RESUMO
Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR)/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc) as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Neurônios/citologia , Neurônios/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Mutação , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Ligação Proteica , Interferência de RNA , Retina/metabolismo , Transdução de SinaisRESUMO
Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific.
Assuntos
Encéfalo/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neuroglia/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Proliferação de Células , Cruzamentos Genéticos , Drosophila melanogaster , Hibridização In Situ , Insulina/metabolismo , Modelos Biológicos , Interferência de RNA , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de TempoRESUMO
BACKGROUND: In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines. RESULTS: By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped. CONCLUSIONS: In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA transcription cofactor U-shaped as a key regulator of the plasmatocyte to lamellocyte transformation.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Homeostase , Animais , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Genes Supressores de Tumor , Hematopoese , Hemócitos/citologia , Hemócitos/imunologia , Interferência de RNARESUMO
Glial cells constitute a large proportion of the central nervous system (CNS) and are critical for the correct development and function of the adult CNS. Recent studies have shown that specific subtypes of glia are generated through the proliferation of differentiated glial cells in both the developing invertebrate and vertebrate nervous systems. However, the factors that regulate glial proliferation in specific glial subtypes are poorly understood. To address this we have performed global gene expression analysis of Drosophila post-embryonic CNS tissue enriched in glial cells, through glial specific overexpression of either the FGF or insulin receptor. Analysis of the differentially regulated genes in these tissues shows that the expression of known glial genes is significantly increased in both cases. Conversely, the expression of neuronal genes is significantly decreased. FGF and insulin signalling drive the expression of overlapping sets of genes in glial cells that then activate proliferation. We then used these data to identify novel transcription factors that are expressed in glia in the brain. We show that two of the transcription factors identified in the glial enriched gene expression profiles, foxO and tramtrack69, have novel roles in regulating the proliferation of cortex and perineurial glia. These studies provide new insight into the genes and molecular pathways that regulate the proliferation of specific glial subtypes in the Drosophila post-embryonic brain.
Assuntos
Encéfalo/metabolismo , Drosophila/fisiologia , Neuroglia/metabolismo , Animais , Encéfalo/citologia , Proliferação de Células/fisiologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Receptor de Insulina/genética , Fatores de Transcrição/genéticaRESUMO
Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-kappaB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36-expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/imunologia , Endopeptidases/fisiologia , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Dosagem de Genes , Inativação Gênica , Vida Livre de Germes/imunologia , Bactérias Gram-Negativas/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , NF-kappa B/biossíntese , Mapeamento de Interação de ProteínasRESUMO
Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2-mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Drosophila melanogaster/microbiologia , Pseudomonas aeruginosa/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/química , Drosophila melanogaster/metabolismo , Bactérias Gram-Negativas/metabolismo , Macrófagos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Fagocitose , Plasmócitos/metabolismo , Plasmócitos/microbiologia , Sepse , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.
Assuntos
ADP Ribose Transferases/biossíntese , Toxinas Bacterianas/biossíntese , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Pseudomonas aeruginosa/genética , ADP Ribose Transferases/genética , Animais , Animais Geneticamente Modificados , Toxinas Bacterianas/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Olho/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Proteínas Ativadoras de GTPase/genética , Hemócitos/metabolismo , Microscopia Eletrônica de Varredura , Morfogênese , Fagocitose , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/fisiologia , Transdução de Sinais , Staphylococcus aureus/patogenicidade , Virulência , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In Drosophila, RotundRacGAP/RacGAP(84C) is critical to retinal organisation and spermatogenesis. We show that eye-directed expression of RacGAP(84C) or its GTPase activating protein (GAP) domain induces a dominant rough eye phenotype which we used as a starting point in a gain-of-function screen to identify new partners of RacGAP(84C). Proteins known to function in Ras, Rho and Rac signalling were identified confirming the essential role of RacGAP(84C) in crosstalk between GTPases. Other potential RacGAP(84C) partners identified by the screen are implicated in signal transduction, DNA remodelling, cytoskeletal organisation, membrane trafficking and spermatogenesis. This latter class includes the serine/threonine kinase Center divider (Cdi), which is homologous to the human LIM kinase, Testis specific kinase 1 (TESK1), involved in cytoskeleton control through Cofilin phosphorylation. Eye-directed expression of cdi strongly suppressed the phenotypes induced by either RacGAP(84C) gain-of-function or by the dominant negative form of Rac1, Rac1N17. These results are consistent with Cdi being a specific downstream target of Rac1. We showed that Rac1 and cdi are both expressed in Drosophila testis and that homozygous Rac1 mutants exhibit poor fertility that is further reduced by introducing a cdi loss-of-function mutation in trans. Thus, results from a misexpression screen in the eye led us to a putative novel Rac1-Cdi-Cofilin pathway, regulated by RacGAP(84C), coordinating Drosophila spermatogenesis.