Larval Wing Imaginal Discs Research Articles

A genome-wide RNAi screen for genes important for proliferation of cultured Drosophila cells at low temperature identifies the Ball/VRK protein kinase

A change in ambient temperature is predicted to disrupt cellular homeostasis by affecting all cellular processes in an albeit non-uniform manner. Diffusion is generally less temperature-sensitive than enzymes, for example, and each enzyme has a characteristic individual temperature profile. The actual effects of temperature variation on cells are still poorly understood at the molecular level. Towards an improved understanding, we have performed a genome-wide RNA interference screen with S2R + cells. This Drosophila cell line proliferates over a temperature range comparable to that tolerated by the parental ectothermic organism. Based on effects on cell counts and cell cycle profile after knockdown at 27 and 17 °C, respectively, genes were identified with an apparent greater physiological significance at one or the other temperature. While 27 °C is close to the temperature optimum, the substantially lower 17 °C was chosen to identify genes important at low temperatures, which have received less attention compared to the heat shock response. Among a substantial number of screen hits, we validated a set successfully in cell culture and selected ballchen for further evaluation in the organism. This gene encodes the conserved metazoan VRK protein kinase that is crucial for the release of chromosomes from the nuclear envelope during mitosis. Our analyses in early embryos and larval wing imaginal discs confirmed a higher requirement for ballchen function at temperatures below the optimum. Overall, our experiments validate the genome-wide screen as a basis for future characterizations of genes with increased physiological significance at the lower end of the readily tolerated temperature range.

Regulation of spatial distribution of BMP ligands for pattern formation.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, have been shown to contribute to embryogenesis and organogenesis during animal development. Relevant studies provide support for the following concepts: (a) BMP signals are evolutionarily highly conserved as a genetic toolkit; (b) spatiotemporal distributions of BMP signals are precisely controlled at the post-translational level; and (c) the BMP signaling network has been co-opted to adapt to diversified animal development. These concepts originated from the historical findings of the Spemann-Mangold organizer and the subsequent studies about how this organizer functions at the molecular level. In this Commentary, we focus on two topics. First, we review how the BMP morphogen gradient is formed to sustain larval wing imaginal disc and early embryo growth and patterning in Drosophila. Second, we discuss how BMP signal is tightly controlled in a context-dependent manner, and how the signal and tissue dynamics are coupled to facilitate complex tissue structure formation. Finally, we argue how these concepts might be developed in the future for further understanding the significance of BMP signaling in animal development.

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Hydroxyapatite nanoparticle causes sensory organ defects by targeting the retromer complex in Drosophila melanogaster

Hydroxyapatite nanoparticles are utilized extensively in the era of nanotechnology to deliver various drugs to the right area. However, the molecular mechanism behind the transportation of these nanoparticles and its effect on the development and maintenance of sensory organs is not known. The current study aims to check the effect of hydroxyapatite nanoparticle (HApNP) on sensory organs of Drosophila. Various concentrations of HApNP (10 mg·L−1, 20 mg·L−1, 40 mg·L−1 and 80 mg·L−1) were mixed with fly food and the developmental defects were monitored. Sensory organs like the eye and the mechanosensory organs were studied in a developmental time window. The larval eye-antennae and wing imaginal discs were checked for apoptotic cell death. In pupae, the damage becomes more prominent and it persists till adult. In the adult fly, the eye with a fused facet, defective cone and rhabdomere appear. Similarly, the wing with wing spots, macrochaetae, and defective alignment of hairs were also observed. All these phenotypic defects suggest the mistargeting of various proteins involved in the development. The current study correlates the phenotypic defects observed in the eye and wing is due to the down-regulation of the genes associated with the retromer complex.

A Unique Histone 3 Lysine 14 Chromatin Signature Underlies Tissue-Specific Gene Regulation

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac) and H3K4 methylation, histone modifications common to most active genes. Here, we describe a chromatin state featuring unique H3K14ac peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 with arginine in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defects in wing patterning. We find that the bromodomain in the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Multiple Functions of the Essential Gene PpV in Drosophila Early Development.

Protein phosphatase V (PpV) encodes the Drosophila homolog of the evolutionarily conserved Protein Phosphatase 6 (PP6). The physiological and developmental functions of PpV/PP6 have not been well characterized due to lack of a genetically defined mutant. Here, we identified a PpV non-sense mutation and describe multiple mutant phenotypes in oogenesis and early embryogenesis. Specifically, we found that the defects in chromosome segregation during nuclear cycles are related to AuroraA function, which is consistent with the interaction of PP6 and AuroraA in mammalian cells. Surprisingly, we also identified a PpV function specifically in blastoderm cell cycle but not in cell proliferation in the follicle epithelium or larval wing imaginal discs. Embryos from PpV germline clones frequently undergo an extra nuclear division cycle. By epistasis analysis, we found that PpV functions in parallel with tribbles, but independently of auroraA for the remodeling of the nuclear cycles. Taken together, this study reports novel developmental functions of PpV and provides a framework for further genetic analysis under physiological conditions.

