RESUMEN
How complex 3D tissue shape emerges during animal development remains an important open question in biology and biophysics. Here, we discover a mechanism for 3D epithelial shape change based on active, in-plane cellular events that is analogous to inanimate "shape programmable" materials, which undergo blueprinted 3D shape transformations from in-plane gradients of spontaneous strains. We study eversion of the Drosophila wing disc pouch, when the epithelium transforms from a dome into a curved fold, quantifying 3D tissue shape changes and mapping spatial patterns of cellular behaviors on the evolving geometry using cellular topology. Using a physical model inspired by shape programming, we find that active cell rearrangements are the major contributor to pouch eversion and validate this conclusion using a knockdown of MyoVI, which reduces rearrangements and disrupts morphogenesis. This work shows that shape programming is a mechanism for animal tissue morphogenesis and suggests that patterns in nature could present design strategies for shape-programmable materials.
Asunto(s)
Morfogénesis , Alas de Animales , Animales , Alas de Animales/crecimiento & desarrollo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila , Modelos Biológicos , Discos Imaginales/metabolismo , Discos Imaginales/crecimiento & desarrolloRESUMEN
The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster , Receptores ErbB , Regulación del Desarrollo de la Expresión Génica , Neuronas , Transducción de Señal , Factores de Transcripción , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Neuronas/citología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Diferenciación Celular , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Discos Imaginales/metabolismo , Discos Imaginales/citología , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas , Receptores de Péptidos de InvertebradosRESUMEN
Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.
Asunto(s)
Acetilcoenzima A , Núcleo Celular , Cromatina , Drosophila melanogaster , Animales , Acetato CoA Ligasa/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Discos Imaginales/citología , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Lisina/metabolismo , Oxidación-Reducción , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Morphogenetic programs coordinate cell signaling and mechanical interactions to shape organs. In systems and synthetic biology, a key challenge is determining optimal cellular interactions for predicting organ shape, size, and function. Physics-based models defining the subcellular force distribution facilitate this, but it is challenging to calibrate parameters in these models from data. To solve this inverse problem, we created a Bayesian optimization framework to determine the optimal cellular force distribution such that the predicted organ shapes match the experimentally observed organ shapes. This integrative framework employs Gaussian Process Regression, a non-parametric kernel-based probabilistic machine learning modeling paradigm, to learn the mapping functions relating to the morphogenetic programs that maintain the final organ shape. We calibrated and tested the method on Drosophila wing imaginal discs to study mechanisms that regulate epithelial processes ranging from development to cancer. The parameter estimation framework successfully infers the underlying changes in core parameters needed to match simulation data with imaging data of wing discs perturbed with collagenase. The computational pipeline identifies distinct parameter sets mimicking wild-type shapes. It enables a global sensitivity analysis to support the regulation of actomyosin contractility and basal ECM stiffness to generate and maintain the curved shape of the wing imaginal disc. The optimization framework, combined with experimental imaging, identified that Piezo, a mechanosensitive ion channel, impacts fold formation by regulating the apical-basal balance of actomyosin contractility and elasticity of ECM. This workflow is extensible toward reverse-engineering morphogenesis across organ systems and for real-time control of complex multicellular systems.
Asunto(s)
Teorema de Bayes , Morfogénesis , Alas de Animales , Animales , Modelos Biológicos , Drosophila melanogaster , Discos Imaginales , Simulación por Computador , DrosophilaRESUMEN
The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are unknow. Here, we analyze a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and we identified a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Discos Imaginales/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/metabolismo , Alas de AnimalesRESUMEN
Automated image quantification workflows have dramatically improved over the past decade, enriching image analysis and enhancing the ability to achieve statistical power. These analyses have proved especially useful for studies in organisms such as Drosophila melanogaster, where it is relatively simple to obtain high sample numbers for downstream analyses. However, the developing wing, an intensively utilized structure in developmental biology, has eluded efficient cell counting workflows due to its highly dense cellular population. Here, we present efficient automated cell counting workflows capable of quantifying cells in the developing wing. Our workflows can count the total number of cells or count cells in clones labeled with a fluorescent nuclear marker in imaginal discs. Moreover, by training a machine-learning algorithm we have developed a workflow capable of segmenting and counting twin-spot labeled nuclei, a challenging problem requiring distinguishing heterozygous and homozygous cells in a background of regionally varying intensity. Our workflows could potentially be applied to any tissue with high cellular density, as they are structure-agnostic, and only require a nuclear label to segment and count cells.
Asunto(s)
Drosophila , Discos Imaginales , Animales , Drosophila melanogaster , Larva , Núcleo CelularRESUMEN
Some animals respond to injury by inducing new growth to regenerate the lost structures. This regenerative growth must be carefully controlled and constrained to prevent aberrant growth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. Using a genetic ablation system in the Drosophila wing imaginal disc, we have identified one mechanism that constrains regenerative growth, impairment of which also leads to erroneous patterning of the final appendage. Regenerating discs with reduced levels of the RNA-regulator Brain tumor (Brat) exhibit enhanced regeneration, but produce adult wings with disrupted margins that are missing extensive tracts of sensory bristles. In these mutants, aberrantly high expression of the pro-growth factor Myc and its downstream targets likely contributes to this loss of cell-fate specification. Thus, Brat constrains the expression of pro-regeneration genes and ensures that the regenerating tissue forms the proper final structure.
Asunto(s)
Proteínas de Drosophila , Regeneración , Animales , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Discos Imaginales/metabolismo , Regeneración/genética , Alas de Animales , Proteínas de Unión al ADN/genéticaRESUMEN
engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal discs; in contrast, small IDE-reporter transgenes are expressed mainly in the anterior compartment. We found that En binds to the IDEs and suggest that it may directly repress IDE function and modulate En expression levels. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from an endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data suggest that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic memory set by the embryonic enhancers. This epigenetic regulation is similar to that of the Ultrabithorax IDEs and we suggest that the activity of late-acting enhancers in other genes may be similarly regulated.
Asunto(s)
Proteínas de Drosophila , Discos Imaginales , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Homeostasis , Discos Imaginales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022).1,2,3.
Asunto(s)
Drosophila , Discos Imaginales , Puromicina/análogos & derivados , Animales , Larva/metabolismo , Puromicina/farmacologíaRESUMEN
Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. We previously showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, we found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. We propose a model in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Diferenciación Celular , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/metabolismo , Transducción de Señal , Alas de Animales/metabolismoRESUMEN
A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.
Asunto(s)
Proteínas de Drosophila , Discos Imaginales , Animales , Proteínas de Drosophila/genética , Drosophila , Genómica , Hiperplasia , Proteínas del Grupo Polycomb/genéticaRESUMEN
Apicobasal polarity determinants are potential tumor suppressors that have been extensively studied. However, the precise mechanisms by which their misregulation disrupts tissue homeostasis are not fully understood. Here, we present a comprehensive protocol for establishing a conditional RNAi knockdown of scribble in Drosophila wing imaginal disc. We describe steps for generating fly lines, conditional knockdown in host stocks, and sample preparation. We then detail procedures for imaging, image analysis, and verification of wing disc phenotypes by various antibodies. For complete details on the use and execution of this protocol, please refer to Huang et al.1.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Discos Imaginales , Proteínas de Drosophila/genética , Drosophila , ComunicaciónRESUMEN
wingless expression is exquisitely regulated by different factors and enhancers in the imaginal wing discs of Drosophila melanogaster in four domains: the dorsal band, the dorso-ventral boundary, and the inner and outer ring domains. tonalli is a trithorax group gene that encodes a putative SUMO E3 ligase that binds to chromatin to regulate the expression of its targets, including the Hox genes. However, its role in modulating gene expression is barely known. Here, we show that TnaA modulates the wingless expression at two domains of the wing disc, the dorso-ventral boundary and the inner ring. At first, tonalli interacts genetically with Notch to form the wing margin. In the inner ring domain, TnaA modulates wingless transcription. When the dosage of TnaA increases in or near the inner ring since early larval stages, this domain expands with a rapid increase in wingless expression. TnaA occupies the wingless Inner Ring Enhancer at the wing disc, meanwhile it does not affect wingless expression directed by the Ventral Disc Enhancer in leg discs, suggesting that TnaA acts as a wingless enhancer-specific factor. We describe for the first time the presence of TnaA at the Inner Ring Enhancer as a specific regulator of wingless in the development of wing boundaries.
Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Drosophila melanogaster/genética , Discos Imaginales , Larva , Ubiquitina-Proteína LigasasRESUMEN
Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. Here, we identify the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). Our findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, we identify the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands our understanding of NF-YC's role from cell cycle control to encompass developmental processes.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Discos Imaginales/metabolismo , Proteínas Hedgehog/metabolismo , Ojo/metabolismo , Ciclo Celular/genética , Puntos de Control del Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Ciclina B/metabolismoRESUMEN
Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Here, using the Drosophila wing imaginal disc, we tested whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization. To this end, we genetically reduced Myosin II levels in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. We found that the recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, we conclude that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation.
Asunto(s)
Miosina Tipo II , Animales , Células Clonales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Morfogénesis , Miosina Tipo II/metabolismo , Discos Imaginales/metabolismoRESUMEN
Cleavage Under Targets & Release Using Nucleases (CUT&RUN) sequencing is a technique used to study gene regulation. The protocol presented here has been used successfully to identify the pattern of histone modifications within the genome of the eye-antennal disc of the fruit fly, Drosophila melanogaster. In its present form, it can be used to analyze genomic features of other imaginal discs. It can be modified for use with other tissues and applications including identifying the pattern of transcription factor occupancy.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Discos Imaginales/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigénesis Genética/genéticaRESUMEN
The imaginal disc growth factor (IDGF), belonging to the glycoside hydrolase 18 family, plays an important role in various physiological processes in insects. However, the detail physiological function of IDGF is still unclear. In this study, transcriptome analysis was performed on the fatbody isolated from staged control and BmIDGF mutant silkworm larvae. Transcriptional profiling revealed that the absence of BmIDGF significantly affected differentially expressed genes involved in tyrosine and purine metabolism, as well as multiple energy metabolism pathways, including glycolysis, galactose, starch, and sucrose metabolism. The interruption of BmIDGF caused similar and specific gene expression changes to male and female fatbody. Furthermore, a genome-scale metabolic network integrating metabolomic and transcriptomic datasets revealed 11 pathways significantly altered at the transcriptional and metabolic levels, including amino acid, carbohydrate, uric acid metabolism pathways, insect hormone biosynthesis, and ABC transporters. In conclusion, this multiomics analysis suggests that IDGF is involved in gene-metabolism interactions, revealing its unique role in melanin synthesis and energy metabolism. This study provides new insights into the physiological function of IDGF in insects.
Asunto(s)
Bombyx , Masculino , Animales , Femenino , Bombyx/metabolismo , Melaninas/metabolismo , Discos Imaginales/metabolismo , Perfilación de la Expresión Génica , Metabolismo Energético , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
Chitinase-like proteins (CLPs) are members of the family 18 glycosyl hydrolases, which include chitinases and the enzymatically inactive CLPs. A mutation in the enzyme's catalytic site, conserved in vertebrates and invertebrates, allowed CLPs to evolve independently with functions that do not require chitinase activity. CLPs normally function during inflammatory responses, wound healing, and host defense, but when they persist at excessive levels at sites of chronic inflammation and in tissue-remodeling disorders, they correlate positively with disease progression and poor prognosis. Little is known, however, about their physiological function. Drosophila melanogaster has 6 CLPs, termed Imaginal disk growth factors (Idgfs), encoded by Idgf1, Idgf2, Idgf3, Idgf4, Idgf5, and Idgf6. In this study, we developed tools to facilitate characterization of the physiological roles of the Idgfs by deleting each of the Idgf genes using the CRISPR/Cas9 system and assessing loss-of-function phenotypes. Using null lines, we showed that loss of function for all 6 Idgf proteins significantly lowers viability and fertility. We also showed that Idgfs play roles in epithelial morphogenesis, maintaining proper epithelial architecture and cell shape, regulating E-cadherin and cortical actin, and remarkably, protecting these tissues against CO2 exposure. Defining the normal molecular mechanisms of CLPs is a key to understanding how deviations tip the balance from a physiological to a pathological state.
Asunto(s)
Quitinasas , Proteínas de Drosophila , Animales , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Quitinasas/genética , Quitinasas/metabolismo , Dióxido de Carbono , Discos Imaginales/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Morfogénesis/genética , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Genetic ablation is a highly efficient method to study regeneration in vivo by stimulating tissue-specific cell death that subsequently induces regrowth and repair in a developing organism. This approach has been particularly successful in Drosophila, for which various temperature-based genetic ablation tools have been developed to explore the complexities of regeneration in larval imaginal discs. Here, we describe the use of a recently established ablation system called DUAL Control, which can be used to both characterize the damage response and genetically manipulate blastema cells to identify novel regulators of regeneration.
Asunto(s)
Proteínas de Drosophila , Discos Imaginales , Animales , Discos Imaginales/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Cicatrización de Heridas/fisiología , Larva/metabolismoRESUMEN
The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin-inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways.