RESUMEN
Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.
Asunto(s)
Núcleo Celular/patología , Drosophila melanogaster/crecimiento & desarrollo , Proteína Huntingtina/genética , Mutación , Neuronas/patología , Péptidos/genética , Tráquea/patología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Cuerpos de Inclusión , Masculino , Neuronas/metabolismo , Tráquea/metabolismoRESUMEN
The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w + mW.hs ), or rosy (ry+ ) and neomycin (neoR ) transgenes are in common use. Parallel ovoD lines were developed using w + mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as ry+ , neoR FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry+ , neoR FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w + mW.hs FRT. Here we report the creation of a new ovoD line for the ry+ , neoR FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Infertilidad Femenina/genética , N-Acetilglucosaminiltransferasas/genética , Alelos , Animales , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Mutación de Línea Germinal/genética , Humanos , Masculino , Mosaicismo/embriología , Fenotipo , Mutaciones Letales Sintéticas/genética , TransgenesRESUMEN
Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. Here we describe the isolation and phenotypic characterization of hdac3 mutations. We find that both hdac3 and hdac1 mutations are dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. Our results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue.
Asunto(s)
Apoptosis/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Histona Desacetilasas/metabolismo , Alelos , Animales , Apoptosis/genética , Proliferación Celular , Drosophila/citología , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Silenciador del Gen , Genes de Insecto , Genes Letales , Genes Recesivos , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Humanos , Larva/citología , Larva/enzimología , Larva/crecimiento & desarrollo , Mutación , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Procesamiento Proteico-Postraduccional , Alas de Animales/citología , Alas de Animales/enzimología , Alas de Animales/crecimiento & desarrolloRESUMEN
Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion, and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Furthermore, we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes.
Asunto(s)
Acetiltransferasas/metabolismo , Dominio Catalítico/fisiología , Proteínas de Drosophila/metabolismo , Ovario/citología , Ovario/enzimología , Acetiltransferasas/genética , Alelos , Animales , Western Blotting , Dominio Catalítico/genética , Drosophila , Proteínas de Drosophila/genética , Femenino , Inmunohistoquímica , Reacción en Cadena de la PolimerasaRESUMEN
Heparan sulfate proteoglycans (HSPGs) are extracellular macromolecules found on virtually every cell type in eumetazoans. HSPGs are composed of a core protein covalently linked to glycosaminoglycan (GAG) sugar chains that bind and modulate the signaling efficiency of many ligands, including Hedgehog (Hh), Wingless (Wg) and Bone morphogenetic proteins (BMPs). Here, we show that, in Drosophila, loss of HSPGs differentially affects embryonic Hh, Wg and BMP signaling. We find that a stage-specific block to GAG synthesis prevents HSPG expression during establishment of the BMP activity gradient that is crucial for dorsal embryonic patterning. Subsequently, GAG synthesis is initiated coincident with the onset of Hh and Wg signaling which require HSPGs. This temporal regulation is achieved by the translational control of HSPG synthetic enzymes through internal ribosome entry sites (IRESs). IRES-like features are conserved in GAG enzyme transcripts from diverse organisms, suggesting that this represents a novel evolutionarily conserved mechanism for regulating GAG synthesis and modulating growth factor activity.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/metabolismo , Proteoglicanos de Heparán Sulfato/biosíntesis , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Tipificación del Cuerpo/efectos de los fármacos , Cartilla de ADN/genética , Drosophila/efectos de los fármacos , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Heparina/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Wnt1RESUMEN
Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.