Ex vivo Drosophila Wing Imaginal Disc Culture and Furin Inhibitor Assay.

Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are processed by Furin. To quantitatively assess the potential role of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a simple assay by combining genetics, ex vivo organ culture, pharmacological treatment, and imaging analyses. The protocol herein describes how to ex vivo culture Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over a long period of time in the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl parts using microscopy. Although the assay described here is for assessing the effect of Furin inhibition on Bnl cleavage in the Drosophila larval wing imaginal disc, the principle and methodology can easily be adopted for any other signals, tissue systems, or organisms. This strategy and protocol provide an assay for examining Furin activity on a specific substrate by directly visualizing the spatiotemporal distribution of its truncated parts in an ex vivo-cultured organ.

Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster

The eye and wing morphology of Drosophila melanogaster maintain unique, stable pattern of genesis from larval eye and wing imaginal discs. Increased apoptosis in cells of eye and wing discs was found to be associated with flubendiamide (fluoride containing insecticide) exposure (at the range 0.25–10μg/mL) in D. melanogaster larvae. The chemical fed larvae on attaining adulthood revealed alterations in morphology and symmetry of their compound eyes and wings through scanning electron microscopy. Nearly 40% and 30% of flies (P generation) demonstrated alterations in eyes and wings respectively. Transmission electron microscopic study (at the range 1–20μg/mL) also established variation in the rhabdomere and pigment cell orientation as well as in the shape of the ommatidium. Subsequent SEM study with F1 and F2 generation flies also revealed structural variation in eye and wing. Decrease in percentage of altered eye and wing phenotype was noted in subsequent generations (P> F1>F2). Thus, the diamide insecticide, flubendiamide, expected to be environmentally safe at sub-lethal concentrations was found to increase apoptosis in larvae and thereby cause morphological alteration in the adult D. melanogaster. This study further demonstrated trans-generational transmission of altered phenotype in three subsequent generations of a non-target insect model, D. melanogaster.

STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila.

We report here a study of regeneration in Drosophila larval wing imaginal discs after damage by ionizing radiation. We detected faithful regeneration that restored a wing disc and abnormal regeneration that produced an extra wing disc. We describe a sequence of changes in cell number, location and fate that occur to produce an ectopic disc. We identified a group of cells that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.

Regulation of Notch Signaling by the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex in Drosophila melanogaster.

Notch signaling is an evolutionarily conserved pathway that is found to be involved in a number of cellular events throughout development. The deployment of the Notch signaling pathway in numerous cellular contexts is possible due to its regulation at multiple levels. In an effort to identify the novel components integrated into the molecular circuitry affecting Notch signaling, we carried out a protein-protein interaction screen based on the identification of cellular protein complexes using co-immunoprecipitation followed by mass-spectrometry. We identified Hrp48, a heterogeneous nuclear ribonucleoprotein in Drosophila, as a novel interacting partner of Deltex (Dx), a cytoplasmic modulator of Notch signaling. Immunocytochemical analysis revealed that Dx and Hrp48 colocalize in cytoplasmic vesicles. The dx mutant also showed strong genetic interactions with hrp48 mutant alleles. The coexpression of Dx and Hrp48 resulted in the depletion of cytoplasmic Notch in larval wing imaginal discs and downregulation of Notch targets cut and wingless Previously, it has been shown that Sex-lethal (Sxl), on binding with Notch mRNA, negatively regulates Notch signaling. The overexpression of Hrp48 was found to inhibit Sxl expression and consequently rescued Notch signaling activity. In the present study, we observed that Dx together with Hrp48 can regulate Notch signaling in an Sxl-independent manner. In addition, Dx and Hrp48 displayed a synergistic effect on caspase-mediated cell death. Our results suggest that Dx and Hrp48 together negatively regulate Notch signaling in Drosophila melanogaster.

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway.

Abstract B13: EGF/Ras/Erk signaling controls growth and proliferation through regulation of tRNA synthesis

