The actin polymerization factor Diaphanous and the actin severing protein Flightless I collaborate to regulate sarcomere size.

The sarcomere is the basic contractile unit of muscle, composed of repeated sets of actin thin filaments and myosin thick filaments. During muscle development, sarcomeres grow in size to accommodate the growth and function of muscle fibers. Failure in regulating sarcomere size results in muscle dysfunction; yet, it is unclear how the size and uniformity of sarcomeres are controlled. Here we show that the formin Diaphanous is critical for the growth and maintenance of sarcomere size: Dia sets sarcomere length and width through regulation of the number and length of the actin thin filaments in the Drosophila flight muscle. To regulate thin filament length and sarcomere size, Dia interacts with the Gelsolin superfamily member Flightless I (FliI). We suggest that these actin regulators, by controlling actin dynamics and turnover, generate uniformly sized sarcomeres tuned for the muscle contractions required for flight.

Drosophila myogenesis.

The skeletal muscle system is the largest organ in motile animals, constituting between 35 and 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.

Diaphanous regulates SCAR complex localization during Drosophila myoblast fusion.

From Drosophila to man, multinucleated muscle cells form through cell-cell fusion. Using Drosophila as a model system, researchers first identified, and then demonstrated, the importance of actin cytoskeletal rearrangements at the site of fusion. These actin rearrangements at the fusion site are regulated by SCAR and WASp mediated Arp2/3 activation, which nucleates branched actin networks. Loss of SCAR, WASp or both leads to defects in myoblast fusion. Recently, we have found that the actin regulator Diaphanous (Dia) also plays a role both in organizing actin and in regulating Arp2/3 activity at the fusion site. In this Extra View article, we provide additional data showing that the Abi-SCAR complex accumulates at the fusion site and that excessive SCAR activity impairs myoblast fusion. Using constitutively active Dia constructs, we provide additional evidence that Dia functions upstream of SCAR activity to regulate actin dynamics at the fusion site and to localize the Abi-SCAR complex.

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

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.

PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site.

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.

RGMa and RGMb expression pattern during chicken development suggest unexpected roles for these repulsive guidance molecules in notochord formation, somitogenesis, and myogenesis.

BACKGROUND: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues. RESULTS: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo. CONCLUSIONS: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development.

Dynamic control of head mesoderm patterning.

The 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.

Extrinsic versus intrinsic cues in avian paraxial mesoderm patterning and differentiation.

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.

The molecular setup of the avian head mesoderm and its implication for craniofacial myogenesis.

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.

Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of alpha2beta1 integrin.

The alpha2beta1 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, alpha2beta1 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 alpha2-deficient mice, which we generated by targeting the integrin alpha2 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 alpha2-deficient mice, antibodies blocking integrin function and downregulation of integrin alpha2 expression. The results show that the alpha2beta1 integrin is absolutely required for keratinocyte adhesion to collagens whereas for fibroblasts other collagen-binding integrins partially back-up the lack of alpha2beta1 in simple adhesion to collagen monomers. A prominent requirement for alpha2beta1 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 alpha2-deficient fibroblasts appeared to be linked to alterations in the distribution of force-bearing focal adhesions and deregulation of Rho-GTPase activation.

Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development.

The related bHLH transcription factors MyoR and Capsulin control craniofacial myogenesis and the development of a number of mesoderm-derived organs in the mouse. However, their molecular function as regulators of differentiation processes is conversely debated. One approach to clarify the roles of these genes is to comparatively analyse their biological and molecular function in various vertebrate models. For this, a prerequisite is the determination of their similarity and their expression patterns. Here we show that vertebrate MyoR and Capsulin are paralogous genes with a high level of conservation regarding their protein sequence, their cDNA sequence and their chromosomal organisation. In the chick, both genes are co-expressed in the developing branchiomeric muscles, the anterior heart field and the splanchnopleura lining the foregut. However, both genes show unique expression domains in trunk skeletal muscle precursors, in the lateral and intermediate mesoderm.