RESUMO
Axon guidance at choice points depends on the precise regulation of guidance receptors on the growth cone surface. Upon arrival at the intermediate target or choice point, a switch from attraction to repulsion is required for the axon to move on. Dorsal commissural (dI1) axons crossing the ventral midline of the spinal cord in the floor plate represent a convenient model for the analysis of the molecular mechanism underlying the switch in axonal behavior. We identified in chick a role for calsyntenin 1 in the regulation of vesicular trafficking of guidance receptors in dI1 axons at choice points. In cooperation with RabGDI, calsyntenin 1 shuttles Rab11-positive vesicles containing Robo1 to the growth cone surface in a precisely regulated manner. By contrast, calsyntenin 1-mediated trafficking of frizzled 3, a guidance receptor in the Wnt pathway, is independent of RabGDI. Thus, tightly regulated insertion of guidance receptors, which is required for midline crossing and the subsequent turn into the longitudinal axis, is achieved by specific trafficking.
Assuntos
Axônios/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas Aviárias/metabolismo , Células COS , Galinhas , Chlorocebus aethiops , Inativação Gênica , Cones de Crescimento/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fenótipo , Transporte ProteicoRESUMO
The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This "egg chamber autonomous" ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/enzimologia , Ecdisona/biossíntese , Ecdisterona/metabolismo , Oxigenases de Função Mista/metabolismo , Animais , Proteínas de Drosophila/genética , Ecdisona/genética , Microscopia de Fluorescência , Oxigenases de Função Mista/genética , Mutação/genéticaRESUMO
The Notch signaling pathway plays important roles in a variety of developmental events. The context-dependent activities of positive and negative modulators dramatically increase the diversity of cellular responses to Notch signaling. In a screen for mutations affecting the Drosophila melanogaster follicular epithelium, we isolated a mutation in CoREST that disrupts the Notch-dependent mitotic-to-endocycle switch of follicle cells at stage 6 of oogenesis. We show that Drosophila CoREST positively regulates Notch signaling, acting downstream of the proteolytic cleavage of Notch but upstream of Hindsight activity; the Hindsight gene is a Notch target that coordinates responses in the follicle cells. We show that CoREST genetically interacts with components of the Notch repressor complex, Hairless, C-terminal Binding Protein and Groucho. In addition, we demonstrate that levels of H3K27me3 and H4K16 acetylation are dramatically increased in CoREST mutant follicle cells. Our data indicate that CoREST acts as a positive modulator of the Notch pathway in the follicular epithelium as well as in wing tissue, and suggests a previously unidentified role for CoREST in the regulation of Notch signaling. Given its high degree of conservation among species, CoREST probably also functions as a regulator of Notch-dependent cellular events in other organisms.
Assuntos
Proteínas Correpressoras/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Animais , Proteínas Correpressoras/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Feminino , Dosagem de Genes , Histonas/metabolismo , Mitose , Mutação , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oogênese/genética , Fenótipo , Proteólise , Deleção de Sequência , Transdução de Sinais , Supressão Genética , Fatores de Transcrição/metabolismo , Asas de Animais/metabolismoRESUMO
After midline crossing, axons of dorsolateral commissural neurons turn rostrally into the longitudinal axis of the spinal cord. In mouse, the graded distribution of Wnt4 attracts post-crossing axons rostrally. In contrast, in the chicken embryo, the graded distribution of Sonic hedgehog (Shh) guides post-crossing axons by a repulsive mechanism mediated by hedgehog-interacting protein. Based on these observations, we tested for a possible cooperation between the two types of morphogens. Indeed, we found that Wnts also act as axon guidance cues in the chicken spinal cord. However, in contrast to the mouse, Wnt transcription did not differ along the anteroposterior axis of the spinal cord. Rather, Wnt function was regulated by a gradient of the Wnt antagonist Sfrp1 (Secreted frizzled-related protein 1) that in turn was shaped by the Shh gradient. Thus, Shh affects post-crossing axon guidance both directly and indirectly by regulating Wnt function.
Assuntos
Proteínas Aviárias/metabolismo , Axônios/fisiologia , Proteínas Hedgehog/metabolismo , Medula Espinal/embriologia , Medula Espinal/fisiologia , Proteínas Wnt/metabolismo , Animais , Células COS , Movimento Celular/fisiologia , Quimiotaxia , Embrião de Galinha , Chlorocebus aethiops , Técnicas de CoculturaRESUMO
Mating and immunity are two major components of fitness and links between them have been demonstrated in a number of recent investigations. In Drosophila melanogaster, a seminal fluid protein, sex-peptide (SP), up-regulates a number of antimicrobial peptide (AMP) genes in females after mating but the resulting effect on pathogen resistance is unclear. In this study, we tested (1) whether SP-induced changes in gene expression affect the ability of females to kill injected non-pathogenic bacteria and (2) how the injection process per se affects the expression of AMP genes relative to SP. The ability of virgin females and females mated to SP lacking or control males to clear bacteria was assayed using an established technique in which Escherichia coli are injected directly into the fly body and the rate of clearance of the injected bacteria is determined. We found no repeatable differences in clearance rates between virgin females and females mated to SP producing or SP lacking males. However, we found that the piercing of the integument, as occurs during injection, up-regulates AMP gene expression much more strongly than SP. Thus, assays that involve piercing, which are commonly used in immunity studies, can mask more subtle and biologically relevant changes in immunity, such as those induced by mating.
