RESUMEN
The ability of secreted Transforming Growth Factor ß (TGFß) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFß signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.
Asunto(s)
Proteínas de Drosophila/metabolismo , Endopeptidasas/metabolismo , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Cruzamientos Genéticos , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Modelos Genéticos , Mutación , Fenotipo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
CORL proteins (FUSSEL/SKOR proteins in humans) are related to Sno/Ski oncogenes but their developmental roles are unknown. We have cloned Drosophila CORL and show that its expression is restricted to distinct subsets of cells in the central nervous system. We generated a deletion of CORL and noted that homozygous individuals rarely survive to adulthood. Df(4)dCORL adult escapers display mushroom body (MB) defects and Df(4)dCORL larvae are lacking Ecdysone Receptor (EcR-B1) expression in MB neurons. This is phenocopied in CORL-RNAi and Smad2-RNAi clones in wild-type larvae. Furthermore, constitutively active Baboon (type I receptor upstream of Smad2) cannot stimulate EcR-B1 MB expression in Df(4)dCORL larvae, which demonstrates a formal requirement for CORL in Smad2 signaling. Studies of mouse Corl1 (Skor1) revealed that it binds specifically to Smad3. Overall, the data suggest that CORL facilitates Smad2 activity upstream of EcR-B1 in the MB. The conservation of neural expression and strong sequence homology of all CORL proteins suggests that this is a new family of Smad co-factors.
Asunto(s)
Proteínas de Drosophila/metabolismo , Cuerpos Pedunculados/metabolismo , Receptores de Esteroides/metabolismo , Proteína Smad2/metabolismo , Animales , Biología Computacional , Drosophila , Proteínas de Drosophila/genética , Receptores de Esteroides/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteína Smad2/genéticaRESUMEN
A previous genetic analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Here we report that within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, our results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/metabolismo , Proteínas Represoras/metabolismo , Sistema Respiratorio/embriología , Sistema Respiratorio/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sitios de Unión , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Drosophila/genética , Proteínas de Drosophila/genética , Embrión no Mamífero , Elementos de Facilitación Genéticos , Eliminación de Gen , Genes Reporteros , Inmunohistoquímica , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética , beta-Galactosidasa/metabolismoRESUMEN
CORL proteins (SKOR in mice and Fussel in humans) are a subfamily of central nervous system (CNS) specific proteins related to Sno/Ski oncogenes. Their developmental and homeostatic roles are largely unknown. We previously showed that Drosophila CORL (dCORL; fussel in Flybase) functions between the Activin receptor Baboon and Ecdysone Receptor-B1 (EcR-B1) activation in mushroom body neurons of third instar larval brains. To better understand dCORL regulation and function we generated a series of reporter genes. We examined the embryonic and larval CNS and found that dCORL is regulated by stage specific interactions between intertwined activators and repressors spanning numerous reporters. The reporter AH.lacZ, which contains sequences 7-11kb upstream of dCORL exon1, reflects dCORL brain expression at all stages. Surprisingly, AH.lacZ was not detected in EcR-B1 expressing mushroom body neurons. In larvae AH.lacZ is coexpressed with Elav and the transcription factor Drifter in dILP2 insulin producing cells of the pars intercerebralis. The presence of dCORL in insulin producing cells suggests that dCORL functions non-autonomously in the regulation of EcR-B1 mushroom body activation via the modulation of insulin signaling. Overall, the high level of sequence conservation seen in all CORL/SKOR/Fussel family members and their common CNS specificity suggest that similarly complex regulation and a potential function in insulin signaling are associated with SKOR/Fussel proteins in mammals.
