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Keywords:

  • dystroglycan;
  • basement membrane;
  • muscle;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Using immunohistochemistry, we have examined β-Dystroglycan protein distribution in the mouse embryo at embryonic stages E9.5 to E11.5. Our data show that Dystroglycan expression correlates with basement membranes in many tissues, such as the notochord, neural tube, promesonephros, and myotome. In the myotome, we describe the timing of Dystroglycan protein re-distribution at the surface of myogenic precursor cells as basement membrane assembles into a continuous sheet. We also report on non-basement-membrane-associated Dystroglycan expression in the floor plate and the myocardium. This distribution often corresponds to sites of expression previously reported in adults, suggesting that Dystroglycan is continuously produced during development. Developmental Dynamics 236:2627–2635, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dystroglycan is a cell-surface multimeric protein encoded by a single gene, Dag-1 (Dystrophin associated glycoprotein) (Ibraghimov-Beskrovnaya et al.,1992). Translation of Dag-1 produces a 895–amino acid propeptide, which is subsequently cleaved by an unknown protease at a conserved serine residue (Ser 655) (Esapa et al.,2003) to generate a 72-kDa N-terminal product that contains a signal peptide, α-Dystroglycan, and a 26-kDa C-terminal product, β-Dystroglycan (Barresi and Campbell,2006). The mature α-Dystroglycan protein is a 120–156-kDa extra-cellular protein that lacks the signal peptide and contains numerous N-glycosylations on its N and C terminal domains and O-glycosylations in the central Mucin-like domain (Ibraghimov-Beskrovnaya et al.,1993). α-Dystroglycan is non-covalently linked to the 43-kDa mature trans-membrane β-Dystroglycan protein, also glycosylated in its N-terminal domain (Ibraghimov-Beskrovnaya et al.,1993).

The pattern of glycosylation of α-Dystroglycan varies between tissues, and dictates the specificity of ligand binding (Ervasti and Campbell,1993; Michele and Campbell,2003). Indeed, both α-Dystroglycan and β-Dystroglycan associate with numerous proteins (Winder,2001). Binding of β-Dystroglycan to Dystrophin was a key factor in the cloning of α- and β-Dystroglycan and their identification as components of the large Dystrophin Glycoprotein Complex (DGC) in skeletal muscle cells (Ibraghimov-Beskrovnaya et al.,1992). β-Dystroglycan binds to the C-terminal domain of Dystrophin via a PPXY motif situated at its C-terminus (Jung et al.,1995; Huang et al.,2000). In non-muscle cells, this motif is involved in the binding to Utrophin, a homolog of Dystrophin (Chung and Campanelli,1999; Ishikawa-Sakurai et al.,2004; Hnia et al.,2007). Other partners of β-Dystroglycan include: α-dystrobrevin, a dystrophin-related protein (Chung and Campanelli,1999); Caveolin-3, a muscle-specific Caveolin, localised to the sarcolemma (Sotgia et al.,2000); Grb2, MEK, and ERK downstream components of the ERK-MAP kinase cascade (Yang et al.,1995; Spence et al.,2004b); Dynamin-1, a GTPase implicated in endocytosis (Zhan et al.,2005); Rapsyn, a protein located at the post-synaptic membrane in neuromuscular junctions (Cartaud et al.,1998; Bartoli et al.,2001); Dbl and Ezrin, involved in regulation and organisation of the actin cytoskeleton (Spence et al.,2004b; Batchelor et al.,2007); and Plectin, a widely expressed cytolinker protein with diverse functions (Rezniczek et al.,2007). α-Dystroglycan associates to several partners via its extra-cellular domain, thus ensuring a connection between the extra-cellular matrix (ECM) and the cytoskeleton. The principal ECM components are Laminin 1 and 2, Agrin and Perlecan (Gee et al.,1994; Talts et al.,1999). Binding occurs on the mucin-like domain of α-Dystroglycan via globular Laminin G-like (LG) domains located on the alpha chain of Laminins (Tisi et al.,2000). More recently, Biglycan, a proteoglycan expressed in muscle cells, and Neurexin, a neural-specific cell surface protein, were also shown to bind to α-Dystroglycan (Bowe et al.,2000; Sugita et al.,2001).

