RESUMEN
Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7â bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Corazón , Regiones Promotoras Genéticas , Factores de Transcripción , Animales , Regiones Promotoras Genéticas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Corazón/embriología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Drosophila melanogaster/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Mutación/genética , Desarrollo Embrionario/genética , Humanos , Transcripción Genética , Proteínas Represoras , TransactivadoresRESUMEN
Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. Our study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. We identify Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites.
Asunto(s)
Drosophila , Fibras Musculares Esqueléticas , Animales , Diferenciación Celular/genética , Endodermo , MesodermoRESUMEN
BACKGROUND: Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative-to some degree-for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. RESULTS: Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). CONCLUSIONS: We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.
Asunto(s)
Escarabajos , Tribolium , Animales , Escarabajos/genética , Drosophila , Drosophila melanogaster/genética , Pupa , Interferencia de ARN , Tribolium/genéticaRESUMEN
The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.
Asunto(s)
Músculos Abdominales/citología , Linaje de la Célula/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/anatomía & histología , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Anatómicos , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Músculos Abdominales/fisiología , Animales , Animales Modificados Genéticamente , Inmunoprecipitación de Cromatina , Drosophila/genética , Drosophila/fisiología , Inmunohistoquímica , Larva/anatomía & histología , Larva/fisiología , Imagen de Lapso de Tiempo , Vísceras/anatomía & histologíaRESUMEN
Flying insects oscillate their wings at high frequencies of up to 1,000 Hz and produce large mechanical forces of 80 W per kilogram of muscle. They utilize a pair of perpendicularly oriented indirect flight muscles that contain fibrillar, stretch-activated myofibres. In contrast, all other, more slowly contracting, insect body muscles have a tubular muscle morphology. Here we identify the transcription factor Spalt major (Salm) as a master regulator of fibrillar flight muscle fate in Drosophila. salm is necessary and sufficient to induce fibrillar muscle fate. salm switches the entire transcriptional program from tubular to fibrillar fate by regulating the expression and splicing of key sarcomeric components specific to each muscle type. Spalt function is conserved in insects evolutionarily separated by 280 million years. We propose that Spalt proteins switch myofibres from tubular to fibrillar fate during development, a function potentially conserved in the vertebrate heart--a stretch-activated muscle sharing features with insect flight muscle.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Músculos/anatomía & histología , Músculos/fisiología , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ~50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.
Asunto(s)
Sitios de Unión , Proteínas de Drosophila , Drosophila melanogaster , Miocardio , Proteínas Represoras , Transactivadores , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Corazón/crecimiento & desarrollo , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Miocardio/citología , Miocardio/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/fisiología , Proteínas de Dominio T Box/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Linaje de la Célula , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Músculos/citología , Músculos/embriología , Músculos/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Madre/citología , Células Madre/fisiología , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/fisiología , Animales , Tipificación del Cuerpo , Diferenciación Celular , Movimiento Celular , Drosophila melanogaster , Elementos de Facilitación Genéticos , Genes Reporteros , Inmunohistoquímica/métodos , Mesodermo/metabolismo , Microscopía Fluorescente/métodos , Modelos Genéticos , Morfogénesis , Transducción de Señal , Transcripción GenéticaRESUMEN
BACKGROUND: The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. RESULTS: Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin ß and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. CONCLUSIONS: Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the ß6/ß3/ß8 interface of the laminin ß LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.
Asunto(s)
Colágeno Tipo IV/genética , Proteínas de Drosophila/genética , Matriz Extracelular/metabolismo , Laminina/genética , Miocardio/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Membrana Basal/embriología , Membrana Basal/metabolismo , Sitios de Unión/genética , Codón sin Sentido , Colágeno Tipo IV/metabolismo , Vasos Coronarios/embriología , Vasos Coronarios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Metanosulfonato de Etilo/toxicidad , Corazón/embriología , Laminina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis/efectos de los fármacos , Mutágenos/toxicidad , Miocardio/citología , Homología de Secuencia de AminoácidoRESUMEN
Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.
Asunto(s)
Proteínas de Drosophila/metabolismo , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Células Musculares/metabolismo , Transducción de Señal , Animales , Animales Modificados Genéticamente , Adhesión Celular , Movimiento Celular , Supervivencia Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , Prueba de Complementación Genética , Inmunohistoquímica , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Microscopía Confocal , Mutación , Vísceras/citología , Vísceras/embriología , Vísceras/metabolismoRESUMEN
HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. We show that HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. We generated HLH54F mutations and show that this gene is crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by our analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. We conclude that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Movimiento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Mesodermo/metabolismo , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Diferenciación Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Mesodermo/citología , Datos de Secuencia Molecular , Músculo Esquelético/citología , Filogenia , Alineación de SecuenciaRESUMEN
Little is known about the genetic program that generates synaptic specificity. Here we show that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. We focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. We found that Toll, which encodes a transmembrane protein with leucine-rich repeats, was preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) formed smaller synapses on M12 and instead appeared to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. We identified Tey as a negative regulator of Toll expression in M12. In tey mutants, Toll was strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Represoras/metabolismo , Receptores Toll-Like/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Neuronas Motoras/metabolismo , Mutación , Proteínas Represoras/genética , Sinapsis/metabolismoRESUMEN
Although the Drosophila longitudinal visceral muscles have been shown to undergo major morphological changes during the transition from larval to adult gut musculature, there have been conflicting views as to whether these muscles persist as such during metamorphosis or whether they are built anew (Klapper 2000; Aghajanian et al. 2016). Here we present our independent analysis using HLH54Fb-eGFP as a cell type specific marker, which strengthens the proposition by Aghajanian et al. (2016) that the syncytial larval longitudinal gut muscles completely dedifferentiate and fragment into mononucleated myoblasts during pupariation before they fuse again and redifferentiate to form the adult longitudinal gut muscles.
RESUMEN
Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.
RESUMEN
We report that the Drosophila mind bomb2 (mib2) gene is a novel regulator of muscle development. Unlike its paralogue, mib1, zygotic expression of mib2 is restricted to somatic and visceral muscle progenitors, and their respective differentiated musculatures. We demonstrate that in embryos that lack functional Mib2, muscle detachment is observed beginning in mid stage 15 and progresses rapidly, culminating in catastrophic degeneration and loss of most somatic muscles by stage 17. Notably, the degenerating muscles are positive for apoptosis markers, and inhibition of apoptosis in muscles prevents to a significant degree the muscle defects. Rescue experiments with Mib1 and Neuralized show further that these E3 ubiquitin ligases are not capable of ameliorating the muscle mutant phenotype of mib2. Our data suggest strongly that mib2 is involved in a novel Notch- and integrin-independent pathway that maintains the integrity of fully differentiated muscles and prevents their apoptotic degeneration.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Desarrollo de Músculos , Músculos/citología , Músculos/embriología , Alelos , Animales , Apoptosis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Supervivencia Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Integrinas/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Músculos/enzimología , Músculos/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Transporte de Proteínas , Receptores Notch/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Vísceras/citología , Vísceras/metabolismoRESUMEN
The mesoderm- and muscle-specific expression of microRNAs observed in a wide range of organisms suggests that post-transcriptional regulation by microRNAs can contribute significantly to the regulation of muscle development and physiology. One of these microRNAs, miR-1, is among the most widely conserved microRNAs during evolution. Genetic inactivation of miR-1 in Drosophila has shown that miR-1 is essential for maintaining the development and integrity of body wall muscles during phases of rapid growth, whereas it is not needed for normal mesoderm patterning and muscle specification. Expression analysis of a large set of potential miR-1 target mRNAs has revealed that these mRNAs tend to be expressed in non-muscle tissues, in patterns that are mutually exclusive with miR-1. Together, these findings lend support to the hypothesis that miR-1 exerts 'quality control' during muscle development by blocking detrimental mRNAs that are promiscuously expressed. Other miRNAs might promote specific developmental switches during the development and regeneration of muscles.
Asunto(s)
Diferenciación Celular , Drosophila/citología , Drosophila/genética , MicroARNs/genética , Músculos/citología , Músculos/metabolismo , Animales , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismoRESUMEN
The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. Here we show that the Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as org-1, a Drosophila homologue of mammalian TBX1, in these cells.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana , Proteínas Musculares , Músculos/citología , Músculos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Sistema de Señalización de MAP Quinasas , Mesodermo/citología , Mesodermo/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculos/embriología , Fosforilación , Proto-Oncogenes Mas , Proteínas Tirosina Quinasas Receptoras , Proteínas de Dominio T Box/metabolismoRESUMEN
The linear heart tube of the fruit fly Drosophila has served as a very valuable model for studying the regulation of early heart development. In the past, regulatory genes of Drosophila cardiogenesis have been identified largely through candidate approaches. The vast genetic toolkit available in this organism has made it possible to determine their functions and build regulatory networks of transcription factors and signaling inputs that control heart development. In this review, we summarize the major findings from this study and present current approaches aiming to identify additional players in the specification, morphogenesis, and differentiation of the heart by forward genetic screens. We also discuss various genomic and bioinformatic approaches that are currently being developed to extend the known transcriptional networks more globally which, in combination with the genetic approaches, will provide a comprehensive picture of the regulatory circuits during cardiogenesis.
Asunto(s)
Drosophila melanogaster/genética , Corazón/embriología , Morfogénesis , Miocitos Cardíacos , Factores de Transcripción/genética , Transcripción Genética , Animales , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Humanos , Modelos AnimalesRESUMEN
In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Our genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. We propose that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.
Asunto(s)
Drosophila/genética , Proteínas de Insectos/fisiología , Mioblastos/citología , Tribolium/genética , Animales , Comunicación Celular/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Metamorfosis Biológica , Morfogénesis/genética , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Fenotipo , Interferencia de ARN , Tribolium/crecimiento & desarrollo , Tribolium/metabolismoRESUMEN
Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. Recently, we reported that the ventral longitudinal musculature of the adult Drosophila heart arises in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). Here, we shed light on the events occurring downstream of Org-1 in this first step of transdifferentiation and show that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.