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Three types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bataillé, Lebreton, Boukhatmi and Vincent.)

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells., (© 2022 The Authors. genesis published by Wiley Periodicals LLC.)

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis., Competing Interests: The authors declare that they have no conflict of interest.

The Drosophila lymph gland is the larval hematopoietic organ and is aligned along the anterior part of the cardiovascular system, composed of cardiac cells, that form the cardiac tube and its associated pericardial cells or nephrocytes. By the end of embryogenesis the lymph gland is composed of a single pair of lobes. Two additional pairs of posterior lobes develop during larval development to contribute to the mature lymph gland. In this study we describe the ontogeny of lymph gland posterior lobes during larval development and identify the genetic basis of the process. By lineage tracing we show here that each posterior lobe originates from three embryonic pericardial cells, thus establishing a bivalent blood cell/nephrocyte potential for a subset of embryonic pericardial cells. The posterior lobes of L3 larvae posterior lobes are composed of heterogeneous blood progenitors and their diversity is progressively built during larval development. We further establish that in larvae, homeotic genes and the transcription factor Klf15 regulate the choice between blood cell and nephrocyte fates. Our data underline the sequential production of blood cell progenitors during larval development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morin-Poulard, Destalminil-Letourneau, Bataillé, Frendo, Lebreton, Vanzo and Crozatier.)

Myogenesis is an evolutionarily conserved process. Little known, however, is how the morphology of each muscle is determined, such that movements relying upon contraction of many muscles are both precise and coordinated. Each Drosophila larval muscle is a single multinucleated fibre whose morphology reflects expression of distinctive identity Transcription Factors (iTFs). By deleting transcription cis-regulatory modules of one iTF, Collier, we generated viable muscle identity mutants, allowing live imaging and locomotion assays. We show that both selection of muscle attachment sites and muscle/muscle matching is intrinsic to muscle identity and requires transcriptional reprogramming of syncytial nuclei. Live-imaging shows that the staggered muscle pattern involves attraction to tendon cells and heterotypic muscle-muscle adhesion. Unbalance leads to formation of branched muscles, and this correlates with locomotor behavior deficit. Thus, engineering Drosophila muscle identity mutants allows to investigate, in vivo, physiological and mechanical properties of abnormal muscles., Competing Interests: AC, LB, GL, LD, AP, YC, AW, AV, JF No competing interests declared, (© 2020, Carayon et al.)

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) expressed by its FC. Here, we addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation?, Results: The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently., Conclusions: Transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.

The body plan of arthropods and vertebrates involves the formation of repetitive segments, which subsequently diversify to give rise to different body parts along the antero-posterior/rostro-caudal body axis. Anatomical variations between body segments are crucial for organ function and organismal fitness. Pioneering work in Drosophila has established that Hox transcription factors play key roles both in endowing initially identical segments with distinct identities and organogenesis. The focus of this review is on Alary Muscles (AMs) and the newly discovered Thoracic Alary-Related Muscles (TARMs). AMs and TARMs are thin muscles which together connect the circulatory system and different midgut regions to the exoskeleton, while intertwining with the respiratory tubular network. They were hypothesized to represent a new type of muscles with spring-like properties, maintaining internal organs in proper anatomical positions during larval locomotion. Both the morphology of TARMs relative to AMs, and morphogenesis of connected tissues is under Hox control, emphasizing the key role of Hox proteins in coordinating the anatomical development of the larva., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles.

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates., (© 2014. Published by The Company of Biologists Ltd.)