Asunto(s)
Sistema Nervioso Central/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Animales , Drosophila/embriología , Expresión Génica , Genes Reporteros , Fenotipo , Unión ProteicaRESUMEN
CORL proteins (known as SKOR in mice, Fussel in humans and fussel in Flybase) are a family of CNS specific proteins related to Sno/Ski oncogenes. Their developmental and adult roles are largely unknown. A Drosophila CORL (dCORL) reporter gene is expressed in all Drosophila insulin-like peptide 2 (dILP2) neurons of the pars intercerebralis (PI) of the larval and adult brain. The transcription factor Drifter is also expressed in the PI in a subset of dCORL and dILP2 expressing neurons and in several non-dILP2 neurons. dCORL mutant virgin adult brains are missing all dILP2 neurons that do not also express Drifter. This phenotype is also seen when expressing dCORL-RNAi in neurosecretory cells of the PI. dCORL mutant virgin adults of both sexes have a significantly shorter lifespan than their parental strain. This longevity defect is completely reversed by mating (lifespan increases over 50% for males and females). Analyses of dCORL mutant mated adult brains revealed a complete rescue of dILP2 neurons without Drifter. Taken together, the data suggest that dCORL participates in a neural network connecting the insulin signaling pathway, longevity and mating. The conserved sequence and CNS specificity of all CORL proteins imply that this network may be operating in mammals.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Regulación de la Expresión Génica/fisiología , Insulina/metabolismo , Longevidad/fisiología , Neuronas/metabolismo , Neurosecreción/fisiología , Animales , Drosophila melanogaster , Femenino , Masculino , Red Nerviosa/citología , Red Nerviosa/metabolismo , Neuronas/citologíaRESUMEN
A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno). The dSno locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for dSno function, an intriguing possibility from the perspective of developmental evolution. dSno is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. dSno mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and dSmad2 mutants. This suggests that dSno is a mediator of Baboon signaling. dSno binds to Medea and Medea/dSno complexes have enhanced affinity for dSmad2. Alternatively, Medea/dSno complexes have reduced affinity for Mad such that, in the presence of dSno, Dpp signaling is antagonized. We propose that dSno functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFbeta signaling and a novel function for Sno/Ski family proteins.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Drosophila , Drosophila/fisiología , Proteínas de Insectos/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Asparagina/metabolismo , Secuencia de Bases , Drosophila/embriología , Drosophila/genética , Embrión no Mamífero , Eliminación de Gen , Inmunoprecipitación , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-beta signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genotipo , Inmunohistoquímica , Microscopía Confocal , Proteínas Nucleares/genética , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. At this time, how the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, we expressed five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes in flies. We found that all of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified - dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4(100T) appears to be capable of activating the expression of WG target genes. These results imply that SMAD4(100T) may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes - an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. Our results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4(100T) is likely to be different from those with other SMAD mutations. From a larger perspective, our study shows that the genetic characterization of missense mutations, particularly in modular proteins, requires experimental verification.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Smad/genética , Proteína Smad4/fisiología , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Humanos , Neoplasias/etiología , Proteína Smad4/genética , Alas de Animales/crecimiento & desarrollo , Proteína Wnt1RESUMEN
The experimental validation of genes predicted from genomic sequence and the identification of functions for these genes is an increasingly important task. We report a multidisciplinary analysis of CG3488, a predicted gene adjacent to Mothers against dpp in Drosophila melanogaster. We cloned and sequenced a cDNA corresponding to CG3488 and we show that it is expressed in embryos. A computational analysis shows that CG3488 contains a number of conserved domains present in enzymes capable of lipid hydrolysis. A phylogenetic analysis shows that CG3488 is the homolog of human alpha/beta hydrolase2 and that these genes belong to a novel multigene family with members in animals, plants, fungi, and bacteria. A genetic analysis shows that heterozygosity for a chromosomal deletion that removes CG3488 dominantly enhances the excess lipid phenotype associated with a mutation in adipose, an uncloned obesity gene. Further, overexpression of a CG3488 transgene rescues this obesity phenotype. Overall, the data suggests that CG3488 functions as a lipase and that analyses of its homologs will provide unique insights into lipid metabolism in many species.