The identity of Dystroglycan's associated proteins has suggested a role for Dystroglycan in the assembly of basement membranes and in the function of synaptic and neuromuscular junctions. Consistent with this prediction, Dag-1 mutant mice die at embryonic day 5.5 and display defects in the formation of the Reichert's membrane, an extra-embryonic basement membrane, and Dag-1 null embryonic bodies fail to assemble Laminin-1, suggesting a requirement for basement membrane assembly (Williamson et al.,1997; Henry and Campbell,1998). Furthermore, conditional knock-out mice that lack Dag-1 in the brain (in GFAP-expressing cells) display pial basement membrane disruption (Moore et al.,2002). However, recent studies indicate that Dystroglycan also functions in cell signalling and control processes such as cell proliferation and cell survival (Winder,2001). Establishing sites of Dystroglycan expression that are not associated to basement membranes should help identify tissues/cells in which Dystroglycan has a signalling function. Yet, the early lethality of Dag-1-deficient mice has precluded studies on the role of Dystroglycans in embryogenesis beyond implantation. In addition, little information is available on the distribution of α and β-Dystroglycan during embryogenesis, although Dag-1 mRNA expression pattern was reported in the early mouse embryo, and in the embryonic brain and kidneys (Schofield et al.,1995; Yotsumoto et al.,1996). α/β-Dystroglycan protein expression has essentially been reported in adult tissues such as skeletal muscles and kidneys (Ibraghimov-Beskrovnaya et al.,1992; Durbeej et al.,1995,1998).

In this study, we investigated the distribution of β-Dystroglycan proteins between E9.5 and E11.5 during mouse embryogenesis, its co-localization with Laminin, and its relationship to Myf5-expressing muscle cells. Our analysis reveals that β-Dystroglycan co-localises with Laminin in basement membranes during mouse embryogenesis, although it is also expressed in tissues independently of basement membranes. Together, our data provide a framework for future studies investigating the role of β-Dystroglycan in basement membrane assembly and/or tissue function.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

β-Dystroglycan Expression in the Developing Mouse CNS

At E9.5, β-Dystroglycan is strongly expressed in the ectoderm of the developing brain and in the basement membrane delineating the neuroepithelium of forebrain, hindbrain, and spinal cord, and the otic and optic vesicles (Fig. 1B–D). Interestingly, β-Dystroglycan is also expressed in the Rathke's pouch, a structure that will give rise to the anterior pituitary (Fig. 1B). By E11.5, β-Dystroglycan expression in the brain clearly marks the neuroepithelium of nasal pits, forebrain, midbrain, the cerebellar primordium, and the hindbrain (Fig. 1A). In addition, strong expression is observed in the developing pituitary (Fig. 1A). Expression of β-Dystroglycan in the otic vesicle is maintained at all stages examined (Fig. 1C,K). In these structures, expression of β-Dystroglycan co-localises with Laminin, consistent with its association to basement membranes (Fig. 1K, and data not shown). In the developing eye, both β-Dystroglycan and Laminin are present in the neuroepithelium of the optic vesicle at E9.5 (Fig. 1D) and lining the outer pigmented layer of optic cup (retina), lens, corneal ectoderm, and optic stalk at E10.5 and E11.5 (Fig. 1E,F). Interestingly, we observe that although Laminin is expressed on both sides of the pigmented layer of the future retina, β-Dystroglycan appears restricted to the basal side of the retinal pigmented epithelium (green and yellow arrows in Fig. 1E). In the spinal cord, both β-Dystroglycan and Laminin co-localise in the basement membrane surrounding the neural tube (Figs. 1G, 2E, I, 4F). However, we noticed that floor plate cells, although not expressing Dystroglycan in the posterior neural tube (see Fig. 4E,F), begin to preferentially express β-Dystroglycan at the thoracic level of the neural tube at E9.5 (Fig. 1H). This expression is detected more caudally in E10.5 and E11.5 embryos and extends to the hindbrain (white and red arrows in Fig. 1G,I,J,K). From E10.5 onward, β-Dystroglycan is also observed in motoneuron projections leaving the ventral horn of the spinal cord (white arrowhead in Fig. 1G). Noticeably, β-Dystroglycan and Laminin are also detected in the notochord throughout the anterio-posterior axis from E9.5 to E11.5 (Fig. 1G, and 2E,H,I).

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Figure 1. β-Dystroglycan expression in the mouse CNS. Immunofluorescence detection of β-dystroglycan (β-dys, red) and Laminin (Lm, green) on sagittal (A) and transverse (B–K) sections of E9.5 (B,C,D,H), E10.5 (E,G,I,K), and E11.5 (A,F,J) mouse embryos. A: β-dystroglycan is strongly expressed in the ectoderm, the brain neuroepithelium, and in the developing pituitary gland at E11.5. B,C: Transverse sections at E9.5 reveal β-dystroglycan distribution in the neuroepithelium of forebrain, hindbrain, Rathke's pouch, floor plate, and optic and otic vesicles. DF: β-dystroglycan co-localizes with Laminin in the neuroectoderm of the optic cup, the lens (le), in the corneal ectoderm (c), and in the outer layer of the optic cup (oc) of the developing eye between E9.5 and E11.5. GK: Co-labelling of β-dystroglycan and Laminin in the notochord and the neural tube basement membrane, whereas only β-dystroglycan labels floor plate cells of the spinal cord and hindbrain (white arrow), and motoneuron projections (white arrowhead). Np, nasal pit; pi, developing pituitary; fb, forebrain; hb, hindbrain; e, optic vesicle; ov, otic vesicle; rp, Rathke's pouch; nc, notochord. Red and green arrows indicate regions where Laminin (green) and β-dystroglycan (red) do not co-localise, whereas yellow arrows show sites of co-expression. Magnification: 100× (A,K), 200× (B,C,D,E,F,G), 400× (H–J).

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Figure 2. β-Dystroglycan expression in the developing heart and gut. Immunofluorescence detection of β-dystroglycan (β-dys, red) and Laminin (Lm, green) on sagittal (A,D,F) and transverse (B,C,E,G–I') sections of E9.5 (A–C), E10.5 (D,E), and E11.5 (F–I,I') mouse embryos. A: At E9.5, β-dystroglycan is detected in the branchial arch ectoderm, the trabeculated myocardium, and the pericardium. B,C: It is also strongly expressed in the hindgut (C) and foregut (B). D: At E10.5, β-dystroglycan proteins are present in the ectoderm of all branchial arches, and (E) in the foregut, atrial myocardium, and the ventricular myocardium. F: By E11.5, β-dystroglycan strongly labels the trabeculae of the ventricular myocardium, the atrial myocardium, and the epithelial lining of the stomach. GI: Laminin and β-dystroglycan are co-expressed in the atrial walls, the main bronchus of lung buds, the oesophagus, and the stomach. β-dystroglycan is also found in the cystic primordium of the gallbladder (G). I': High magnification of ventricle myocardium. Inset in I' shows that β-dystroglycan is present both within the trabeculae and in the trabeculae walls, whereas Laminin is only expressed at the surface of trabeculae. My, myocardium; pe, pericardium; ec, endocardium; st, stomach; fg, foregut; ba, branchial arch; hg, hindgut; a, atrium; ve, ventricle; h, heart; li, liver; cp, cystic primordium; gr, genital ridge; cp, cystic primordium; oe, oesophagus; lu, lung bud. Magnification: 100× (A,B,D–I), 200× (C), 400× (I'), 630× (inset in I').

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Figure 4. β-Dystroglycan expression in the myotomal basement membrane. Immunofluorescence detection of β-dystroglycan (β-dys, red), Laminin (Lm, green) and Myf5 (green) on sagittal (B–D) and transverse (E–P) sections of E9.5 (B–I), E10.5 (J–M), and E11.5 (N–P) mouse embryos. A: Schematic representation of E9.5, E10.5, and E11.5 embryos. The position of sagittal and transverse sections is indicated by grey arrows. BD: Sagittal sections of E9.5 embryos at an interlimb level show β-dystroglycan expression in Myf5-expressing myotomal cells, as well as associated with the myotomal basement membrane. E: Laminin is synthesised and deposited in the somitocoele of E9.5 caudal somites, and appears associated with low levels of β-dystroglycan (white arrowhead). F,G: Adjacent sections of E9.5 caudal interlimb somites illustrating that β-dystroglycan has a pericellular distribution in Myf5-positive cells entering the myotome (see inset in G). Note that Myf5-expressing dorsal medial lip (dml) cells do not express β-dystroglycan. At this stage, no Laminin-containing basement membrane has formed. H,I: Adjacent sections of E9.5 rostral somites showing polarised expression of β-dystroglycan at the time that a Laminin-containing myotomal basement membrane is present (white arrowheads and inset in I). J,K: E10.5 caudal somites display co-localisation of β-dystroglycan and Laminin in the epaxial myotomal basement membrane, and strong β-dystroglycan expression by all Myf5-positive cells in the myotome. L,M: Adjacent sections of E10.5 rostral somites showing co-localisation of β-dystroglycan and Laminin in the dml, in addition to the myotomal basement membrane. NP: By E11.5 at the hindlimb level, β-dystroglycan remains closely associated to the Laminin-containing basement membrane in the epaxial and hypaxial myotome. However, β-dystroglycan expression is down-regulated in the central myotome (white arrowhead). P: β-dystroglycan down-regulation in the central myotome coincides with myotomal growth as indicated by Myf5-positive cell ventral dispersion (white arrowheads). Dm, dermomyotome; sc, sclerotome; nt, neural tube; my, myotome; dml, dorsal medial lip of dermomyotome; vll, ventral lateral lip of dermomyotome. Magnification: 200× (D,E,M–Q), 400× (A–C,F–L), 1,000× (insets in G, I).

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β-Dystroglycan Expression in the Developing Heart and Gut

At E9.5, β-Dystroglycan is already expressed in columnar muscle cells of the trabeculated myocardium and pericardium (Fig. 2A,B), and its expression increases gradually until E11.5 (Fig. 2I). At this stage, β-Dystroglycan can be seen in the ventricular trabeculations, whereas Laminin is found essentially in the trabeculation walls (Fig. 2I'). β-Dystroglycan also marks the atrial myocardium, but not the endocardium (Fig. 2E,H). β-Dystroglycan proteins are present in the basement membrane surrounding the developing hindgut (Fig. 2C), midgut (not shown), and foregut (Fig. 2B), where they co-localise with Laminin (Fig. 2E). By E11.5, β-Dystroglycan and Laminin are both found in the basement membrane underlying the epithelial lining of the oesophagus and in the main bronchus of the lung buds (Fig. 2H), as well as in the epithelial lining of the stomach (Fig. 1F,G,I). β-Dystroglycan is also found in the cystic primordium of the gallbladder (Fig. 2G). Finally, β-Dystroglycan is expressed in the branchial arch ectoderm at all stages examined (Fig. 2A,D).

β-Dystroglycan Expression in the Developing Urogenital System

At E9.5, β-Dystroglycan is already strongly detected in the mesonephric duct (Fig. 3A,A'), where it co-localises with Laminin in the basement membrane (data not shown). This expression continues at E10.5 (Fig. 3B,B'), and low levels of β-Dystroglycan can be detected in the urogenital ridge (Fig. 3B). By E11.5, β-Dystroglycan and Laminin expression is robust in the mesonephric duct, and more anteriorly is now clearly visible in the mesonephric tubules and in the gonadal ridge, associated with the formation of primitive sex cords (Figs. 2G,I and 3C,D).

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Figure 3. β-Dystroglycan expression in the developing urogenital system. Immunofluorescence detection of β-dystroglycan (β-dys, red) and Laminin (Lm, green) on transverse sections of E9.5 (A,A'), E10.5 (B,B'), and E11.5 (C,D) mouse embryos. A,A': At E9.5, β-dystroglycan is strongly expressed in the mesonephric duct. B,B': At E10.5, β-dystroglycan remains co-localised with Laminin in the mesonephric duct, and begins to be expressed in the urogenital ridge. C,D: By E11.5, β-dystroglycan is clearly detected in the mesonephric duct and tubules, as well as the urogenital ridge. C is posterior to D. Gr, urogenital ridge; md, mesonephric duct; mt, mesonephric tubules. Magnification: 400× (A–D), 630× (A',B').

