RESUMEN
RNA sequencing is an approach to transcriptomic profiling that enables the detection of differentially expressed genes in response to genetic mutation or experimental treatment, among other uses. Here we describe a method for the use of a customizable, user-friendly bioinformatic pipeline to identify differentially expressed genes in RNA sequencing data obtained from C. elegans, with attention to the improvement in reproducibility and accuracy of results.
Asunto(s)
Caenorhabditis elegans , Biología Computacional , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Programas Informáticos , Flujo de Trabajo , Caenorhabditis elegans/genética , Animales , Biología Computacional/métodos , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Transcriptoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reproducibilidad de los ResultadosRESUMEN
Reproductive organs and developing tissues have high energy demands that require metabolic functions primarily supported by mitochondria function. The highly conserved CISD/NEET iron-sulfur (Fe-S) protein family regulates iron and reactive oxygen homeostasis, both of which are important for mitochondrial function. Disruption of iron and reactive oxygen homeostasis typically leads to detrimental effects. In humans, CISD dysfunction is associated with human health issues including Wolfram syndrome 2. Using C. elegans, we previously determined that the cisd-1, cisd-3.1 and cisd-3.2 have an overlapping role in the regulation of physiological germline apoptosis through the canonical programmed cell death pathway. Here, we isolated the cisd-3.2(pnIs68) mutant that resulted in physiological and fitness defects including germline abnormalities that are associated with abnormal stem cell niche and disrupted formation of bivalent chromosomes. The cisd-3.2(pnIs68) mutation led to complete disruption of the cisd-3.2 gene expression and a decrease in expression of genetically intact cisd-1 and cisd-3.1 genes suggesting an indirect impact of the cisd-3.2(pnIs68) allele. The CISD-3.2 and CISD-3.1 proteins localize to the mitochondria in many tissues throughout development. The cisd-3.2(pnIs68) mutant displays phenotypes associated with mitochondrial dysfunction, including disruption of the mitochondrial network within the germline. These results further support the idea that the CISD protein family is required for mitochondrial function that supports important functions in animals including overall fitness and germline viability.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas Hierro-Azufre/genética , Mitocondrias/genética , Proteínas Mitocondriales/genéticaRESUMEN
Programmed cell death, which occurs through a conserved core molecular pathway, is important for fundamental developmental and homeostatic processes. The human iron-sulfur binding protein NAF-1/CISD2 binds to Bcl-2 and its disruption in cells leads to an increase in apoptosis. Other members of the CDGSH iron sulfur domain (CISD) family include mitoNEET/CISD1 and Miner2/CISD3. In humans, mutations in CISD2 result in Wolfram syndrome 2, a disease in which the patients display juvenile diabetes, neuropsychiatric disorders and defective platelet aggregation. The C. elegans genome contains three previously uncharacterized cisd genes that code for CISD-1, which has homology to mitoNEET/CISD1 and NAF-1/CISD2, and CISD-3.1 and CISD-3.2, both of which have homology to Miner2/CISD3. Disrupting the function of the cisd genes resulted in various germline abnormalities including distal tip cell migration defects and a significant increase in the number of cell corpses within the adult germline. This increased germ cell death is blocked by a gain-of-function mutation of the Bcl-2 homolog CED-9 and requires functional caspase CED-3 and the APAF-1 homolog CED-4. Furthermore, the increased germ cell death is facilitated by the pro-apoptotic, CED-9-binding protein CED-13, but not the related EGL-1 protein. This work is significant because it places the CISD family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery.