Abstract Ras signaling promotes growth and proliferation in many tissues throughout animal development. An important challenge is to identify how the Ras-Erk pathway alters cellular metabolism to drive growth. Here we report on the control of tRNA synthesis as growth effector of EGF/Ras/Erk signaling in Drosophila. I find that overexpression of oncogenic Ras (RasV12) leads to increased mRNA translation and protein content in Drosophila S2 cells, suggesting that Ras may promote growth through enhanced protein synthesis. The conventional view is that the Ras pathway functions by controlling translation initiation factor activity. However I have identified an alternate mechanism involving control of tRNA synthesis. My data suggest that overexpression of RasV12 or the activated versions of EGFR and the Raf1 in wing imaginal discs increases tRNA synthesis. Similarly, expression of RasV12 in S2 cells increases tRNA levels, while blocking Ras/Erk signaling using the MEK inhibitor, U0126 or RNAi reduces tRNA synthesis. We previously identified the RNA polymerase III (Pol III) factor, Brf, as regulator of cell and tissue growth in Drosophila. Here we show that knockdown of either Brf blocks the effects of Ras signaling on growth and proliferation in larval wing imaginal discs, adult midgut progenitor cells and adult intestinal stem cells. Several transcription factors have been shown to link Ras signaling to changes in mRNA expression and growth. I have identified Myc is required but not sufficient for Ras-induced cell proliferation and growth through tRNA synthesis. Previously we have shown that TOR signaling regulates protein synthesis through RNA Pol III repressor, Maf1. I found that Maf1 RNAi increases tRNA synthesis in S2 cells. In addition, the decrease in tRNA synthesis induced by the MEK inhibitor is blocked in the presence of Maf1 RNAi suggesting Maf1 is downstream of Ras signaling pathway. My data point to control of tRNA synthesis possibly through Myc and/or Maf1 as a new mechanisms by which Ras signaling enhances protein synthesis to promote cell and tissue growth. Citation Format: Shrivani Sriskanthadevan-Pirahas, Savraj S. Grewal. EGF/Ras/Erk signaling controls growth and proliferation through regulation of tRNA synthesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B13.

Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System

Cell ablation is a strategy to study cell lineage andfunction during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool forcell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ’s final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal–distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue.

NK2-specific domain is responsible for cell death upon ectopic expression of VND in various Drosophila tissues

Drosophila ventral nervous system defective (VND) is a transcription factor containing tinman domain, homeodomain, and NK2-specific domain. The VND plays an essential role during the development of embryonic nervous system, however, little is known about its roles beyond embryonic stage. In this study, the expression patterns and levels of the VND during larval and adult stages were examined by using GAL4/UAS binary expression system and real-time PCR. The VND protein was expressed in larval brain, eye, and wing imaginal discs, implying possible roles of this protein in the larval tissues other than embryonic nervous system. Gain-of-function phenotypes of vnd were analyzed by crossing various GAL4 drivers with UAS-vnd transgenic line. Interestingly, we observed the massive cell death in the tissues including eye and wing discs where GAL4 is expressed. To down-regulate endogenous vnd transcripts in the eye disc, UAS-vnd-IR (inverted repeat) was crossed with ey3.5-GAL4 line. Loss-of-function phenotypes of vnd also revealed the black ommatidial spots indicating apoptotic cell death in the transgenic eye tissues. To identify a domain within the VND involved in apoptosis, VND mutant proteins were ectopically expressed in the eye disc. In contrast to VND-Δtinman and VND-Y54M mutant proteins, overexpression of VND-ΔNK2 did not lead to cell death, indicating that NK2-specific domain is responsible for apoptosis. Taken together, our results suggest that VND might be involved in the development of the various tissues after embryonic stages and in apoptosis via NK2-specific domain.

Transgenic Expression of the Helicobacter pylori Virulence Factor CagA Promotes Apoptosis or Tumorigenesis through JNK Activation in Drosophila

Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

In-Vivo Imaging of the Drosophila Wing Imaginal Disc over Time: Novel Insights on Growth and Boundary Formation

In developmental biology, the sequence of gene induction and pattern formation is best studied over time as an organism develops. However, in the model system of Drosophila larvae this oftentimes proves difficult due to limitations in imaging capabilities. Using the larval wing imaginal disc, we show that both overall growth, as well as the creation of patterns such as the distinction between the anterior(A) and posterior(P) compartments and the dorsal(D) and ventral(V) compartments can be studied directly by imaging the wing disc as it develops inside a larva. Imaged larvae develop normally, as can be seen by the overall growth curve of the wing disc. Yet, the fact that we can follow the development of individual discs through time provides the opportunity to simultaneously assess individual variability. We for instance find that growth rates can vary greatly over time. In addition, we observe that mechanical forces act on the wing disc within the larva at times when there is an increase in growth rates. Moreover, we observe that A/P boundary formation follows the established sequence and a smooth boundary is present from the first larval instar on. The division of the wing disc into a dorsal and a ventral compartment, on the other hand, develops quite differently. Contrary to expectation, the specification of the dorsal compartment starts with only one or two cells in the second larval instar and a smooth boundary is not formed until the third larval instar.

Detection of Genetically Altered Copper Levels in Drosophila Tissues by Synchrotron X-Ray Fluorescence Microscopy

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes.

Duration of the cell cycle phases in mutants for the tumor suppressor Merlin in Drosophila melanogaster

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulse-labeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.

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

BackgroundPatterning 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 FindingsThe 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/SignificanceWe 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.