RESUMEN
Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments we demonstrate that pasiflora proteins are core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Secuencia de Aminoácidos , Animales , Barrera Hematoencefálica , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Epitelio/embriología , Genes de Insecto , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Morfogénesis , Tamaño de los Órganos , Tráquea/anatomía & histologíaRESUMEN
Male and female germ cells follow distinct developmental paths with respect to germline stem cell (GSC) production and the types of differentiated progeny they produce (sperm versus egg). An essential aspect of germline development is how sexual identity is used to differentially regulate the male and female germ cell genomes to allow for these distinct outcomes. Here, we identify a gene, no child left behind (nclb), that plays very different roles in the male versus female germline in Drosophila. In particular, nclb is required for GSC maintenance in males, but not in females. Male GSCs mutant for nclb are rapidly lost from the niche, and begin to differentiate but cannot complete spermatogenesis. We further find that nclb encodes a member of a new family of conserved chromatin-associated proteins. NCLB interacts with chromatin in a specific manner and is associated with sites of active transcription. Thus, NCLB appears to be a novel chromatin regulator that exhibits very different effects on the male and female germ cell genomes.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Espermatozoides/metabolismo , Células Madre/metabolismo , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Mutación , Oogénesis , Óvulo/citología , Óvulo/metabolismo , Espermatogénesis , Espermatozoides/citología , Células Madre/citología , Transcripción GenéticaRESUMEN
The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, we first conducted a molecular screen for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. We also find that the genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with our findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination.
Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/fisiología , Células Germinativas/fisiología , Procesos de Determinación del Sexo , Animales , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas/citología , Gónadas/fisiología , Masculino , Factores de Transcripción/fisiologíaRESUMEN
Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, the sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue (for example, refs 1, 2, reviewed in refs 3, 4); however, little is known about how the soma controls germline sex determination. Here we show that the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway provides a sex-specific signal from the soma to the germ line in Drosophila embryonic gonads. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behaviour during early gonad development, and is sufficient to activate aspects of male germ cell behaviour in female germ cells. Our findings provide direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development.