RESUMO
The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell-cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group - Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) - play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work from our laboratory had identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, we investigated the role of the IRM molecules Rst, Kirre and Hbs in maintaining the functionality of the ovarian muscle sheaths. We found that knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Moléculas de Adesão Celular , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Membrana , Contração Muscular , Oogênese , Ovário , ÓvuloRESUMO
The Irre cell-recognition module (IRM) is a group of evolutionarily conserved and structurally related transmembrane glycoproteins of the immunoglobulin superfamily. In Drosophila melanogaster, it comprises the products of the genes roughest (rst; also known as irreC-rst), kin-of-irre (kirre; also known as duf), sticks-and-stones (sns), and hibris (hbs). In this model organism, the behavior of this group of proteins as a partly redundant functional unit mediating selective cell recognition was demonstrated in a variety of developmental contexts, but their possible involvement in ovarian development and oogenesis has not been investigated, notwithstanding the fact that some rst mutant alleles are also female sterile. Here, we show that IRM genes are dynamically and, to some extent, coordinately transcribed in both pupal and adult ovaries. Additionally, the spatial distribution of Hbs, Kirre, and Rst proteins indicates that they perform cooperative, although largely nonredundant, functions. Finally, phenotypical characterization of three different female sterile rst alleles uncovered two temporally separated and functionally distinct requirements for this locus in ovarian development: one in pupa, essential for the organization of peritoneal and epithelial sheaths that maintain the structural integrity of the adult organ and another, in mature ovarioles, needed for the progression of oogenesis beyond stage 10.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Oogênese/genética , Ovário/crescimento & desenvolvimento , Animais , Drosophila melanogaster/citologia , Feminino , Expressão Gênica , MutaçãoRESUMO
Cell adhesion molecules play a central role in morphogenesis, as they mediate the complex range of interactions between different cell types that result in their arrangement in multicellular organs and tissues. How their coordinated dynamic expression in space and time - an essential requirement for their function - is regulated at the genomic and transcriptional levels constitutes an important, albeit still little understood question. The Irre Cell Recognition Module (IRM) is a highly conserved phylogenetically group of structurally related single pass transmembrane glycoproteins belonging to the immunoglobulin superfamily that in Drosophila melanogaster are encoded by the genes roughest (rst), kin-of-irre (kirre), sticks-and-stones (sns) and hibris (hbs). Their cooperative and often partly redundant action are crucial to major developmental processes such axonal pathfinding, myoblast fusion and patterning of the pupal retina. In this latter system rst and kirre display a tightly regulated complementary transcriptional pattern so that lowering rst mRNA levels leads to a concomitant increase in kirre mRNA concentration. Here we investigated whether other IRM components are similarly co-regulated and the extent changes in their mRNA levels affect each other as well as their collective function in retinal patterning. Our results demonstrate that silencing any of the four IRM genes in 24% APF retinae changes the levels all other group members although only kirre and hbs mRNA levels are increased. Furthermore, expression, in a rst null background, of truncated versions of rst cDNA in which the portion encoding the intracellular domain has been partially or completely removed not only can still induce changes in mRNA levels of other IRM members but also result in Kirre mislocalization. Taken together, our data point to the presence of a highly precise and fine-tuned control mechanism coordinating IRM expression that may be crucial to the functional redundancy shown by its components during the patterning of the pupal retina.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Pupa/genética , Retina/fisiologia , Transcrição Gênica/genética , Animais , Moléculas de Adesão Celular/genética , Regulação da Expressão Gênica/genética , Glicoproteínas/genética , Proteínas de Membrana/genética , Morfogênese/genética , RNA Mensageiro/genéticaRESUMO
Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila.
Assuntos
Autofagia/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisona/genética , Metamorfose Biológica/genética , Proteínas dos Microfilamentos/genética , Glândulas Salivares/fisiologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Anticorpos , Autofagia/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Ecdisona/metabolismo , Feminino , Forminas , Regulação da Expressão Gênica no Desenvolvimento , Larva , Proteínas dos Microfilamentos/metabolismo , Mutagênese Insercional , Especificidade de Órgãos , Fenótipo , Coelhos , Proteínas Recombinantes , Glândulas Salivares/citologia , Glândulas Salivares/crescimento & desenvolvimentoRESUMO
The ability to determine the expression dynamics of individual genes "in situ" by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogaster, as well as methods for fluorescent visualization of specific subcellular structures in these tissues.
Assuntos
Drosophila melanogaster/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/química , Imuno-Histoquímica/métodos , Microscopia de Fluorescência/métodos , Animais , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Olho/química , Corantes Fluorescentes/análise , Indóis/análise , Larva/anatomia & histologia , Faloidina/análise , Pupa/anatomia & histologia , Retina/química , Glândulas Salivares/química , Tubulina (Proteína)/análiseRESUMO
Since the first evidence that physiological cell death is a normal feature of cell life, there has been considerable effort in trying to define the mechanisms, regulation and morphology of cell death events. In nearly four decades of investigation, several types of cell death have been described in a wide range of organisms and cells, and this has led to great deal of confusion regarding the morphology and regulation of these processes. Historically, cell death has been characterized as physiological or accidental (necrosis). However, in the past fi ve years, several attempts have been made to define the types of cell death based on mechanistic and morphological criteria. Currently, at least three types of cell death apoptosis, autophagy and necrosis are recognized and share some mechanisms in common. Thus, cell death is more than simply being a caspase-mediated phenomenon. The aim of this review is to discuss recent findings on how cells choose to die in different biological contexts, and to consider the morphological changes associated with each cell type of cell death.