Assuntos
Drosophila melanogaster/imunologia , Comportamento Sexual Animal/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Escherichia coli , Feminino , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Peptídeos/genética , Peptídeos/metabolismo , Fatores de Tempo , Ferimentos e LesõesRESUMO
Seminal fluid elicits a variety of physiological and behavioral changes in insect females. In Drosophila melanogaster females, sex peptide (SP) is the major seminal agent eliciting oviposition and reduction of receptivity. But SP also has many other effects; for example, it stimulates food intake, egg production, ovulation, juvenile hormone production and antimicrobial peptide synthesis. Thus, SP very probably has several receptors. To identify putative targets and signaling cascades, we studied the genome-wide regulation of genes by microarray analysis of RNA isolated from females after mating with wild-type males or males lacking SP, respectively. In addition, we studied the effects of SP on the proteome of females. Sex peptide regulates gene activity differentially in the head and in the abdomen. Genes coding for unspecific antimicrobial peptides are specifically transcribed in the abdomen, e.g. the antimicrobial peptide drosocin in epithelial tissues of the female genital tract (oviduct and calyx). Hence, SP elicits a systemic [Peng J, Zipperlen P & Kubli E (2005) Curr Biol15, 1690-1694] and an epithelial immune response. Ectopic expression of SP in the fat body of transgenic virgin females (with subsequent secretion into the hemolymph) does not elicit drosocin synthesis in the genital tract. Thus, the receptors for the stimulation of the systemic and the epithelial responses by SP are compartmentalized. The hydroxyproline (P*) motif of SP, P*TKFP*IP*SP*NP*, is identified as a novel elicitor of the innate immune response. We suggest that SP acts by chemical mimicry of sugar components of the bacterial cell wall. Thus, SP may induce the immune system via pattern recognition receptors.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/imunologia , Drosophila/imunologia , Hidroxiprolina/química , Imunidade Inata , Peptídeos/química , Peptídeos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica , Glicopeptídeos/biossíntese , Hidroxiprolina/metabolismo , Masculino , Dados de Sequência Molecular , Oviductos/metabolismo , Comportamento Sexual Animal , Relação Estrutura-AtividadeRESUMO
BACKGROUND: During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS. RESULTS: Downregulation of Sema6A in boundary cap cells by in ovo RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point. CONCLUSION: Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.
Assuntos
Padronização Corporal/genética , Sistema Nervoso Central/embriologia , Neuroglia/metabolismo , Sistema Nervoso Periférico/embriologia , Semaforinas/metabolismo , Animais , Células COS , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/genética , Movimento Celular/genética , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Embrião de Galinha , Chlorocebus aethiops , Regulação para Baixo/genética , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Neuroglia/citologia , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Interferência de RNA , Semaforinas/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Raízes Nervosas Espinhais/citologia , Raízes Nervosas Espinhais/embriologia , Raízes Nervosas Espinhais/metabolismoRESUMO
Heterochromatic DNA sequences in the polytene chromosomes of Drosophila melanogaster salivary glands are under-replicated in wild-type strains. In salivary glands of SuUR and in the nurse cells of otu mutants, under-replication is partly suppressed and a banded structure appears within the centric heterochromatin of chromosome 3. This novel banded structure in salivary gland chromosomes was called Plato Atlantis. In order to characterize the heterochromatic component of Plato Atlantis, we constructed a fine-scale cytogenetic map of deletions with break points within centric heterochromatin (Df(3L)1-16, Df(3L)2-66, Df(3R)10-65, Df(3R)4-75 and Df(3L)6B-29 + Df(3R)6B-29). Salivary gland chromosomes show that Df(3L)1-16 removes the complete Plato Atlantis, while Df(3L)2-66 deletes the most proximal 3L regions. These deletions therefore show a substantial cytological overlap. However, in the chromosomes of nurse cells, the same deficiencies remove distinct heterochromatic blocks, with the region of overlap being almost invisible. Satellite (AATAACATAG, AAGAG) and dodecasatellite DNAs mapped in a narrow interval in salivary glands but were found in three clearly distinguishable blocks in nurse cells. The 1.688 satellite was found at a single site in salivary glands but at two sites in nurse cells. We show that newly polytenized heterochromatic structures include blocks h47-h50d of mitotic heterochromatin in salivary glands, but the additional blocks h50p, h53 and h57 are also included in nurse cell chromosomes. Tissue specificity of the patterns of abnormal heterochromatic polytenization implies differential control of DNA replication in somatic and germline cells.