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β-Dystroglycan Expression in the Myotomal Basement Membrane

We next examined β-Dystroglycan distribution in somites along the antero-posterior axis of E9.5 to E11.5 mouse embryos (Fig. 4A). We find that Laminin is secreted and deposited in the somitocoele of caudal epithelial somites, and low levels of β-Dystroglycan are observed in a pattern overlapping that of Laminin (white arrowhead in Fig. 4E). As somites mature, Myf5-positive muscle progenitor cells delaminate first from the dorsal medial lip (dml) of the dermomyotome and populate the nascent myotome (Fig. 4G). This first myogenic population will be followed by a second population of Myf5-positive cells delaminating from the lateral dermomyotome (ventral lateral lip: vll) in rostral interlimb somites. In caudal interlimb somites, newly delaminated Myf5-positive cells express β-Dystroglycan, and β-Dystroglycan peptide distribution is pericellular (see inset in Fig. 4G). Notably, we observe that this first population of myogenic precursor cells enters the myotome and organises itself into a coherent sheet in the absence of basement membrane (compare Fig. 4F to 4G). However, Laminin organisation into a continuous sheet will rapidly occur following this initial step, and the myotomal basement membrane can be detected in anterior interlimb somites at E9.5 (data not shown). Interestingly, we notice that in rostral somites of E9.5 embryos following the formation of the myotomal basement membrane, β-Dystroglycan protein relocates from a predominantly pericellular distribution to a polarised expression tightly associated with the myotomal basement membrane (white arrowheads in Fig. 4H,I and inset in Fig. 4I). At rostral levels, all Myf5-positive cells that have delaminated from the dml and vll of the dermomyotome and entered the myotome express β-Dystroglycan in a pattern that overlaps with that of basement membrane-associated Laminin (Fig. 4B–D and compare Fig. 4H to 4I). At E10.5, more robust β-Dystroglycan expression is observed in Myf5-positive muscle cells in caudal somites (Fig. 4K), and laminin deposition into the basement membrane has been initiated (Fig. 4J). In E10.5 rostral somites, polarised β-Dystroglycan expression is observed in the basement membrane that underlies the myotome, as well as in the basement membrane that surrounds the dml (Fig. 4L,M). As embryonic development proceeds, redistribution of β-Dystroglycan from a pericellular to a polarised pattern takes place in younger somites and is readily detectable in hindlimb somites of E11.5 embryos (Fig. 4N,O). This dynamic pattern of expression correlates with the timing of myotomal basement membrane formation, which is already detected as a continuous sheet in caudal somites at older embryonic stages. By E11.5, β-Dystroglycan expression in myotomal cells of hindlimb somites decreases, in line with their differentiation (Fig. 4N,P). β-Dystroglycan association with the myotomal basement membrane remains intense in the most medial and lateral domains of the myotome (Fig. 4N,O). However, expression decreases and becomes patchy in the central myotomal domain (white arrowhead in Fig. 4N,P), although Laminin assembly is still visible (Fig. 4O). This expression pattern corresponds to a stage at which the myotome expands, as shown by the enlargement of the Myf5-expressing domain (Fig. 4P).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dystroglycan was initially cloned as a trans-membrane protein component of the dystrophin-glycoprotein complex. In agreement with this association, Dystroglycan is expressed at high levels in muscle cells and mutations that affect Dystroglycan activity cause muscular dystrophy (Cote et al.,1999). Dystroglycan is also a major player in basement membrane assembly, through α-Dystroglycan binding to the basement membrane components Laminin and Perlecan. Consistent with this function, Dag-1-deficient mice fail to form an embryonal basement membrane and in this study, we show that most sites of Dystroglycan expression during embryogenesis coincide with basement membranes. However, we also report tissues in which Dystroglycan distribution is not tightly correlated to basement membranes, suggesting that Dystroglycan has an additional function in these cells.

β-Dystroglycan in Epithelia and Basement Membranes

Our data report β-Dystroglycan association to epithelia and basement membranes in several embryonic organs, including notochord, spinal cord, lung buds, sex cords, and mesonephric duct and tubules. This distribution corroborates previous reports on Dystroglycan expression pattern at foetal stages, including the presence of transcripts in lung bud, urogenital, and gut epithelia, and the presence of proteins in the ureter, glomeruli, and tubules of developing kidneys (Durbeej et al.,1995). Notably, many tissues that express β-Dystroglycan in the embryo continue to do so in the adult, as is the case for kidneys, which express β-Dystroglycan in podocytes and glomerular basement membrane, and for testis (Durbeej et al.,1998). Continuous expression throughout development suggests that Dystroglycan is required at multiple stages during development, initially in basement membrane assembly (Williamson et al.,1997; Henry and Campbell,1998), and later in other processes essential to organ function. Analyses of these possible later roles in organogenesis have been pre-empted by the early lethality of Dag-1 mutant mice (Williamson et al.,1997), and will require the generation of conditional mutant animals. Although it is clear that some of these later roles will relate to basement membrane integrity, there is already evidence that Dystroglycan may be required for cell survival and cell migration (Li et al.,2002; Moore et al.,2002). In many instances, it may be difficult to ensure that these defects are not the consequence of a primary basement membrane defect (Montanaro and Carbonetto,2003). However, morpholino knockdown of Dystroglycan in the zebrafish embryo does not mimic Laminin mutant zebrafish that lack basement membrane and present a defect in notochord cell differentiation (Parsons et al.,2002a,b), indicating that defects can be distinguished.

β-Dystroglycan and Neurogenesis

We found that β-Dystroglycan is present at the basal side of the pigmented outer layer of the retina at E11.5. At the stages examined, we did not observe significant expression in the neural layer of the retina, although β-Dystroglycan is expressed by photoreceptors of the outer plexiform layer of the adult retina and also transiently in neural ganglia of the inner plexiform layer during foetal development (Montanaro et al.,1995; Blank et al.,2002).

Previous studies reported Dystroglycan mRNA expression in ependymal cells of the developing spinal cord and brain in the mouse embryo (Schofield et al.,1995). Here, we show that β-Dystroglycan is abundant in the brain neuroepithelium, and in the basement membranes surrounding the optic vesicle and the neural tube. This distribution is essential for the future formation of pial basement membrane, which plays a critical role in neuronal migration (Moore et al.,2002). Interestingly, β-Dystroglycan is also found in motoneuron axons and displays a pericellular distribution in floor plate cells of the spinal cord and hindbrain. This expression suggests that Dystroglycan may play a role in axon growth and guidance. In agreement with this hypothesis, knock-down of Dystroglycan in C. elegans causes motoneuron and commissural axon guidance defects (Johnson et al.,2006). In this context, it was suggested that β-Dystroglycan acts mainly as a Laminin receptor rather than a component of the Dystrophin Glycoprotein Complex. Considering the relatedness of Laminins and Netrins (Yurchenco and Wadsworth,2004), the known function of Netrins in axon guidance together with recent studies showing that Netrins can bind Integrin receptors (Hinck,2004), one could hypothesize that Dystroglycan expression in axons and floor plate cells mediate axon guidance through a Netrin/Integrin/Dystroglycan complex.

β-Dystroglycan and Muscle Differentiation

Our data show expression of β-Dystroglycan in the myocardium, specifically in the ventricular trabeculations. This distribution correlates with previous data reporting that Integrins α5, α6, and β1 are detected at high levels in the trabeculated component of ventricles and down-regulated or absent in the compacted myocardium (Carver et al.,1994; Collo et al.,1995; Pow and Hendrickx,1995). Our analysis does not allow us to address whether β-Dystroglycan is down-regulated in the compacted compartment of the myocardium at later stages. However, the co-localisation of β-Dystroglycan and integrins, together with our observation that Laminin is mainly expressed on the trabeculae walls, raise the possibility that β-Dystroglycan participates to processes involving ECM–cell interaction in the embryonic myocardium. Such a role has already been reported in cardiac remodeling of the pathological heart (Manso et al.,2006) and is in line with the suggestion that the ventricular conduction system originates from the trabeculated ventricles (Franco et al.,1998).

Another muscle cell population expressing Dystroglycan is that of skeletal muscle cells (Ibraghimov-Beskrovnaya et al.,1992). Its role in adult muscles as a Dystrophin-associated glycoprotein in the maintenance of myofiber integrity and the consequences of its loss in muscular dystrophies (Winder,2001), and its role as an Agrin-associated protein facilitating Acetyl choline receptor clustering at the neuromuscular junction (Sunada and Campbell,1995) are well established. Much less is known about its role in early skeletal myogenesis. Here, we show that Dystroglycan is synthesised by Myf5-expressing myogenic precursor cells entering the myotome. The initial distribution of Dystroglycan at the surface of myogenic precursor cells is reminiscent of that of another Laminin receptor, α6β1 Integrin (Bajanca et al.,2004). However, Dystroglycan proteins lose their pericelullar distribution and become polarised during myotome formation, such that most expression is associated with the Laminin-containing basement membrane in E9.5 rostral somites. This contrasts with α6β1 Integrin, whose distribution remains pericellular throughout myotome formation. The significance of this re-distribution is uncertain. One possibility is that it plays a role in myotomal basement assembly. Consistent with this possiblity, Dystroglycan mutations have been previously incriminated in defects in basement membrane assembly in several tissues (Williamson et al.,1997; Henry and Campbell,1998; Moore et al.,2002). However, we have found that Dystroglycan protein polarisation occurs after, rather than before, the formation of a continuous myotomal basement membrane. Thus, Dystroglycan re-distribution is unlikely to initiate basement membrane assembly, although Dystroglycan itself may still be essential for myotomal basement membrane formation. Moreover, this distribution is consistent with a possible role in maintaining basement membrane stability. Supporting this hypothesis is our observation that polarised Dystroglycan expression at the myotomal basement membrane disappears prior to Laminin disassembly in the central myotome at E11.5. This suggests that Dystroglycan could play an active role in initiating basement membrane disassembly through disengagement from the basement membrane. It has been proposed that myotome growth, which occurs in the central compartment of the myotome (Gros et al.,2005; Relaix et al.,2005), requires first the disruption of the underlying myotomal basement membrane (Bajanca et al.,2006). Indeed, one suggested role for the myotomal basement membrane is to promote differentiation, and act as a scaffold allowing migration and orientation of muscle progenitor cells entering the myotome (Bajanca et al.,2006).

β-Dystroglycan Distribution Is Conserved During Evolution

There is a remarkable conservation of Dystroglycan expression during evolution. Indeed, Dystroglycan expression in the developing urogenital system and CNS has been reported in C. elegans, Drosophila, and X. laevis embryos (Lunardi and Dente,2002; Moreau et al.,2003; Dekkers et al.,2004; Johnson et al.,2006). Interestingly, myogenic expression is conserved in the invertebrate Drosophila, in amphibians and fishes, but not in the nematode (Lunardi and Dente,2002; Parsons et al.,2002; Moreau et al.,2003; Dekkers et al.,2004; Johnson et al.,2006). Such conservation of Dystroglycan distribution during evolution suggests a crucial role for Dystroglycan in organogenesis.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Mouse Lines

CD1 mice (Charles River) were bred. Embryonic day 0.5 was the day vaginal plugs were found. Embryos were harvested at indicated time (between E9.0 and E12.5) by caesarian section.

Immunohistochemistry

Embryos were fixed in 0.2% PFA in 0.2M phosphate buffer + 4% sucrose overnight at 4°C, then washed in the same solution without PFA. Embryos were then washed in 15% sucrose in 0.12M phosphate buffer and incubated at 37°C for 1 hr in the same solution with 7.5% Gelatin. Embryos were embedded in the gelatin solution using chilled iso-pentane, then stored at −80°C. Twelve-micrometer-thick sections were collected on SuperFrost slides (BDH). Sections were permeabilised and blocked using PBS + 1% HINGS (heat inactivated goat serum) + 0.05% TritonX-100 (PBT) for approximately 1 hr. Primary antibodies diluted in PBT were applied and incubated overnight at 4°C. Slides were washed in PBT, then secondary antibodies were applied and incubated for 1 hr at room temperature followed by PBT washes. Slides were mounted with Mowiol + 2.5% DABCO. Control immunohistochemistry was performed by incubating slides directly with secondary antibodies, before proceeding with detection. No specific staining was observed with controls.

Primary antibodies used in this study were mouse anti-β-Dystroglycan MANDAG-2 (Pereboev et al.,2001) at a dilution of 1:50, rabbit anti-Laminin (L 9393, Sigma) at 1:100, and rabbit anti-Myf5 (sc-302, Santa Cruz) at 1:800. Secondary antibodies included Alexa Fluor-594 conjugated goat anti-mouse (A11005, Molecular Probes) diluted at 1:500 and goat anti-rabbit Cy2 (111-225-144, Jackson ImmunoResearch) at 1:200. Images were captured on a SP1 laser-scanning confocal microscope (Leica), and treated using ImageJ and Photoshop (Adobe) software.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from the European Commission Sixth Framework Programme (Cells into Organs contract: LSHM-CT-2003-504468, and MYORES contract: 511978) to A.G.B., and the Wellcome Trust (grant Number: GR077544AIA) to the Sheffield Light Microscopy Facility.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES