468, 79–83.
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Kachinsky, A.M., Froehner, S.C., Milgram, S.L., 1999. A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells. J. Cell Biol. 145, 391–402. Leschziner, A., Moukhles, H., Lindenbaum, M., Gee, S.H., Butterworth, J., Campbell, K.P., Carbonetto, S., 2000. Neural regulation of alphadystroglycan biosynthesis and glycosylation in skeletal muscle. J. Neurochem. 74, 70–80. Mayor, R., Morgan, R., Sargent, M., 1995. Induction of the prospective neural crest of Xenopus. Development 121, 767–777. Moore, S.A., Saito, F., Chen, J., Michele, D.E., Henry, D.E., Messing, A., Cohn, R.D., Ross-Barta, S.E., Westra, S., Williamson, R.A., Hoshi, T., Cambell, K.P., 2002. Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature 418 (6896), 422–425. Pannese, M., Lupo, G., Kablar, B., Boncinelli, E., Barsacchi, G., Vignali, R.,
1998. The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73, 73–83. Parsons, M.J., Campos, I., Hirst, E.M.A., Stemple, D.L., 2002. Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos. Development 129, 3505–3512. Ross, M.E., 2002. Full circle to cobbled brain. Nature 418, 376–377. Smalheiser, N.R., Kim, E., 1995. Purification of cranin, a laminin binding membrane protein. Identity with dystroglycan and reassessment of its carbohydrate moieties. J. Biol. Chem. 270, 15425–15433. Williamson, R.A., Henry, M.D., Daniels, K.J., Hrstka, R.F., Lee, J.C., Sunada, Y., Ibraghimov-Beskrovnaya, O., Campbell, K.P., 1997. Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dag1-null mice. Hum. Mol. Genet. 6, 831– 841.
Molecular cloning and expression analysis of dystroglycan during Xenopus laevis embryogenesis Andrea Lunardi, Luciana Dente* Dipartimento di Fisiologia e Biochimica, Sezione di Biologia Cellulare e dello Sviluppo, University of Pisa, Via G. Carducci 13, Ghezzano, Pisa 56010, Italy Received 26 July 2002; received in revised form 6 September 2002; accepted 11 September 2002
Abstract Dystroglycan is a transmembrane receptor protein that provides a structural linkage between extracellular matrix components and cytoskeletal proteins. It was originally characterized as a member of dystrophin associated protein complex in muscle but, unlike other proteins of this complex, mutations in the dystroglycan gene have not been implicated as a cause of muscular dystrophies. Indeed, dystroglycan is an essential gene, expressed early in development that, if removed in knockout mice, provokes lethal defects before the onset of myogenesis. Dystroglycan is synthesized as a precursor propeptide that is post-translationally cleaved and glycosylated to yield alpha and beta subunits. We have cloned and characterized a cDNA clone, containing the complete coding region of the dystroglycan precursor, from a Xenopus laevis cDNA library. We have performed a spatial and temporal analysis of its expression in X. laevis embryos, using whole-mount in situ hybridization and reverse transcription-polymerase chain reaction analysis. Early expression of dystroglycan in a variety of tissues of different embryological derivation suggests a crucial role in morphogenetic events, especially during central nervous system differentiation. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xenopus; Expression pattern; Dystroglycan; Muscular dystrophy; Dystrophin; Dystrophin associated proteins; DAP complex; CNS; Extracellular matrix; Basement membrane; Ectodermal layer; Eye; Ganglional cells
1. Results Search in NCBI-EST GenBank for dystroglycan homologs in Xenopus laevis provided two EST clones (acc. no. BE506803 and BG037666) showing considerable homology to 5 0 and 3 0 extremities of the human dystroglycan coding region (acc. no. L19711). We designed specific primers to isolate the complete coding region of dystroglycan from a stage 28/30 X. laevis head cDNA library, using polymerase chain reaction amplification. Sequence analysis of a 2661 bases cDNA fragment revealed a 886 amino acids long putative protein that shares high homology with human dystroglycan. In particular, the percentage of identity is 95% in the cytoplasmic and transmembrane regions of the beta subunits, 56% in the extracellular region of beta and 60% in the alpha subunits, respectively. A comparison between Xenopus dystroglycan sequence and those of human (L19711.1), mouse (AAH07150.1), bovine (BAA23650.1) and rabbit (X64393.1), is shown in Fig. 1. The highest homology among different species is always found in the beta-subunits, confirming the biological relevance of this * Corresponding author. E-mail address: [email protected] (L. Dente).
