Regulation and Functions of the lms Homeobox Gene during Development of Embryonic Lateral Transverse Muscles and Direct Flight Muscles in Drosophila

BACKGROUND: Patterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development. METHODOLOGY/PRINCIPAL FINDINGS: The newly described homeobox gene, termed lateral muscles scarcer (lms), which has yet uncharacterized orthologs in other invertebrates and primitive chordates but not in vertebrates, is expressed exclusively in subsets of developing muscle tissues. In embryos, lms is expressed specifically in the four lateral transverse (LT) muscles and their founder cells in each hemisegment, whereas in larval wing imaginal discs, it is expressed in myoblasts that develop into direct flight muscles (DFMs), which are important for proper wing positioning. We have analyzed the regulatory inputs of various other muscle identity genes with overlapping or complementary expression patterns towards the cell type specific regulation of lms expression. Further we demonstrate that lms null mutants exhibit reduced numbers of embryonic LT muscles, and null mutant adults feature held-out-wing phenotypes. We provide a detailed description of the pattern and morphology of the direct flight muscles in the wild type and lms mutant flies by using the recently-developed ultramicroscopy and show that, in the mutants, all DFMs are present and present normal morphologies. CONCLUSIONS/SIGNIFICANCE: We have identified the homeobox gene lms as a new muscle identity gene and show that it interacts with various previously-characterized muscle identity genes to regulate normal formation of embryonic lateral transverse muscles. In addition, the direct flight muscles in the adults require lms for reliably exerting their functions in controlling wing postures

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

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

AbstractIn Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell–cell interactions within the developing Drosophila ventral nerve cord

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis

ETUDE DE LA FONCTION DES GENES A HOMEOBOITE, LADYBIRD, DANS LA MYOGENESE CHEZ DROSOPHILA MELANOGASTER (DOCTORAT)

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Contrôle génétique et propriétés de cellules souches musculaires chez Drosophila melanogaster

Ces dernières années, des cellules souches musculaires ont été identifiées chez la drosophile. Ces cellules, nommées les précurseurs de muscles adultes (AMPs) ont des caractéristiques similaires aux cellules souches musculaires (cellules satellites) présentent chez les vertébrés. L'analyse des cellules AMP chez la drosophile, un modèle simple et adapté aux analyses génétiques est une alternative afin d'apporter de nouvelles données sur les cellules souches musculaires. Au cours de ma thèse, je me suis intéressé à déterminer l'origine, la morphologie ainsi que les voies de signalisation impliquées dans la détermination des cellules AMP. Cette étude a permis d'établir l'origine d'une sous-population de cellules AMP, les cellules AMP latérales (LAMP). Celles-ci sont issues de progéniteurs musculaires spécifiques caractérisés par l'expression des gènes ladybird (lb) et krüppel. L'analyse morphologique des cellules AMP a ensuite montré l'existence de prolongements membranaires permettant l'établissement de connexions physiques entre elles. Enfin, une analyse détaillée de la voie de signalisation EGFR (Epidermal Growth Factor Receptor) a montré son rôle essentiel dans la spécification de la majorité des cellules AMP. Au niveau des cellules LAMP, la fonction de la voie EGFR s'effectue via la régulation directe des gènes lb nécessaires à leur identité cellulaire. Un effet protecteur plus tardif de la voie EGFR vis-à-vis des cellules AMP est également envisagé. La régulation des cellules satellites impliquant également la voie de signalisation EGFR et les gènes ladybird, nos résultats montrent l'existence de voies de signalisation communes entre les cellules souches musculaires présentes chez la drosophile et chez les vertébrés. Dans une 2ème partie, je me suis intéressé à déterminer la fonction musculaire d'un nouveau gène (CG10576) dont l'homologue vertébré, Ebp1 (ErbB3 binding protein 1) est impliqué dans la voie de signalisation EGFR. Des résultats préliminaires suggèrent une fonction de la protéine CG10576 au niveau du maintien de l'intégrité du tissu musculaire. Cette fonction pouvant être liée à son interaction avec le facteur de transcription SO (Sine Oculis) dont l'homologue vertébré Six1 est essentiel à la myogenèse. Des analyses complémentaires devraient permettre de définir la fonction précise des gènes CG10576 et SO au cours de la mygenèse.Recently, muscle stem cells with properties similar to satellite cells have been identified in Drosophila. These cells called adult muscle precursors (AMPs) represent a new model well adapted for genetic analyses and well suited for providing new insights into biology of muscle stem cells. During my PhD trainning I have analysed the origin, the morphology and signaling pathways implicated in specification of AMPs. My work has led to the identification of the progenitor cell expressing two genes ladybird (lb) and Kruppel (Kr) from which arise lateral AMPs (LAMPs). Morphological analyses allowed to identify long cellular processes sent by AMP cells that make them interconnected. Finally, detailed functional analysis of EGFR pathways components has revealed their critical role in specification of the majority of AMPs. In LAMPs, EGFR functions via direct regulation of lb genes, which are required for cellular identity of lateral AMPs. In later stages EGFR pathways is likely to protect AMPs from apoptosis. Interestingly, the antiapoptotic activity of EGFR and the specific expression of ib orthologue, Lbx1 have also been reported in satellite cells, strongly suggesting that common genetic mechanisms operate in Drosophila and in vertebrate muscle stem cells. A second part of my PhD project was dedicated to analyse the role of a new gene (CG10576) whose vertebrate homologue, Ebp1 (ErbB3 binding protein 1) is involved in EGF signaling pathway. In situ hybridisation and antibody staining showed that CG10576 is expressed exclusively in differentiating muscle fibers. Preliminary data revealed that CG10576 is required for late aspects of muscle differentiation and muscle patterning. This function is likely to be due to the interactions with transcription factor Sine Oculis (SO) with which CG10576 is coexpressed and whose vertebrate homologue Six1 plays an essential role in myogenesis. Additional analyses are required to define the precise role of CG10576 and SO during myogenesis and their link with the EGFR pathway.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Molecular mechanisms underlying appendicular myogenesis in Drosophila melanogasaster

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 specialized appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognized as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultra structure 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 (duf). 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 ultra structure, 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 signalling. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fiber formation. A key role for ladybird in leg myogenesis is further supported by its ability to repress vestigial in myoblasts and to down-regulate the vestigial-governed flight muscle developmental program. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialized 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 marketing an appendage adapted for locomotion.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Genetic Control of Muscle Diversification and Homeostasis: Insights from Drosophila

In the fruit fly, Drosophila melanogaster, the larval somatic muscles or the adult thoracic flight and leg muscles are the major voluntary locomotory organs. They share several developmental and structural similarities with vertebrate skeletal muscles. To ensure appropriate activity levels for their functions such as hatching in the embryo, crawling in the larva, and jumping and flying in adult flies all muscle components need to be maintained in a functionally stable or homeostatic state despite constant strain. This requires that the muscles develop in a coordinated manner with appropriate connections to other cell types they communicate with. Various signaling pathways as well as extrinsic and intrinsic factors are known to play a role during Drosophila muscle development, diversification, and homeostasis. In this review, we discuss genetic control mechanisms of muscle contraction, development, and homeostasis with particular emphasis on the contractile unit of the muscle, the sarcomere

Diversification of muscle types in Drosophila embryos

International audienceDrosophila embryonic somatic muscles represent a simple and tractable model system to study the gene regulatory networks that control diversification of cell types. Somatic myogenesis in Drosophila is initiated by intrinsic action of the mesodermal master gene twist, which activates a cascade of transcriptional outputs including myogenic differentiation factor Mef2, which triggers all aspects of the myogenic differentiation program. In parallel, the expression of a combinatorial code of identity transcription factors (iTFs) defines discrete particular features of each muscle fiber, such as number of fusion events, and specific attachment to tendon cells or innervation, thus ensuring diversification of muscle types. Here, we take the example of a subset of lateral transverse (LT) muscles and discuss how the iTF code and downstream effector genes progressively define individual LT properties such as fusion program, attachment and innervation. We discuss new challenges in the field including the contribution of posttranscriptional and epitranscriptomic regulation of gene expression in the diversification of cell types

La formation des muscles de la patte chez Drosophila melanogaster (un nouveau modèle d'étude de la myogenèse appendiculaire)

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF