The head mesoderm is the mesodermal tissue on either side of the brain, from forebrain to hindbrain levels, and gives rise to the genuine head muscles. Its relatedness to the more posterior paraxial mesoderm, the somites, which generate the muscles of the trunk, is conversely debated. To gain insight into the molecular setup of the head mesoderm, its similarity or dissimilarity to the somitic mesoderm, and the implications of its setup for the progress of muscle formation, we investigated the expression of markers (1) for mesoderm segmentation and boundary formation, (2) for regional specification and somitogenesis and (3) for the positive and negative control of myogenic differentiation. We show that the head mesoderm is molecularly distinct from somites. It is not segmented; even the boundary to the first somite is ill-defined. Importantly, the head mesoderm lacks the transcription factors driving muscle differentiation while genes suppressing differentiation and promoting cell proliferation are expressed. These factors show anteroposteriorly and dorsoventrally regionalised but overlapping expression. Notably, expression extends into the areas that actively contribute to the heart, overlapping with the expression of cardiac markers.

The α2β1 integrin is a collagen-binding protein with very high affinity for collagen I. It also binds several other collagens and laminins and it is expressed by many cells, including keratinocytes and fibroblasts in the skin. In the past, α2β1 integrin was suggested to be responsible for cell attachment, spreading and migration on monomeric collagen I and contraction of three-dimensional collagen lattices. In view of these functions, normal development and fertility in integrin α2-deficient mice, which we generated by targeting the integrin α2 gene, came as a surprise. This suggested the existence of compensatory mechanisms that we investigate here using primary fibroblasts and keratinocytes isolated from wild-type and α2-deficient mice, antibodies blocking integrin function and downregulation of integrin α2 expression. The results show that the α2β1 integrin is absolutely required for keratinocyte adhesion to collagens whereas for fibroblasts other collagen-binding integrins partially back-up the lack of α2β1 in simple adhesion to collagen monomers. A prominent requirement for α2β1 integrins became apparent when fibroblasts executed mechanical tasks of high complexity in three-dimensional surroundings, such as contracting free-floating collagen gels and developing isometric forces in tethered lattices. The deficits observed for α2-deficient fibroblasts appeared to be linked to alterations in the distribution of force-bearing focal adhesions and deregulation of Rho-GTPase activation.

SUMMARYThe embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. We show that this pattern is established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, we found that the process is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of MyoR and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart.

Somitic and head mesoderm contribute to cartilage and bone and deliver the entire skeletal musculature. Studies on avian somite patterning and cell differentiation led to the view that these processes depend solely on cues from surrounding tissues. However, evidence is accumulating that some developmental decisions depend on information within the somitic tissue itself. Moreover, recent studies established that head and somitic mesoderm, though delivering the same tissue types, are set up to follow their own, distinct developmental programmes. With a particular focus on the chicken embryo, we review the current understanding of how extrinsic signalling, operating in a framework of intrinsically regulated constraints, controls paraxial mesoderm patterning and cell differentiation.

No abstract

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization.

The skeletal muscle system is the largest organ in motile animals, constituting between 35 to 55% of the human body mass, and up to 75% of the body mass in flying organisms like Drosophila. The flight muscles alone in flying insects comprise up to 65% of total body mass. Not only is the musculature the largest organ system, it is also exquisitely complex, with single muscles existing in different shapes and sizes. These different morphologies allow for such different functions as the high frequency beating of a wing in a hummingbird, the dilation of the pupil in a human eye, or the maintenance of posture in a giraffe's neck.Myogenesis, the development of the musculature, has received considerable attention for its unveiling of basic mechanisms including signaling, transcriptional and posttranscriptional control of cell fate, cell-cell fusion, cellular differentiation and cellular syncytium repair. An increased focus in the field is application of these basic mechanisms to congenital muscle diseases, aging, and cancer-induced muscle wasting (cachexia). The fruit fly Drosophila melanogaster has been the model system of choice for many aspects of myogenesis, successfully leading the field by identifying mechanisms for signal integration on specific promoters, the site for myoblast fusion site, the connection of aberrant myonuclear position to muscle function, and how forces sculpt myofibril formation, among many others. These paradigms have provided novel genes and mechanisms and have shaped studies in other model systems. Moreover the fly model system continues to be adapted to address novel challenges, especially in disease modeling, and no doubt will influence and be influenced by other models. Myogenesis research in Drosophila makes use of Drosophila's short generation span, ease of genetic manipulation, simplicity of the muscle pattern, and optical tractability with the help of fluorescent reporters. Both basic cell biological processes, such as cell-cell fusion and organelle positioning, and systemic processes, such as muscle growth and atrophy, can be effectively studied in this system. This primer introduces the key steps and notable variations during Drosophila myogenesis, including gastrulation and muscle formation in the embryo, muscle growth in the larva, and stem cell based muscle remodeling in the pupa, to give rise to a walking and flying adult. We will illustrate these relevant processes using the Dorsal Oblique 1 (DO1) muscle through the life of the organism as our example (Figure 1).