RESUMO
The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/citologia , Olho/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/genética , Serina/metabolismo , Transativadores/genética , Asas de Animais/citologia , Asas de Animais/metabolismo , Proteínas de Sinalização YAPRESUMO
Developing organisms may contain billions of cells destined to differentiate in numerous different ways. One strategy organisms use to simplify the orchestration of development is the separation of cell populations into distinct functional units. Our expanding knowledge of boundary formation and function in different systems is beginning to reveal general principles of this process. Fields of cells are subdivided by the interpretation of morphogen gradients, and these subdivisions are then maintained and refined by local cell-cell interactions. Sharp and stable separation between cell populations requires special mechanisms to keep cells segregated, which in many cases appear to involve the regulation of cell affinity. Once cell populations become distinct, specialized cells are often induced along the borders between them. These boundary cells can then influence the patterning of surrounding cells, which can result in progressively finer subdivisions of a tissue. Much has been learned about the signaling pathways that establish boundaries, but a key challenge for the future remains to elucidate the cellular and molecular mechanisms that actually keep cell populations separated.
Assuntos
Padronização Corporal/fisiologia , Adesão Celular/fisiologia , Morfogênese , Animais , Padronização Corporal/genética , Compartimento Celular , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Separação Celular , Drosophila , Previsões , Expressão Gênica , Organizadores Embrionários , Transdução de Sinais , Asas de Animais/citologia , Asas de Animais/fisiologiaRESUMO
fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles.
Assuntos
Glicosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases , Oogênese/fisiologia , Ovário/citologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Feminino , Expressão Gênica , Glicosiltransferases/genética , Mutagênese , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/metabolismo , Óvulo/citologia , Óvulo/metabolismo , Receptores NotchRESUMO
The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Divisão Celular , Dimerização , Drosophila melanogaster/citologia , Genes Reporter , Proteínas de Fluorescência Verde , Hormônios de Inseto/fisiologia , Larva , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genética , Asas de Animais/citologia , Proteína Wnt1RESUMO
Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.
Assuntos
Glicosiltransferases , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas/metabolismo , Animais , Células CHO , Catálise , Linhagem Celular , Cricetinae , Drosophila , Proteínas de Drosophila , Fator de Crescimento Epidérmico/metabolismo , Fucose/metabolismo , Mutagênese Sítio-Dirigida , Polissacarídeos/metabolismo , Proteínas/genética , Receptores Notch , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
The separation of cells into populations that do not intermix, termed compartments, is a fundamental organizing principle during development. Dorsal-ventral compartmentalization of the Drosophila wing is regulated downstream of the apterous (ap) gene, which encodes a transcription factor that specifies dorsal wing fate. fringe (fng) is normally expressed by dorsal cells downstream of ap; here we show that fng plays a key role in dorsal-ventral compartmentalization. Loss of fng function causes dorsal cells to violate the compartment boundary, and ectopic expression of the Fng protein causes ventral cells to violate thecompartment boundary. Fng modulates signalling through the Notch receptor. Notch and its ligands are essential for formation of the dorsal-ventral compartment border, and repositioning the stripe of Notch activation that is normally established there appears to reposition the compartment border. However, activation of Notch does not itself confer either dorsal or ventral cell location, and fng can influence compartmentalization even within regions of ubiquitous Notch activation. Our results indicate that the primary mechanism by which fng establishes a compartment border is by positioning a stripe of Notch activation, but also that fng may exert additional influences on compartmentalization.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Homeodomínio , Proteínas de Insetos/fisiologia , N-Acetilglucosaminiltransferases , Transdução de Sinais , Asas de Animais/embriologia , Animais , Proteínas de Ligação ao Cálcio , Adesão Celular , Células Clonais , Drosophila/citologia , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Proteínas com Homeodomínio LIM , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Mutação , Receptores Notch , Recombinação Genética , Proteínas Serrate-Jagged , Fatores de Transcrição/metabolismo , Asas de Animais/citologiaRESUMO
Multiple mechanisms are involved in positioning and restricting specialized dorsal-ventral border cells in the Drosophila wing, including modulation of Notch signaling by Fringe, autonomous inhibition by Notch ligands, and inhibition of Notch target genes by Nubbin. Recent studies have revealed that Fringe also modulates a Notch-mediated signaling process between dorsal and ventral cells in the Drosophila eye, establishing an organizer of eye growth and patterning along the dorsal-ventral midline. Fringe-dependent modulation of Notch signaling also plays a key role in Drosophila leg segmentation and growth. Lunatic Fringe has been shown to be required for vertebrate somitogenesis, where it appears to act as a crucial link between a molecular clock and the regulation of Notch signaling.
