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1.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34323270

RESUMO

The formation of the cardiac tube is a remarkable example of complex morphogenetic processes conserved from invertebrates to humans. It involves coordinated collective migration of contralateral rows of cardiac cells. The molecular processes underlying the specification of cardioblasts (CBs) prior to migration are well established and significant advances have been made in understanding the process of lumen formation. However, the mechanisms of collective cardiac cells migration remain elusive. Here, we have identified CAP and MSP300 as novel actors involved during CB migration. They both exhibit highly similar temporal and spatial expression patterns in Drosophila migrating cardiac cells, and are necessary for the correct number and alignment of CBs, a prerequisite for the coordination of their collective migration. Our data suggest that CAP and MSP300 are part of a protein complex linking focal adhesion sites to nuclei via the actin cytoskeleton that maintains post-mitotic state and correct alignment of CBs.


Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Organogênese/fisiologia , Animais , Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia
2.
Mol Cell ; 81(16): 3356-3367.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34297910

RESUMO

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.


Assuntos
Proteínas de Drosophila/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Polimerase II/genética , Transcrição Genética , Animais , Drosophila melanogaster/genética , Metiltransferases/genética , Regiões Promotoras Genéticas/genética
3.
Sci Rep ; 11(1): 13197, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162956

RESUMO

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

4.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32001438

RESUMO

Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, we provide evidence that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, we report the previously undetected expression of the guidance molecule-encoding genes sidestep and side IV in AMPs. Altogether, our data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.


Assuntos
Axônios/fisiologia , Neurônios Motores/fisiologia , Músculos/embriologia , Músculos/inervação , Mioblastos/fisiologia , Animais , Apoptose , Orientação de Axônios , Movimento Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Genótipo , Proteínas de Fluorescência Verde , Cones de Crescimento/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/fisiologia , Microscopia de Fluorescência , Pseudópodes/fisiologia , Transdução de Sinais , Células-Tronco/citologia
5.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829940

RESUMO

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.


Assuntos
Canais de Cálcio/biossíntese , Doença do Sistema de Condução Cardíaco/genética , Doença do Sistema de Condução Cardíaco/fisiopatologia , Regulação da Expressão Gênica , Distrofia Miotônica/complicações , Animais , Canais de Cálcio/genética , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Humanos , Camundongos
6.
Development ; 143(23): 4533-4542, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27899510

RESUMO

Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, we examined H3K4me3 and H3K27ac chromatin landscapes to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. Our results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which we show is essential for differentiated heart function.


Assuntos
Drosophila/embriologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/embriologia , Miócitos Cardíacos/citologia , Organogênese/genética , Regiões Promotoras Genéticas/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Cromatina , Drosophila/genética , Redes Reguladoras de Genes/genética , Histonas/metabolismo , Organogênese/fisiologia , Fatores de Transcrição/genética
7.
PLoS Comput Biol ; 12(9): e1005073, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27599298

RESUMO

Given the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. Here, we delineate a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature. The four main tissues derived from mesoderm correspond to alternative stable states. We demonstrate that the model can predict known mutant phenotypes and use it to systematically predict the effects of over 300 new, often non-intuitive, loss- and gain-of-function mutations, and combinations thereof. We further validated several novel predictions experimentally, thereby demonstrating the robustness of model. Logical modelling can thus contribute to formally explain and predict regulatory outcomes underlying cell fate decisions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/fisiologia , Modelos Biológicos , Transdução de Sinais , Animais , Biologia Computacional , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/fisiologia , Mutação , Fenótipo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
8.
J Vis Exp ; (103)2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26381166

RESUMO

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.


Assuntos
RNA Mensageiro/isolamento & purificação , Animais , Animais Geneticamente Modificados , Cromatografia de Afinidade/métodos , Drosophila , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Magnetismo/métodos , Músculos/química , Músculos/citologia , Músculos/fisiologia , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribossomos/química , Ribossomos/genética
9.
Development ; 142(5): 994-1005, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25715399

RESUMO

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.


Assuntos
Proteínas de Drosophila/metabolismo , Coração/embriologia , Proteínas de Choque Térmico Pequenas/metabolismo , Músculos/embriologia , Músculos/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Filaminas/genética , Filaminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Choque Térmico Pequenas/genética , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia
10.
Mol Biosyst ; 9(9): 2248-58, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23868318

RESUMO

A limited number of signalling pathways are involved in the specification of cell fate during the development of all animals. Several of these pathways were originally identified in Drosophila. To clarify their roles, and possible cross-talk, we have built a logical model for the nine key signalling pathways recurrently used in metazoan development. In each case, we considered the associated ligands, receptors, signal transducers, modulators, and transcription factors reported in the literature. Implemented using the logical modelling software GINsim, the resulting models qualitatively recapitulate the main characteristics of each pathway, in wild type as well as in various mutant situations (e.g. loss-of-function or gain-of-function). These models constitute pluggable modules that can be used to assemble comprehensive models of complex developmental processes. Moreover, these models of Drosophila pathways could serve as scaffolds for more complicated models of orthologous mammalian pathways. Comprehensive model annotations and GINsim files are provided for each of the nine considered pathways.


Assuntos
Drosophila/metabolismo , Modelos Biológicos , Transdução de Sinais , Animais , Drosophila/embriologia , Proteínas de Drosophila/metabolismo
11.
Cell ; 148(3): 473-86, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22304916

RESUMO

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. Here, we demonstrate that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this "dormant" TF binding signature may represent a molecular footprint of these cells' developmental lineage.


Assuntos
Drosophila melanogaster/citologia , Redes Reguladoras de Genes , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Mesoderma/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo
12.
Methods Mol Biol ; 798: 543-53, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22130860

RESUMO

Mapping the cis-regulatory modules (CRMs) to which bind myogenic transcription factors is an -obligatory step towards understanding gene regulatory networks governing muscle development and function. This can be achieved in silico or by chromatin immunoprecipitation (ChIP) approaches. We have developed a ChIP-enriched in silico targets (ChEST) strategy designed for mapping the CRMs by combining in silico and ChIP methods. ChEST involves a software-assisted prediction of transcription factor (TF) - specific CRMs, which are spotted to produce a computed genomic CRM microarray. In parallel, the in vivo pool of targets of a given TF is isolated by ChIP and used as a probe for hybridization with the array generated. Here we describe ChEST strategy applied to identify direct targets of Myogenic Enhancer Factor, Dmef2 in Drosophila embryos.


Assuntos
Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica/métodos , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Biologia Computacional , Sondas de DNA/síntese química , Drosophila/genética , Embrião não Mamífero/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodos
13.
Genes Dev ; 21(23): 3163-80, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18056427

RESUMO

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.


Assuntos
Drosophila/citologia , Drosophila/genética , Genes Homeobox , Genes de Insetos , Mioblastos Cardíacos/citologia , Mioblastos/citologia , Animais , Animais Geneticamente Modificados , Adesão Celular/genética , Diferenciação Celular/genética , Fusão Celular , Movimento Celular/genética , Imunoprecipitação da Cromatina , Drosophila/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genoma de Inseto , Modelos Biológicos , Interferência de RNA
14.
Proc Natl Acad Sci U S A ; 102(51): 18479-84, 2005 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-16339902

RESUMO

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. We have developed a chromatin immunoprecipitation (ChIP)-chip strategy for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. We report the use of ChEST in Drosophila to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. Our approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome.


Assuntos
Imunoprecipitação da Cromatina , Biologia Computacional , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Fatores de Transcrição MEF2 , Ligação Proteica
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