Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell-ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell-ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell-ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this "extracellular matrix dimension" should be added to our conceptual network of factors contributing to skeletal myogenesis.

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellular organisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, we describe the expression patterns of the ␣1, ␣4, ␣5, ␣6, and ␣7 integrin subunits in the mouse myotome and correlate them with the expression of several differentiation markers. Our results indicate that these integrin subunits may be differentially involved in the various phases of myogenic determination and differentiation. A detailed characterization of the myogenic cell types expressing the ␣4 and ␣6 subunits showed a regionalization of the myotome and dermomyotome based on cell-adhesion properties. We conclude that ␣6␤1 may be an early marker of epaxial myogenic progenitor cells. In contrast, ␣4␤1 is up-regulated in the intercalated myotome after myocyte differentiation. Furthermore, ␣4␤1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome. Developmental Dynamics 231:402-415, 2004.

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogeninnegative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5 nlacZ/nlacZ (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the ␣6␤1 integrin, a laminin receptor, suggesting that integrin ␣6␤1-laminin interactions are required for myotomal laminin matrix assembly. Blocking ␣6␤1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5 nlacZ/nlacZ embryos. Furthermore, inhibition of ␣6␤1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that ␣6␤1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining ␣6␤1 expression on myogenic precursor cells, and that ␣6␤1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by ␣6␤1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.

Here, we determined ␣ subunit expression patterns at embryonic day (E) 11.5-E14.5. Differentiated myotomal myocytes express all ␣ subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5-E12.5), laminin receptors ␣6␤1 and ␣7␤1 are undetectable, and an assembled laminin matrix is absent. Instead ␣1␤1, ␣4␤1, ␣5␤1, and an ␣v-containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5-E14.5, ␣6␤1 and ␣7␤1 are expressed, and a laminin matrix forms around individual myotubes. Thus, myogenic cells change their integrin expression pattern during the course of primary myogenesis in the mouse, suggesting different roles for fibronectin-and laminin-containing matrices in this process.

When migrating in vivo, cells are exposed to numerous conflicting signals: chemokines, repellents, extracellular matrix, growth factors. The roles of several of these molecules have been studied individually in vitro or in vivo, but we have yet to understand how cells integrate them. To start addressing this question, we used the cephalic neural crest as a model system and looked at the roles of its best examples of positive and negative signals: stromal-cell derived factor 1 (Sdf1/Cxcl12) and class3-Semaphorins. Here we show that Sdf1 and Sema3A antagonistically control cell-matrix adhesion via opposite effects on Rac1 activity at the single cell level. Directional migration at the population level emerges as a result of global Semaphorin-dependent confinement and broad activation of adhesion by Sdf1 in the context of a biased Fibronectin distribution. These results indicate that uneven in vivo topology renders the need for precise distribution of secreted signals mostly dispensable.

Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin-proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin-proteasome pathway, its biological function is still unknown. In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell-extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of alpha-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls. In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.

Integrins are extracellular matrix receptors composed of α and β subunits involved in cell adhesion, migration and signal transduction. The β1 subunit has two isoforms, β1A ubiquitously expressed and β1D restricted to striated muscle. They are not functionally equivalent. Replacement of β1A by β1D (β1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the β1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The β1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of β1A with β1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in β1D knock-in embryos. Finally, overexpression of β1D in C2C12 cells impaired myotube formation while overexpression of β1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for β1 integrins in primary versus secondary myogenesis and that the β1A and β1D variants are not functionally equivalent in this process.

In vertebrates, the embryonic nervous system is shaped and patterned by a series of temporally and spatially regulated cell divisions, cell specifications, and cell adhesions and movements. Morphogens of the Hedgehog, Wnt, and bone morphogenetic protein families have been shown to play a crucial role in the control of cell division and specification in the trunk neural tube, but their possible implication in the regulation of adhesive events has been poorly documented. In the present study, we demonstrate that Sonic hedgehog regulates neural epithelial cell adhesion and polarity through regulation of integrin activity, cadherin cell-cell contact, and cell polarity genes in immature neural epithelial cells before the specification of neuronal cells. We propose that Sonic hedgehog orchestrates neural tube morphogenesis by coordinating adhesive and motility events with cell proliferation and differentiation.