Dev Genes Evol (2003) 213:355–359 DOI 10.1007/s00427-003-0328-6
EXPRESSION NOTE
Nicole Moreau · Dominique Alfandari · Alban Gaultier · Hlne Cousin · Thierry Darribre
Cloning and expression patterns of dystroglycan during the early development of Xenopus laevis Received: 27 January 2003 / Accepted: 25 March 2003 / Published online: 9 May 2003 Springer-Verlag 2003
Abstract Dystroglycan is a cell surface receptor involved in the pathogenesis of muscular dystrophy, and plays a critical role in the assembly and homeostasis of basement membranes. Since data about the amphibian homologue are limited, we have cloned the full-length dystroglycan cDNAs from the frog Xenopus laevis. Using in situ hybridization, we show that mRNA expression is dynamic, particularly in the notochord at the end of gastrulation and during neurulation, suggesting that the protein might play unexplored roles in the specification and/or formation of this tissue. Subsequently, the transcripts are detected in the otic vesicle, the developing brain, and in mesenchymal cells of the visceral arches, as well as in pharyngeal endoderm, the pronephros, pronephric ducts, proctodaeum and the somites. Keywords Amphibian · Dystroglycan · Early development · Notochord · Xenopus laevis Dystroglycan is a cell surface glycoprotein which binds to the extracellular matrix components laminin, merosin, agrin, neurexin and perlecan. On the cytoplasmic side, dystroglycan is anchored to the cytoskeletal protein dystrophin (Winder 2001). It is also associated with Grb2, which participates in signal transduction pathways. In skeletal muscle cells, dystroglycan serves to stabilize the sarcolemma and is thought to prevent membrane damage during contraction. Patients with muscle-eyebrain disease (MEB) and congenital muscular dystrophy Edited by R.P. Elinson N. Moreau · A. Gaultier · T. Darribre ()) Universit Pierre et Marie Curie, Laboratoire de Biologie du Dveloppement, UMR CNRS 7622, 9 quai Saint Bernard , 75005 Paris, France e-mail:
[email protected] D. Alfandari · A. Gaultier · H. Cousin Department of Cell Biology, School of Medicine, University of Virginia, Box 800732, Charlottesville, Virginia, 22908, USA
possess an abnormally glycosylated form of dystroglycan which disrupts its binding to the extracellular proteins (Michele et al. 2002). In the brain, dystroglycan participates in cell adhesion at the glial–vascular interface and in neuronal migration (Moore et al. 2002). In Drosophila, dystroglycan organizes cellular polarity during both oogenesis and early embryogenesis (Deng et al. 2003). Mice lacking dystroglycan manifest lethal defects early in embryogenesis which are associated with abnormal assembly of the embryonic extracellular matrix (Williamson et al. 1997). All together these data indicate that dystroglycan has different and pivotal roles in embryogenesis as well as in tissue formation and integrity. Finally, it is possible that dystroglycan carries out currently unknown functions during early development. For this reason, and because there are few data concerning the presence, distribution and function of dystroglycan during early development of amphibian, we have cloned the Xenopus dystroglycan cDNA and examined its expression pattern.
Cloning and sequence analysis Partial cDNA were amplified by reverse transcriptionpolymerase chain reaction using the following degenerate primers: sense: 50 YTNGCNYWNGCNTTYGGNGAYMGN30 ; antisense: 50 NSWNSWNGGNGGNGGYTTNSWRTCRTC30 . Amplification products of the expected size, 519 bp, and coding Xenopus dystroglycan were used to screen a stage 45 lZAP cDNA library. Two full-length cDNAs were isolated and sequence comparisons demonstrate that the two clones were identical. The cDNA sequence is 3172 nucleotides long, coding an open reading frame of 2654 bp. The open reading frame starts at nucleotide 181 (ATG) and extends to two termination codons at position 2836. The deduced amino acid sequence starts at the first methionine with a hydrophobic stretch corresponding to a predicted signal sequence. The predicted protein is 886 amino acids long with a molecular weight of 96 kDa for the non-glycosy-
356 Fig. 1 Comparison of the predicted amino acid sequence of Xenopus dystroglycan with human, mouse and zebrafish dystroglycan . The amino acid sequence is shown in single letter code. The signal peptide and the mucin-like domain are double underlined. The b-dystroglycan sequence is underlined. The shaded region indicates the transmembrane domain. The amino acid sequences of human, mouse and zebrafish were obtained from the GenBank database with accession numbers AAH12740, XP135139, and AAM78508 respectively
lated core protein (Fig. 1). It shares 56% amino acid identity with the human (AAH12740), mouse (XP135139), and zebrafish (AAM78508) sequences. The region of amino acids 312–478 is rich in proline, serine and threonine (84 of the 166 amino acids), a feature found in mucin-like proteins. A high degree of glycosylation is predicted for this region of the a-dystroglycan (Net-O-glyc, http://www.cbs.dtu.dk) (Fig. 1), according to the high molecular weight found for Xenopus a-dystro-
glycan in muscle cells (Cohen et al. 1995). The overall sequence similarity to the known dystroglycan homologues, and the presence of both conserved mucin-like and identical cytoplasmic domains indicate that the isolated cDNA corresponds to the Xenopus laevis dystroglycan homologue. The full-length Xenopus dystroglycan cDNA sequence has been submitted to the GenBank (Accession Number is AY188392). It shows amino acid
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Fig. 2a–m Legend see page 358
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identity with the Xenopus dystroglycan sequence reported previously (AJ496325).
Spatio-temporal expression of dystroglycan transcripts during development The temporal expression profile of dystroglycan transcripts was analyzed by reverse transcriptase-polymerase chain reaction using RNAs from different developmental stages. Maternal and zygotic expressions of dystroglycan were detected (not shown). We performed whole-mount in situ hybridization to determine the spatial expression of dystroglycan in the developing Xenopus embryo. Embryos were hybridized by standard procedures (Harland 1991). The antisense or control sense RNA probes corresponding to the encoding sequence of a-dystroglycan (nucleotides 181–2103) or the partial encoding sequence of b-dystroglycan (nucleotides 2155–2589) were constructed and tested with consistent results. Localized expression of dystroglycan transcripts (XDG) was first detected at the end of gastrulation and was thereafter seen throughout embryogenesis (Fig. 2). The XDG expression was detected at stage 12.5 (late gastrula) in the dorso-anterior third of the embryo (Fig. 2a). The staining formed a strip along the midline which extended towards the posterior portion of the embryo during the course of neurulation. The staining was also detected in two distinct patches of cells flanking t
Fig. 2a–m Spatial expression of dystroglycan transcripts during early Xenopus embryogenesis. Whole-mount in situ hybridizations were performed using digoxigenin-labeled antisense RNA probes (nucleotides 2155–2589 in a, b, d; nucleotides 181–2103 in c, e– m). Embryo orientation: anterior to the right, posterior to the left. Section orientation: dorsal to the top, ventral to the bottom. a, b Dorsal view of stage 12.5 (a) and 15 (b) embryos. XDG expression is seen in the anterior third and extends to the posterior region of the embryo. Scale bar: 250 mm. c Stage 19. Inside view of a dissected embryo. Notochord and the anterior part of the embryo are stained (arrowheads). Scale bar: 250 mm. d Lateral view, stage 22. XDG is expressed in the anterior and dorsal region of the embryo. Scale bar: 500 mm. e Transversal section in the trunk, stage 22. The notochord (arrow), the dermatome (arrowheads) and the double-layered epidermis are stained. (ar Archenteron.) Scale bar: 130 mm. f Lateral view, stage 27. XDG transcripts are present in the brain, the otic vesicles (ov), the visceral arches, the somites, the pronephros (p) and the pronephric duct (pd). Scale bar: 550 mm. g Control embryo, stage 27. Scale bar: 550 mm. h Oblique section through the head at the level of the optic vesicles (opv) (stage 27). The dorsal part of the mesencephalon (me) and the ventral pharyngeal endoderm are labeled. (ph Pharynx.) Scale bar: 150 mm. i Section posterior to h. XDG is expressed in the notochord (white arrowhead) and the mesenchyme of the hyoid arch (black arrowhead). Scale bar: 150 mm. j Transverse section of the trunk (stage 27). The notochord, the hypochord (arrowhead) and pronephric ducts (pd) are labeled. (Sc Spinal chord.) Scale bar: 80 mm. k Lateral view, stage 33/34. Note that three arches and the proctodaeum are stained. Scale bar: 50 mm. l Lateral view of a control stage 33/34 embryo. Scale bar: 30 mm. m Close-up view of a stage 40 embryo. The staining is associated with the notochord (arrowheads), the nephrostosmes (arrow) and the somites. Scale bar: 10 mm
the midline in the anterior region of the neurula (Fig. 2b, c). To localize more precisely the domain of XDG expression, we dissected the dorsal part of stained embryos. Figure 2c shows that transcripts were essentially confined to the notochord, in accordance with the expression described in zebrafish (Parsons et al. 2002), and to two lateral patches of ectodermal cells. Subsequently by stage 22, XDG expression was prominent in dorsal and anterior regions of the embryo (Fig. 2d). In transversal sections, the staining was found in the notochord, and lateral to the somites, in the dermatome which forms a continuous subepidermal layer (Fig. 2e). Cells expressing XDG were also detected in the double-layered epidermis. In the anterior region, XDG expression was found to be associated with the neural tube (not shown). By stage 27, XDG expression was visible in the somites, the pronephros, the pronephric ducts and the branchial arches. Within the neural tissue, staining was detected in the brain and otic vesicles (Fig. 2f). XDG expression in the developing ear correlates with otic placode induction in the sensorial layer of epidermis at stage 21 (not shown), invagination at stage 24 (not shown) and separation from the epidermis at stages 24–27 (Fig. 2f). Transversal and oblique sections at the level of optic vesicles revealed XDG expression in the dorsal half of the brain and in the ventral part of the pharyngeal endoderm (Fig. 2h). In sections at the level of branchial arches, XDG transcripts were also associated with the dorsal part of the neural tube, the notochord, the ventral pharyngeal endoderm and mesenchymal cells of the arches (Fig. 2i). By sectioning transversally through the trunk, we noticed that XDG expression was absent in the spinal cord and we observed that cells expressing XDG were restricted to the notochord, the hypochord, the somites and the pronephric ducts (Fig. 2j). By stage 39–40, we noticed prominent stripes of XDG expression associated with the hyoid, mandibular and branchial arches (Fig. 2k). XDG expression remained associated with the excretory system during the extensive coiling of the pronephric tubules (Fig. 2k) and the formation of the nephrostome (Fig. 2m). Both of these areas are also sites of dystroglycan expression in the mouse embryos (Durbeej et al. 1995) whereas they are not expressed in zebrafish embryos (Parsons et al. 2002). In the posterior region of the embryos, XDG-expressing cells are detected in the proctodaeum (Fig. 2k) and further in the rectal diverticulum (not shown). Transversal sections revealed that the labeling remained associated with the dorsal part of the brain and was absent from the spinal chord (not shown). Finally, at stage 39–40, the XDG-expressing cells are still clearly discernible in the notochord (Fig. 2m), in contrast to zebrafish from which it is absent in the mature notochord (Parsons et al. 2002). At all stages, hybridization with the control sense XDG probe did not produce any staining except for the cement gland (Fig. 2g–i; arrows). In conclusion, we have isolated the Xenopus homologue of the mammalian dystroglycan gene. We found that XDG is expressed in the notochord in accordance
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with the expression pattern described for zebrafish embryos and may indicate an unexplored role for dystroglycan in the specification and/or formation of this tissue, in particular in relation to matrix proteins since it has been shown that Xenopus a-dystroglycan is a laminin receptor in muscle cells (Cohen et al. 1997). We also found expression in the pharyngeal endoderm and visceral arches, which contrasts with the reported distribution of dystoglycan in mouse and zebrafish embryos (Durbeej et al. 1998; Parsons et al. 2002) suggesting that in Xenopus dystroglycan may have additional functions that remain to be investigated. Finally, the expression pattern of Xenopus dystroglycan described in this paper is similar to and completes the one recently presented by Lunardi and Dente (2002). Acknowledgements The authors acknowledge Dr Bette Dzamba for critical reading of the manuscript, Claude Brandonne for technical support and Dr M. Delarue for comments on the XDG expression patterns. This work was supported by grants from CNRS, University Pierre and Marie Curie, the Institut Universitaire de France. Drs D. Alfandari, A. Gaultier and H. Cousin are supported by the United States Public Health Grant DE14365.
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