Asunto(s)
Apoptosis/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Animales , Apoptosis/fisiología , Proteínas de Unión al Calcio/metabolismo , Caspasas/metabolismo , Familia de Multigenes , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The iron-sulfur (2Fe-2S) binding motif CDGSH appears in many important plant and animal proteins that regulate iron and reactive oxygen metabolism. In human it is found in CISD1-3 proteins involved in diabetes, obesity, cancer, aging, cardiovascular disease and neurodegeneration. Despite the important biological role of the CDGSH domain, its origin, evolution and diversification, are largely unknown. Here, we report that: (1) the CDGSH domain appeared early in evolution, perhaps linked to the heavy use of iron-sulfur driven metabolism by early organisms; (2) a CISD3-like protein with two CDGSH domains on the same polypeptide appears to represent the ancient archetype of CDGSH proteins; (3) the origin of the human CISD3 protein is linked to the mitochondrial endosymbiotic event; (4) the CISD1/2 type proteins that contain only one CDGSH domain, but function as homodimers, originated after the divergence of bacteria and archaea/eukaryotes from their common ancestor; and (5) the human CISD1 and CISD2 proteins diverged about 650-720 million years ago, and CISD3 and CISD1/2 share their descent from an ancestral CISD about 1-1.1 billion years ago. Our findings reveal that the CDGSH domain is ancient in its origin and shed light on the complex evolutionary path of modern CDGSH proteins.
Asunto(s)
Secuencias de Aminoácidos , Evolución Molecular , Hierro/metabolismo , Filogenia , Azufre/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Humanos , Dominios ProteicosRESUMEN
Individuals with type 2 diabetes display metabolic abnormalities, such as hyperglycemia, increased free fatty acids, insulin resistance, and altered ceramide levels, that contribute to vascular dysfunctions and compromised oxygen delivery. Caenorhabditis elegans fed a glucose-supplemented diet or with altered ceramide metabolism, due to a hyl-2 mutation, are sensitive to oxygen deprivation (anoxia). Our experiments showed that the combination of these factors further decreased the anoxia survival. RNA-sequencing analysis was performed to assess how a glucose-supplemented diet and/or a hyl-2 mutation altered the transcriptome. Comparison analysis of transcripts associated with anoxia-sensitive animals [hyl-2(tm2031) mutation or a glucose diet] revealed 199 common transcripts encoded by genes with known or predicted functions involving innate immunity, cuticle function (collagens), or xenobiotic and endobiotic phase I and II detoxification system. Use of RNA interference (RNAi) to target gene products of the xenobiotic and endobiotic phase I and II detoxification system (UDP-glycosyltransferase and Cytochrome p450 genes; ugt-15, ugt-18, ugt-19, ugt-41, ugt-63, cyp-13A12, cyp-25A1, and cyp-33C8) increased anoxia survival in wild-type animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was suppressed by RNAi of cyp-25A1 or cyp-33C8 genes. A glucose diet fed to the P0 hermaphrodite decreased the anoxia survival of its F1 embryos; however, the RNAi of ugt-63 and cyp-33C8 suppressed anoxia sensitivity. These studies provide evidence that the detoxification system impacts oxygen deprivation responses and that C. elegans can be used to model the conserved detoxification system.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ceramidas/biosíntesis , Perfilación de la Expresión Génica , Glucosa/biosíntesis , Oxígeno/metabolismo , Transducción de Señal , Transcriptoma , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Análisis por Conglomerados , Biología Computacional/métodos , Técnicas de Silenciamiento del Gen , Hipoxia/genética , Hipoxia/metabolismo , Metabolismo de los Lípidos , Masculino , Fase I de la Desintoxicación Metabólica/genética , Fase II de la Desintoxicación Metabólica/genética , Mutación , Fenotipo , Xenobióticos/metabolismoRESUMEN
We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Neoplasias/genética , Nicotina/toxicidad , Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos/genética , Animales , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Fertilidad/genética , Humanos , Locomoción/efectos de los fármacos , Locomoción/genética , Mutación , Neoplasias/inducido químicamente , Neoplasias/patología , Proteínas Proto-Oncogénicas c-met/biosíntesis , Proteínas Proto-Oncogénicas c-met/genética , Proteínas ras/biosíntesisRESUMEN
Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.
Asunto(s)
Histonas/metabolismo , Peces Killi/embriología , Peces Killi/genética , Animales , Cromatina/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Histonas/genética , Peces Killi/metabolismo , Metamorfosis Biológica , Metilación , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases.