Assuntos
Drosophila/embriologia , Proteínas de Insetos/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases , Animais , Proteínas de Drosophila , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Receptores NotchRESUMO
The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, we show that the Notch signaling pathway plays a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. Our studies further show that Notch activation is both necessary and sufficient to promote leg growth. We also identify target genes regulated both positively and negatively downstream of Notch signaling that are required for normal leg development. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues.
Assuntos
Drosophila/crescimento & desenvolvimento , Membro Posterior/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , N-Acetilglucosaminiltransferases , Asas de Animais/crescimento & desenvolvimento , Envelhecimento , Animais , Padronização Corporal , Proteínas de Drosophila , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Larva , Proteínas de Membrana/genética , Oviposição , Pigmentação , Receptores Notch , Transdução de Sinais , Transativadores/fisiologiaRESUMO
Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/embriologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Dimerização , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Deleção de Sequência , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional , LevedurasRESUMO
The development of the Drosophila eye has served as a model system for investigations of tissue patterning and cell-cell communication; however, early eye development has not been well understood. The results presented here indicate that specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells, and that Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning. These observations imply that the developmental homology between Drosophila wings and vertebrate limbs extends to Drosophila eyes.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Drosophila/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , N-Acetilglucosaminiltransferases , Células Fotorreceptoras de Invertebrados/crescimento & desenvolvimento , Fatores de Transcrição , Animais , Proteínas de Ligação ao Cálcio , Drosophila/genética , Drosophila/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Homeodomínio , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Larva/crescimento & desenvolvimento , Ligantes , Proteínas de Membrana/genética , Morfogênese , Mutação , Células Fotorreceptoras de Invertebrados/citologia , Receptores Notch , Proteínas Serrate-Jagged , Transdução de SinaisRESUMO
In addition to the core components of the Notch pathway, a number of proteins have been identified that exert positive or negative influences on Notch signaling. These include extracellular modulators, which may influence binding or activation of Notch by its ligands, cytoplasmic modulators, which presumably influence signal transduction steps after receptor activation, and nuclear modulators, which may influence the transcriptional activity of a Notch-CSL protein complex. Many of the cytoplasmic and nuclear modulators appear to bind directly to discrete domains within the intracellular domain of Notch. Genetic studies indicate that distinct modulators are deployed during distinct modes of Notch signaling.
Assuntos
Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila , Genes de Insetos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores NotchRESUMO
The Notch family of transmembrane receptor proteins mediate developmental cell-fate decisions, and mutations in mammalian Notch genes have been implicated in leukaemia, breast cancer, stroke and dementia. During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe (fng), Serrate (Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. Ser and Dl encode transmembrane ligands for Notch, whereas fng encodes a pioneer protein. We have investigated the relationship between these genes by a combination of expression and coexpression studies in the Drosophila wing. We found that Ser and Dl maintain each other's expression by a positive feedback loop. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein.
Assuntos
Proteínas de Insetos/fisiologia , Proteínas de Membrana/fisiologia , N-Acetilglucosaminiltransferases , Animais , Animais Geneticamente Modificados , Proteínas de Ligação ao Cálcio , Células Cultivadas , Drosophila , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Receptores Notch , Proteínas Serrate-Jagged , Transdução de Sinais , Asas de Animais/embriologiaRESUMO
The formation of boundaries between groups of cells is a universal feature of metazoan development. Drosophila fringe modulates the activation of the Notch signal transduction pathway at the dorsal-ventral boundary of the wing imaginal disc. Three mammalian fringe-related family members have been cloned and characterized: Manic, Radical and Lunatic Fringe. Expression studies in mouse embryos support a conserved role for mammalian Fringe family members in participation in the Notch signaling pathway leading to boundary determination during segmentation. In mammalian cells, Drosophila fringe and the mouse Fringe proteins are subject to posttranslational regulation at the levels of differential secretion and proteolytic processing. When misexpressed in the developing Drosophila wing imaginal disc the mouse Fringe genes exhibit conserved and differential effects on boundary determination.
Assuntos
Padronização Corporal/genética , Glicosiltransferases , Proteínas de Insetos/genética , Proteínas de Membrana/genética , N-Acetilglucosaminiltransferases , Proteínas/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Drosophila , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Glucosiltransferases , Hexosiltransferases , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Família Multigênica , Sistema Nervoso/química , Sistema Nervoso/embriologia , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , RNA Mensageiro/análise , Receptores Notch , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Somitos , Asas de Animais/crescimento & desenvolvimentoRESUMO
In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal-ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal-ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.
Assuntos
Padronização Corporal , Extremidades/crescimento & desenvolvimento , N-Acetilglucosaminiltransferases , Transdução de Sinais , Animais , Comunicação Celular , Drosophila/crescimento & desenvolvimento , Drosophila/fisiologia , Proteínas de Drosophila , Extremidades/embriologia , Proteínas de Insetos/fisiologia , Proteínas de Membrana/fisiologia , Receptores Notch , Vertebrados/embriologia , Vertebrados/fisiologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.
Assuntos
Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Transdução de Sinais/genética , Asas de Animais/embriologia , Animais , Genes de Insetos/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Ativação TranscricionalRESUMO
Wing formation in Drosophila requires interactions between dorsal and ventral cells. We describe a new gene, fringe, which is expressed in dorsal cells and encodes for a novel protein that is predicted to be secreted. Wing margin formation and distal wing outgrowth can be induced by the juxtaposition of cells with and without fringe expression, whether at the normal wing margin, at the boundaries of fringe mutant clones in the dorsal wing, or at sites of fringe misexpression in the ventral wing. By contrast, both loss of fringe expression and uniform fringe expression cause wing loss. These observations suggest that fringe encodes a boundary-specific cell-signaling molecule that is responsible for dorsal-ventral cell interactions during wing development.
Assuntos
Drosophila/fisiologia , N-Acetilglucosaminiltransferases , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cruzamentos Genéticos , Drosophila/genética , Proteínas de Drosophila , Feminino , Expressão Gênica , Larva , Masculino , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Fenótipo , Biossíntese de Proteínas , Transdução de Sinais , Asas de Animais/citologia , Asas de Animais/fisiologiaRESUMO
After the onset of gastrulation, the Drosophila germband undergoes a morphological change in which its length along the anterior-posterior axis increases over two-and-a-half fold while its width along the dorsal-ventral axis simultaneously narrows. The behavior of individual cells during germband extension was investigated by epi-illumination and time-lapse video microscopy of living embryos. Cells intercalate between their dorsal and ventral neighbors during extension, increasing the number of cells along the anterior-posterior axis while decreasing the number of cells along the dorsal-ventral axis. Mutations that reduce segmental subdivision of the embryo along the anterior-posterior axis decrease both germband extension and its associated cell intercalation. In contrast, cell intercalation and germband extension are still detected in embryos that lack dorsal-ventral polarity. Characterization of germband extension and cell intercalation in mutant embryos with altered segmentation gene expression indicates that these processes are regionally autonomous and are dependent upon the establishment of striped expression patterns for certain pair-rule genes. Based on these observations, we propose a model for germband extension in which cell intercalation results from the establishment of adhesive differences between stripes of cells by pair-rule genes.
Assuntos
Drosophila/embriologia , Indução Embrionária/fisiologia , Gástrula/fisiologia , Genes de Insetos , Células Germinativas/fisiologia , Animais , Blastoderma/citologia , Movimento Celular/fisiologia , Drosophila/genética , Gástrula/citologia , Mesoderma/citologia , Microscopia de Vídeo , Modelos Biológicos , Morfogênese/genéticaRESUMO
The Drosophila homeotic gene Ultrabithorax (Ubx) encodes transcriptional regulatory proteins (UBX) that specify thoracic and abdominal segmental identities. Ubx autoregulation was examined by manipulating UBX levels, both genetically and with an inducible transgene, and monitoring the effect of these manipulations on the expression of Ubx and Ubx-lacZ reporter genes. Positive autoregulation by Ubx is restricted to the visceral mesoderm, while in other tissues Ubx negatively autoregulates. In some cases, negative autoregulation stabilizes UBX levels, while in others it modulates the spatial and temporal patterns of UBX expression. This modulation of UBX expression may enable Ubx to specify distinct identities in different segments. The upstream control region of Ubx contains multiple autoregulatory elements for both positive and negative autoregulation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Proteínas de Homeodomínio , Fatores de Transcrição , Animais , Drosophila/embriologia , Hibridização In Situ , Morfogênese/genéticaRESUMO
Ultrabithorax (Ubx) is a Drosophila homeotic gene that determines the segmental identities of parts of the thorax and abdomen. Appropriate Ubx transcription requires a long upstream control region (UCR) that is defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. We have directly analyzed UCR functions by the examination of beta-galactosidase expression in flies containing Ubx-lacZ fusion genes. 35 kb of UCR DNA confers upon beta-galactosidase an expression pattern that closely parallels normal Ubx expression throughout development. In contrast, 22 kb of UCR DNA confers fewer features of normal Ubx expression, and with 5 kb of UCR DNA the expression pattern has no resemblance to Ubx expression except in the visceral mesoderm. We have also shown that bxd chromosome breakpoint mutants form a comparable 5' deletion series in which the severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in the mutant. In Ubx-lacZ fusions containing 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development.
Assuntos
Drosophila/genética , Regulação da Expressão Gênica/genética , Genes Homeobox/fisiologia , Genes Reguladores/fisiologia , Transcrição Gênica/genética , Animais , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/fisiologia , Sistema Nervoso Central/ultraestrutura , Mapeamento Cromossômico , Microscopia Imunoeletrônica , beta-Galactosidase/genéticaRESUMO
Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.