Sólveig Thorsteinsdóttir

EARLY DEVELOPMENT OF THE MYOTOME IN THE MOUSE

The structure and development of the myotome has been extensively studied in birds and amphibians but few studies have systematically addressed its development in mammals. We have used a transgenic mouse carrying an nLacZ marker coupled to a myosin light chain 3F promoter to describe the structure of the developing mammalian myotome. Through studies of transgene expression pattern, coupled with immunohistochemistry for the muscle structural proteins desmin and slow myosin heavy chain we describe a gradient of maturity for the cells within the developing myotome. Our results show that the earliest myocytes of the mammalian myotome span the rostrocaudal extent of the somite and have single large nuclei which localise centrally within the myotome. Throughout the period of study the myotome is more mature ventrally than dorsally and cells comprising the medial aspect of the myotome are younger than those lying laterally. Immunohistochemistry for the earliest expressed muscle regulatory factor (myf‐5) is used to define areas of the myotome contributing new myogenic cells. In the early myotome small, round, myf‐5‐expressing cells are found extensively within the dorsomedial aspect of the dermamyotome and also within the entire rostral and caudal dermamyotomal lips. They subsequently appear within the central zone of the myotome, adjacent to the medially curled rostral and caudal dermamyotomal lips, and there begin to elongate symmetrically. As the myotome enlarges, myf‐5 expression is always restricted to the most medial aspect of the myotome, adjacent to the least mature myocytes, marking the site of addition of new myogenic cells. Together, these results allow development of a model of mammalian myotome formation where growth occurs medially by addition of new cells from both rostral and caudal dermamyotome lips, while more mature myocytes are displaced laterally. Furthermore, early myotomal myocytes differentiate in the absence of MyoD expression, unlike later myotomal myocytes. This, along with their distinct morphology, suggests these cells may form a separate lineage of pioneer myogenic cells. Dev Dyn 1999;216:219–232. ©1999 Wiley‐Liss, Inc.

Spatial and temporal expression of the β1D integrin during mouse development

The β1D protein is a recently characterized isoform of the integrin β1 subunit that is present in cardiac and skeletal muscles. In this study, we have examined the expression of β1D in different types of skeletal muscle and in cardiac muscle and studied its distribution during mouse development, using new monoclonal antibodies specific for β1D. Immunoprecipitation studies revealed that, while β1A is strongly expressed in proliferating C2C12 myoblasts, β1D is only expressed after their differentiation to myotubes. In these myotubes, β1D is associated with different α subunits, namely α3A, α5, α7A, or α7B. Initially, during embryogenesis, the β1A subunit is the only β1 variant expressed in skeletal and cardiac muscle. The β1D subunit is first detected in skeletal muscle at E17.5, whereas in cardiac muscle its expression begins around the time of birth. Later the expression of β1A in skeletal and cardiac muscle becomes restricted to capillary cells, whereas β1D eventually becomes the only variant expressed in adult cardiac and skeletal muscle cells. The switch from the β1A to the β1D subunit in cardiac muscle cells coincides with the expression of α7. In adults there is a distinct concentration of β1D at the myotendinous junctions of muscle fibers and at costameres in both cardiac and skeletal muscle. In addition, β1D is present at intercalated discs in cardiac muscle and at neuromuscular junctions in skeletal muscle cells. The amount of β1D in different types of skeletal muscle (fast, slow, and mixed‐type) was similar, but cardiac muscle expressed almost five times as much of this protein. We suggest that β1D plays a role in the maintenance of the cytoarchitecture of mature muscle and in the functional integrity of the muscle cells. Dev. Dyn. 1997;210:472–486.

A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis.

All skeletal muscle progenitor cells in the body derive from the dermomyotome, the dorsal epithelial domain of developing somites. These multipotent stem cells express Pax3, and this expression is maintained in the myogenic lineage where Pax3 plays an important role. Identification of Pax3 targets is therefore important for understanding the mechanisms that underlie the onset of myogenesis. In a microarray screen of Pax3-GFP sorted cells, with analysis on Pax3 gain and loss of function genetic backgrounds, we identify Dmrt2, expressed in the dermomyotome, as a Pax3 target. In vitro gel shift analysis and chromatin immunoprecipitation with in vivo extracts show that Pax3 binds to a conserved 286 bp sequence, situated at −18 kb from Dmrt2. This sequence directs reporter transgene expression to the somite, and this is severely affected when the Pax3 site is mutated in the context of the locus. In Dmrt2 mutant embryos, somite maturation is perturbed and the skeletal muscle of the myotome is abnormal. We now report that the onset of myogenesis is also affected. This depends on activation, in the epaxial dermomyotome, of the myogenic determination gene, Myf5, through its early epaxial enhancer. This sequence contains sites that bind Dmrt2, which belongs to the DM class of DNA–binding proteins. Mutation of these sites compromises activity of the enhancer in transgenic embryos where the reporter transgene is under the control of the Myf5 epaxial enhancer. Transactivation of this site by Dmrt2 is demonstrated in vitro, and conditional overexpression of Dmrt2 in Pax3 expressing cells in the somite confirms the role of this factor in the activation of Myf5. These results reveal a novel genetic network, comprising a Pax3/Dmrt2/Myf5 regulatory cascade that operates in stem cells of the epaxial dermomyotome to initiate skeletal muscle formation.

Integrin α6β1-laminin interactions regulate early myotome formation in the mouse embryo

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 myogenin-negative 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 Myf5nlacZ/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 α6β1 integrin, a laminin receptor, suggesting that integrin α6β1-laminin interactions are required for myotomal laminin matrix assembly. Blockingα 6β1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5nlacZ/nlacZ embryos. Furthermore, inhibition ofα 6β1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that α6β1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining α6β1 expression on myogenic precursor cells, and that α6β1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin byα 6β1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.

Integrins in the mouse myotome: Developmental changes and differences between the epaxial and hypaxial lineage

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 α1, α4, α5, α6, and α7 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 α4 and α6 subunits showed a regionalization of the myotome and dermomyotome based on cell‐adhesion properties. We conclude that α6β1 may be an early marker of epaxial myogenic progenitor cells. In contrast, α4β1 is up‐regulated in the intercalated myotome after myocyte differentiation. Furthermore, α4β1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome. Developmental Dynamics 231:402–415, 2004.

Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm.

The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrinα 5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal α5β1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse

Cells interact with the extracellular matrix through receptors, most commonly of the integrin family. We (Cachaço et al. [ 2003 ] Development 130:1659–1671) and others (Schwander et al. [ 2003 ] Dev. Cell 4:673–685) have demonstrated a role for β1 integrins in mouse primary myogenesis. However, it is unclear what α subunits pair with β1 during this process in vivo. Here, we determined α subunit expression patterns at embryonic day (E) 11.5–E14.5. Differentiated myotomal myocytes express all α subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5–E12.5), laminin receptors α6β1 and α7β1 are undetectable, and an assembled laminin matrix is absent. Instead α1β1, α4β1, α5β1, and an αv‐containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5–E14.5, α6β1 and α7β1 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. Developmental Dynamics 232:1069–1078, 2005.

Knock-in of integrin β1D affects primary but not secondary myogenesis in mice

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 β 1D (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 β 1A 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 β 1 integrins in primary versus secondary myogenesis and that the β 1A and β1D variants are not functionally equivalent in this process.