Asunto(s)
Caenorhabditis elegans/metabolismo , Glucosa/metabolismo , Hipoxia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos/genética , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Metabolismo de los Hidratos de Carbono/genética , Ceramidas/biosíntesis , Dieta , Ácidos Grasos/biosíntesis , Perfilación de la Expresión Génica , Glucosa/administración & dosificación , Hipoxia/genéticaRESUMEN
The adaptive response to hypoxia is accompanied by widespread transcriptional changes that allow for prolonged survival in low oxygen. Many of these changes are directly regulated by the conserved hypoxia-inducible factor-1 (HIF-1) complex; however, even in its absence, many oxygen-sensitive transcripts in Caenorhabditis elegans are appropriately regulated in hypoxia. To identify mediators of these non-HIF-dependent responses, we established a hif-1 mutant reporter line that expresses GFP in hypoxia or when worms are treated with the hypoxia mimetic cobalt chloride (CoCl2). The reporter is selective and HIF independent, in that it remains insensitive to a number of cellular stresses, but is unaffected by mutation of the prolyl hydroxylase egl-9, suggesting that the regulators of this response pathway are different from those controlling the HIF pathway. We used the HIF-independent reporter to screen a transcription factor RNA interference (RNAi) library and identified genes that are required for hypoxia-sensitive and CoCl2-induced GFP expression. We identified the zinc finger protein BLMP-1 as a mediator of the HIF-independent response. We show that mutation of blmp-1 renders animals sensitive to hypoxic exposure and that blmp-1 is required for appropriate hypoxic-induced expression of HIF-independent transcripts. Further, we demonstrate that BLMP-1 is necessary for an increase of hypoxia-dependent histone acetylation within the promoter of a non-HIF-dependent hypoxia response gene.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Regulación de la Expresión Génica , Factores de Transcripción/fisiología , Transcripción Genética , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Hipoxia de la Célula , Mutación , Proteínas Represoras , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Gamete cells pass on information to the next generation via DNA sequence and also through epigenetic mechanisms such as small RNAs, DNA methylation, or chromatin modifications. Caenorhabditis elegans is a genetic model system that an enormous number of talented researchers have used to understand biological phenomenon and develop molecular tools that have ultimately led to paradigm-shifting ideas in biology. Thus, this model is well poised to further investigate the molecular mechanisms involved with epigenetic modifications and transgenerational epigenetic inheritance. The strengths of this model system include a historical wealth of information regarding genetics, development, germline function, chromosome biology, and the regulation of gene expression. Using this system, one can investigate the mechanisms involved with how the germline passes on heritable epigenetic information to subsequent generations. Here, we highlight aspects about the biology of C. elegans that make it amenable to epigenetic studies, highlight some recent findings in the field of epigenetics, and comment on how this system would be beneficial for future biological studies involving epigenetic processes.
Asunto(s)
Envejecimiento/fisiología , Caenorhabditis elegans/genética , Ensamble y Desensamble de Cromatina/fisiología , Epigénesis Genética/fisiología , Patrón de Herencia/fisiología , Modelos Animales , Estrés Fisiológico/fisiología , Envejecimiento/genética , Animales , Epigénesis Genética/genética , Patrón de Herencia/genética , Membrana Nuclear/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins(1-4). In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; <2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.
Asunto(s)
Caenorhabditis elegans/metabolismo , Hipoxia/patología , Microscopía Confocal/métodos , Microscopía por Video/métodos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Hipoxia/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal/instrumentación , Microscopía por Video/instrumentaciónRESUMEN
Developing organisms require nutrients to support cell division vital for growth and development. An adaptation to stress, used by many organisms, is to reversibly enter an arrested state by reducing energy-requiring processes, such as development and cell division. This "wait it out" approach to survive stress until the environment is conductive for growth and development is used by many metazoans. Much is known about the molecular regulation of cell division, metazoan development and responses to environmental stress. However, how these biological processes intersect is less understood. Here, we review studies conducted in Caenorhabditis elegans that investigate how stresses such as oxygen deprivation (hypoxia and anoxia), exogenous chemicals or starvation affect cellular processes in the embryo, larvae or adult germline. Using C. elegans to identify how stress signals biological arrest can help in our understanding of evolutionary pressures as well as human health-related issues.
