Developmental origin of tendon diversity in Drosophila melanogaster.

Myogenesis is a developmental process that is largely conserved in both Drosophila and higher organisms. Consequently, the fruit fly is an excellent in vivo model for identifying the genes and mechanisms involved in muscle development. Moreover, there is growing evidence indicating that specific conserved genes and signaling pathways govern the formation of tissues that connect the muscles to the skeleton. In this review, we present an overview of the different stages of tendon development, from the specification of tendon progenitors to the assembly of a stable myotendinous junction across three different myogenic contexts in Drosophila: larval, flight and leg muscle development. We underline the different aspects of tendon cell specification and differentiation in embryo and during metamorphosis that result into tendon morphological and functional diversity.

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.

Transcriptomic and Genetic Analyses Identify the Krüppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development.

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor Dar1 as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.

Muscle development : a view from adult myogenesis in Drosophila.

In Drosophila the first wave of myogenesis occurs in the embryo to produce the larval muscles. This musculature undergoes histolysis and largely disappears during metamorphosis, while a second wave of myogenesis begins to generate the muscles of the adult fly. The core myogenic program is highly conserved among both invertebrate and vertebrate species, and Drosophila embryogenic myogenesis is a well-recognized model for identifying genes and pathways governing muscle development. The more diverse and complex adult musculature is also an attractive model to study some aspects of the myogenic process. The more intense research effort focusing on adult myogenesis since early this century has added greatly to our knowledge. We review here what we know about the development of adult muscles, from the specification and diversification of the adult muscle precursors to their final differentiation. The formation of a functional contractile unit requires integrating multiple tissue interactions. We therefore also describe how muscle cells interrelate with the tendons and the nervous and tracheal systems in the course of development.

Odd-skipped and Stripe act downstream of Notch to promote the morphogenesis of long appendicular tendons in Drosophila.

Multiple tissue interactions take place during the development of the limb musculoskeletal system. While appendicular myogenesis has been extensively studied, development of connective tissue associated with muscles has received less attention. In the developing Drosophila leg, tendon-like connective tissue arises from clusters of epithelial cells that invaginate into the leg cavity and then elongate to form internal tube-shape structures along which muscle precursors are distributed. Here we show that stripe-positive appendicular precursors of tendon-like connective tissue are set up among intersegmental leg joint cells expressing odd-skipped genes, and that Notch signaling is necessary and locally sufficient to trigger stripe expression. This study also finds that odd-skipped genes and stripe are both required downstream of Notch to promote morphogenesis of tube-shaped internal tendons of the leg.

Characterization of Drosophila Muscle Stem Cell-Like Adult Muscle Precursors.

Uncovering how muscle stem cells behave in quiescent and activated states is central to understand the basic rules governing normal muscle development and regeneration in pathological conditions. Specification of mesodermal lineages including muscle stemlike adult muscle precursors (AMPs) has been extensively studied in Drosophila providing an attractive framework for investigating muscle stem cell properties. Restricted number of AMP cells, relative ease in following their behavior, and large number of genetic tools available make fruit fly an attractive model system for studying muscle stem cells. In this chapter, we describe the recently developed tools to visualize and target the body wall and imaginal AMPs.

Coordinated Development of Muscles and Tendon-Like Structures: Early Interactions in the Drosophila Leg.

The formation of the musculoskeletal system is a remarkable example of tissue assembly. In both vertebrates and invertebrates, precise connectivity between muscles and skeleton (or exoskeleton) via tendons or equivalent structures is fundamental for movement and stability of the body. The molecular and cellular processes underpinning muscle formation are well-established and significant advances have been made in understanding tendon development. However, the mechanisms contributing to proper connection between these two tissues have received less attention. Observations of coordinated development of tendons and muscles suggest these tissues may interact during the different steps in their development. There is growing evidence that, depending on animal model and muscle type, these interactions can take place from progenitor induction to the final step of the formation of the musculoskeletal system. Here, we briefly review and compare the mechanisms behind muscle and tendon interaction throughout the development of vertebrates and Drosophila before going on to discuss our recent findings on the coordinated development of muscles and tendon-like structures in Drosophila leg. By altering apodeme formation (the functional Drosophila equivalent of tendons in vertebrates) during the early steps of leg development, we affect the spatial localization of subsequent myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon-like precursors, and confirm the appendicular Drosophila muscle system as a valuable model for studying these processes.

The conserved transcription factor Mef2 has multiple roles in adult Drosophila musculature formation.

Muscle is an established paradigm for analysing the cell differentiation programs that underpin the production of specialised tissues during development. These programs are controlled by key transcription factors, and a well-studied regulator of muscle gene expression is the conserved transcription factor Mef2. In vivo, Mef2 is essential for the development of the Drosophila larval musculature: Mef2-null embryos have no differentiated somatic muscle. By contrast, a similar phenotype has not been seen in analyses of the function of Mef2 genes in other examples of myogenesis. These include using conditional mutant mice, using morpholinos in zebrafish and using hypomorphic mutants in Drosophila adult development. However, we show here that Mef2 is absolutely required for a diverse range of Drosophila adult muscle types. These include the dorso-longitudinal muscles (DLMs), the largest flight muscles, which are produced by tissue remodelling. Furthermore, we demonstrate that Mef2 has temporally separable functions in this remodelling and in muscle maintenance. Drosophila adult muscles are multi-fibre and physiologically diverse, in common with vertebrate skeletal muscles, but in contrast to Drosophila larval muscles. These results therefore establish the importance of Mef2 in multiple roles in examples of myogenesis that have parallels in vertebrates and are distinct from that occurring in Drosophila embryogenesis.

The Him gene inhibits the development of Drosophila flight muscles during metamorphosis.

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.

Shaping leg muscles in Drosophila: role of ladybird, a conserved regulator of appendicular myogenesis.

Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. Ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.

Coordinated development of muscles and tendons of the Drosophila leg.

Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors, we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.

The ink4a/arf locus evolution in primates: characterization of three ARF sequences.

The human ink4a/arf locus encodes two cell cycle regulatory proteins - the cyclin-dependent kinase inhibitor (p16(ink4a)), and the p53 activator (ARF) - through the use of alternative first exons. This genomic organization is unique in eukaryotes, with two different proteins obtained using different reading frames. The divergence between mouse or opossum and human ARF is very high, whereas proteins have the same nucleolar localization and function. To gain further insights into the relative importance of ARF in different settings, we characterized here the exon 1beta of ARF in 12 different species of primates. We did not find any polymorphism in studied species (monkeys, apes, and humans). These sequences are very similar, with few amino acids substitutions compared to the human sequence. It is strange to find such a high degree of conservation among primates when there is such a low degree of conservation between the human pig, rat, or mouse, chicken exon 1beta sequences. More surprisingly, we observe a threonine at position 31 in all human sequences, whereas an alanine is always present in other sequences. We suggest that when the radiation human/simian appeared or after, a selection of threonine occurred. Moreover, the modifications detected could play a role in different interactions between ARF and other proteins to stabilize or not these complexes.