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

Understanding gene regulatory pathways underlying diversification of cell types during development is one of the major challenges in developmental biology. Progressive specification of mesodermal lineages that are at the origin of body wall muscles in Drosophila embryos has been extensively studied during past years, providing an attractive framework for dissecting cell type diversification processes. In particular, it has been found that muscle founder cells that are at the origin of individual muscles display specific expression of transcription factors that control diversification of muscle types. These factors, encoded by genes collectively called muscle identity genes, are activated in discrete subsets of muscle founders. As a result, each founder cell is thought to carry a unique combinatorial code of identity gene expression. Considering this, to define temporally and spatially restricted expression of identity genes, a set of coordinated upstream regulatory inputs is required. But also, to realize the identity program and to form specific muscle types with distinct properties, an efficient battery of downstream identity gene targets needs to be activated. Here we review how the specificity of expression and action of muscle identity genes is acquired., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Myogenesis is a highly conserved process ending up by the formation of contracting muscles. In Drosophila embryos, myogenesis gives rise to a segmentally repeated array of thirty distinct fibres, each of which represents an individual muscle. Since Drosophila offers a large range of genetic tools for easily testing gene functions, it has become one of the most studied and consequently best-described model organisms for muscle development. Over the last two decades, the Drosophila model system has enabled major advances in our understanding of how the initially equivalent mesodermal cells become competent for entering myogenic differentiation and how each distinct type of muscle is specified. Here we present an overview of Drosophila muscle development with a special focus on the diversification of muscle types and the genes that control acquisition of distinct muscle properties., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. Here, to understand how cell fate information residing within founders is translated during differentiation, we focus on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Mp20, Pax, and mspo, three genes that regulate actin dynamics and cell adhesion and, as we show here, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation., (2010 Elsevier Inc. All rights reserved.)

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions we performed muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search for direct Ladybird targets. Our data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, we identified bric-a-brac 2 gene as a new component of identity code and inflated encoding alphaPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells.

The differentiation of Drosophila embryonic blood cell progenitors (prohemocytes) into plasmatocytes or crystal cells is controlled by lineage-specific transcription factors. The related proteins Glial cells missing (GCM) and GCM2 control plasmatocyte development, whereas the RUNX factor Lozenge (LZ) is required for crystal cell differentiation. We have investigated the segregation process that leads to the formation of these two cell types, and the interplay between LZ and GCM/GCM2. We show that, surprisingly, gcm is initially expressed in all prohemocytes but is rapidly downregulated in the anterior-most row of prohemocytes, which then initiates lz expression. However, the lz+ progenitors constitute a mixed-lineage population whose fate depends on the relative levels of LZ and GCM/GCM2. Notably, we demonstrate that GCM/GCM2 play a key role in controlling the size of the crystal cell population by inhibiting lz activation and maintenance. Furthermore, we show that prohemocytes are bipotent progenitors, and that downregulation of gcm/gcm2 is required for lz-induced crystal cell formation. These results provide new insight into the mechanisms controlling Drosophila hematopoiesis and establish the basis for an original model for the resolution of the choice of blood cell fate.

Members of the GATA and RUNX families of genes appear to have conserved functions during hematopoiesis from Drosophila to mammals. In Drosophila, the GATA factor Serpent (Srp) is required in blood cell progenitors for the formation of the two populations of blood cells (plasmatocytes and crystal cells), while the RUNX factor Lozenge (Lz) is specifically required for crystal cell development. Here we investigate the function and the mechanisms of action of Lz during hematopoiesis. Our results indicate that Lz can trigger crystal cell development. Interestingly, we show that Lz function is strictly dependent on the presence of functional Srp and that Srp and Lz cooperate to induce crystal cell differentiation in vivo. Furthermore, we show that Srp and Lz directly interact in vitro and that this interaction is conserved between Drosophila and mammals. Moreover, both Srp and mouse GATA1 synergize with mouse RUNX1 to activate transcription. We propose that interaction and cooperation between GATA and RUNX factors may play an important role in regulating blood cell formation from Drosophila to mammals.

serpent (srp) encodes a GATA transcription factor essential for haematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. Here we show that srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). We have examined the respective functions of SrpC and SrpNC during embryonic haematopoiesis. Both isoforms individually rescue blood cell formation that is lacking in an srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. Our results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development.