region. The cleavage site (Ser), the potential membrane targeting signals and the N and O-linked glycosylation sites are identical to those proposed in the human homolog (Deyst et al., 1995; Smalheiser and Kim, 1995; Holt et al., 2000) (Fig. 1). More recently the sequence of zebrafish dystroglycan has been cloned (Parsons et al., 2002). The percentage of identity is 96% in the cytoplasmic and transmembrane regions of the beta subunits, 36% in the extracellular region of the beta subunits and 56% in the alpha subunits, respectively. Temporal expression of X-dystroglycan mRNA (X-dg) during early X. laevis embryogenesis was revealed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. X-dg is present in embryos as a maternal transcript; zygotic expression persists in all the analyzed stages (Fig. 2). Spatial expression pattern was also analyzed using wholemount in situ hybridization and subsequent histological sections. The earliest expression of X-dg is detectable in early neurula stage embryos (st. 13) in the anterior half of the midline (Fig. 3A). At midneurula stage (st. 15) X-dg expression is present in the neural plate, where the neurogenic cells orient medio-laterally to participate in neural tube formation, and in the notochord, as revealed by sections (data not shown). A diffuse and punctate expression
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00091-1
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Fig. 1. Comparison of the amino acid sequences of dystroglycan proteins. Xenopus dystroglycan residues are in bold. Identities are in red colour, gaps are indicated by hyphens. Putative signal and transmembrane domains are boxed. The conserved serine, proposed as post-translational processing site is highlighted in yellow; N- and O-glycosilation sites are shaded in gray. Blue dots indicate two group of residues that have been suggested to be a targeting motif for basolateral surface of epithelial cells (Kachinsky et al., 1999).
A. Lunardi, L. Dente / Mechanisms of Development 119S (2002) S49–S54
Fig. 2. RT-PCR analysis to determine temporal expression of X-dg in whole embryos. X-dg is present in all the analyzed stages (top panel). Ornithine decarboxilase (ODC) amplification was used as control of the reaction (bottom panel). The line marked 2RT indicates all the ingredients of Xdg except for reverse transcriptase.
is also detectable in the epidermal ectoderm (Figs. 3B,C). At late neurula stage (st. 19) a strong expression site is localized in presumptive prosencephalic/mesencephalic region (Fig. 3D, arrowhead). Note the absence of expression in the region from which neural crest cells derive (Fig. 3E, dorsal view). During neural tube closure (st. 22), X-dg transcripts are clearly detectable in prosencephalon evaginations of the eye anlagen, in rhomboencephalon (Fig. 3F, arrowhead) and in the otic vescicle primordium (Fig. 3G, arrowhead). X-dg epidermal ectoderm expression is not present in the most anterior part of the embryo (Fig. 3G) and is limited to the sensorial layer of this tissue, as revealed by sections of this
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stage embryos (Figs. 3J,K). At early tail bud stage (st. 26) Xdg epidermal ectoderm signal disappears, while the expression remains concentrated in prosencephalon, mesencephalon, in a restricted anterior area of the rhomboencephalon and in the dorsomedian portion of eye vesicles, representing the prospective pigmented layer of the retina (Fig. 3H, arrowhead). Finally, at stage 33, X-dg transcripts are present in several tissues derived from different germ layers (Fig. 3I). Enlarged view of the dorsal trunk musculature shows dystroglycan expression at level of myotomes (Fig. 3L). On the other hand, using a cross-reacting antibody raised against human dystroglycan, we have detected immunoreactivity only at level of the transverse myosepta at this stage (Fig. 3M, arrowheads). A similar phenotype has been recently observed also in zebrafish embryos (Parsons et al., 2002). In the adult skeletal muscle the protein has specific sarcolemma localization, as already reported in human and mouse muscles (Fig. 3N). Serial transverse sections of stage 33 embryos from anterior to posterior revealed high expression in the olfactory placode (op), in the telencephalic ventricular zone (Fig. 4A), in the diencephalic ventricular region and in the stomodeum (Fig. 4B, arrowhead). In contrast, in the most posterior portion of the diencephalon X-dg expression is limited to
Fig. 3. Expression of X-dg during Xenopus development (A–M). Whole-mount in situ hybridization was carried out using digoxigenin-labeled cRNA probe. Nieuwkoop-Faber stages of embryogenesis are indicated in higher left corners. Dorsal views, anterior down (A, B, E); postero-lateral view (C); anterior views (D, F, H); lateral views, anterior on the right (G, I). Transverse section of stage 22 (J): arrow head indicates sensorial layer of the ectoderm; parasagittal section of stage 22 (K) shows strong expression in the eye anlage and in the diencephalon/mesencephalon regions. X-dg expression sites at stage 33 (arrow heads in I): central nervous system (black), heart (orange), lens epithelium (turquoise), mandibular, hyoid and branchial arches (yellow and green respectively), myotomes (purple), otic vesicle (red), pronephron (blue). Parasagittal and longitudinal sections of stage 33 showing X-dg transcripts (L) and protein localization (M) in the myotomes. Transverse section of adult skeletal muscle reveals protein localization in the sarcolemma (N).
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Fig. 4. (A–F) Antero-posterior transverse sections of central nervous system at stage 33. Eye expression at level of ganglion cells layer in the neural retina (G); and in the optic stalk (H). Abbreviations: die, diencephalon; ep, epiphysis; gcl, ganglion cells layer; h, hypothalamus; mes, mesencephalon; os, optic stalk; rho, rhombencephalon; st, stomodeum.
the dorsal aspect (Fig. 4C). Furthermore, high signal level is detectable in the epiphysis (ep), hypotalamus (hyp), optic lobe of the mesencephalon (Fig. 4E) and in the dorsal rhomboencephalon (Fig. 4F). In the eye, X-dg is expressed in the ganglion cell layer (gcl) of the neural retina (Fig. 4G) and at level of the optic stalk (os) (Fig. 4H). Neural X-dg expression was further analyzed during early metamorphosis stage (st. 48) by whole-mount in situ hybridization of dissected brains (Fig. 5). This procedure allows to distinguish with more details X-dg specific transcripts, in particular in the ventral territories, where expression is limited to the optic chiasma, hypothalamus, pituitary gland and olfactory bulb. Cloning of the Xenopus dystroglycan coding region allowed a detailed analysis of its expression during embryogenesis. In particular, our analysis revealed for the first time a dynamic pattern of X-dg expression at level of central nervous system that suggests a direct involvement in differentiation of specific structures. A crucial role of dystroglycan in branching epithelial morphogenesis (Durbeej and Ekblom, 1997; Henry and Campbell, 1998) and in muscle differentiation (Brown et al., 1999; Leschziner et al., 2000) has been previously demonstrated. On the other hand, owing to the inability to generate viable dystroglycan-null mice, because of lethal defects in early basal membrane formation, the role of dystroglycan in neuronal cell differentiation has remained elusive (Williamson et al., 1997). Recently Moore et al. made ‘conditional’ knockout mice, where dystroglycan expression was inactivated in glia and brain
Fig. 5. (A–C) Expression of X-dg as detected in dissected brains of stage 48 embryos by whole-mount in situ hybridization. Lateral view, anterior right (A); ventral (B); and dorsal (C) view, anterior up.
A. Lunardi, L. Dente / Mechanisms of Development 119S (2002) S49–S54
neurons only. These mutants have brain malformations, such as disarray of cerebral cortical layering, aberrant migration of granule cells that resemble specific human congenital brain dystrophies (Moore et al., 2002) and give new insight into the role of dystroglycan in neuronal development (Ross, 2002). In contrast, complete removal of dystroglycan in zebrafish embryos causes loss of muscle integrity and necrosis, but not embryo lethality (Parsons et al., 2002). The characterization of the coding region and the expression pattern of Xenopus dystroglycan, presented in this paper, opens the possibility to design specific tools to perturb dystroglycan function and to test its role during morphogenetic events. 2. Methods 2.1. Xenopus dystroglycan cloning Two EST cDNA clones were obtained from IMAGE Consortium and sequenced to confirm and to complete the sequences derived from the NCBI-EST data. The clone BE50680 contains 50 bp of 5 0 untranslated region followed by the first 318 nucleotides of the coding region. The complete sequence of clone BG037666 revealed that it contains the last 600 bp of the coding region and 1168 bp of 3 0 untranslated region, followed by a poly(A) tail 16 bp long. To clone the complete Xenopus dystroglycan coding region, forward primer 5 0 -CTGTAACAGAACGCCAAAATGG-3 0 including the start codon and reverse primer 5 0 AGGCGAATTCTTAAGGAGGTACATAAGGGGGT-3 0 including the stop codon were synthesized and used to amplify the cDNA fragment from st. 28/30 head Xenopus cDNA library. The complete sequence of clone XDAG has been submitted with accession number AJ496325.
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with the corresponding X-dg sense probe. Histological examinations were performed as described in Pannese et al. (1998). Bleaching of pigmented embryos was done after colour reaction as described (Mayor et al., 1995). 2.4. Immunohistochemistry Dissected muscle and embryos were immersion-fixed for 1 h at 4 8C in 4% paraformaldehyde in 100 mM phosphate buffer pH 7.4, then cryoprotected by immersion in 25% sucrose in PBS overnight at 4 8C. Tissues were frozen in freezing medium (OCT compound; Sakura Finetek Europe B.V.), sectioned vertically at 12 mm thickness in a cryostat, collected on slides (SuperFrost Plus; Menzel-Glaser, Germany), air dried for 30 min, and stored at 280 8C until use. Immunohistochemistry labeling: blocking in PBS pH 7.4 containing 10% normal goat serum, 1% bovine serum albumine and 0.05% Triton X-100, sections were incubated with primary monoclonal antibody (43DAG/ 8D5, Novocastra) in PBS supplemented with 3% NGS and 0.05% Triton X-100 for 60 min at room temperature and then with secondary goat anti-mouse antibody coupled to rodamine-isothiocyanate (TRITC; Sigma T7657) or to peroxidase (HRP; Sigma A2304), using 3,3 0 diaminobenzidine (DAB; Sigma D4293) substrate. Acknowledgements We are grateful to Richard Harland for the gift of the st. 28/30 head library. We thank Massimiliano Andreazzoli, Federico Cremisi and Robert Vignali for a critical reading of the manuscript, Gaia Gestri, Michela Ori, Simona Casarosa and Giuseppe Lupo for help and suggestions in the experiments; Marzia Fabbri and Donatella De Matienzo for technical assistance and Salvatore De Maria for frog care. This work was supported by a grant from M.U.R.S.T to L.D.
2.2. RT-PCR analysis RT-PCR analysis was performed using total RNA isolated from embryos at the indicated stages and the SuperScript eII RNase H 2 Reverse Transcriptase kit (Life Technologies). X-dg primers (forward: 5 0 -TATCGGAAGAAGAGGAAAGGCA-3 0 ; reverse: 5 0 -GTGACCCTTTACCTTCCATAGG-3 0 ) and ODC primers (forward: 5 0 -GTCAATGATGGAGTGTATGGATC-3 0 ; reverse: 5 0 -TCCATTCCGCTCTCCTGAGCAC-3 0 ) were annealed at 54 8C for 26 cycles. 2.3. In situ hybridization Whole-mount in situ hybridization on X. leavis embryos was performed as described (Harland, 1991), except that BM purple (Roche) was used as a substrate for the alkaline phosphatase, using a X-dg antisense cRNA 600 bases long. Non-specific hybridization of X-dg riboprobe was evaluated
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