Asunto(s)
Caenorhabditis elegans/citología , Puntos de Control del Ciclo Celular , Oxígeno/metabolismo , Animales , Benomilo/farmacología , Blastómeros/citología , Blastómeros/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Diferenciación Celular , División Celular , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Células Germinativas/metabolismo , Larva/citología , Larva/efectos de los fármacos , Larva/metabolismo , Oocitos/citología , Oocitos/metabolismo , Estrés FisiológicoRESUMEN
BACKGROUND: Preconditioning environments or therapeutics, to suppress the cellular damage associated with severe oxygen deprivation, is of interest to our understanding of diseases associated with oxygen deprivation. Wildtype C. elegans exposed to anoxia enter into a state of suspended animation in which energy-requiring processes reversibly arrest. C. elegans at all developmental stages survive 24-hours of anoxia exposure however, the ability of adult hermaphrodites to survive three days of anoxia significantly decreases. Mutations in the insulin-like signaling receptor (daf-2) and LIN-12/Notch (glp-1) lead to an enhanced long-term anoxia survival phenotype. METHODOLOGY/PRINCIPAL FINDINGS: In this study we show that the combined growth environment of 25°C and a diet of HT115 E. coli will precondition adult hermaphrodites to survive long-term anoxia; many of these survivors have normal movement after anoxia treatment. Animals fed the drug metformin, which induces a dietary-restriction like state in animals and activates AMPK in mammalian cell culture, have a higher survival rate when exposed to long-term anoxia. Mutations in genes encoding components of AMPK (aak-2, aakb-1, aakb-2, aakg-2) suppress the environmentally and genetically induced long-term anoxia survival phenotype. We further determine that there is a correlation between the animals that survive long-term anoxia and increased levels of carminic acid staining, which is a fluorescent dye that incorporates in with carbohydrates such as glycogen. CONCLUSIONS/SIGNIFICANCE: We conclude that small changes in growth conditions such as increased temperature and food source can influence the physiology of the animal thus affecting the responses to stress such as anoxia. Furthermore, this supports the idea that metformin should be further investigated as a therapeutic tool for treatment of oxygen-deprived tissues. Finally, the capacity for an animal to survive long bouts of severe oxygen deprivation is likely dependent on specific subunits of the heterotrimeric protein AMPK and energy stores such as carbohydrates.
Asunto(s)
Adenilato Quinasa/fisiología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Predisposición Genética a la Enfermedad , Hipoxia/genética , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Caenorhabditis elegans/fisiología , Carbohidratos de la Dieta/metabolismo , Carbohidratos de la Dieta/farmacología , Ambiente , Escherichia coli/fisiología , Cadena Alimentaria , Organismos Hermafroditas/genética , Organismos Hermafroditas/metabolismo , Organismos Hermafroditas/fisiología , Hipoglucemiantes/farmacología , Hipoxia/metabolismo , Hipoxia/mortalidad , Hipoxia/patología , Metformina/farmacología , Organismos Modificados Genéticamente , Oxígeno/metabolismo , Oxígeno/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Temperatura , Factores de TiempoRESUMEN
Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type-arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.
Asunto(s)
Blastocisto/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/fisiología , Profase , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica , Hipoxia , Mutación , Nocodazol/farmacología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/fisiología , Oocitos/metabolismo , Interferencia de ARNRESUMEN
Identifying genotypes and phenotypes that enhance an organism's ability to survive stress is of interest. We used Caenorhabditis elegans mutants, RNA interference (RNAi), and the chemical 5-fluorodeoxyuridine (FUDR) to test the hypothesis that a reduction in progeny would increase oxygen deprivation (anoxia) survival. In the hermaphrodite gonad, germ line processes such as spermatogenesis and oogenesis can be simultaneously as well as independently disrupted by genetic mutations. We analyzed genetic mutants [glp-1(q158), glp-4(bn2ts), plc-1(rx1), ksr-1(ku68), fog-2(q71), fem-3(q20), spe-9(hc52ts), fer-15(hc15ts)] with reduced progeny production due to various reproductive defects. Furthermore, we used RNAi to inhibit the function of gene products in the RTK/Ras/MAPK signaling pathway, which is known to be involved in a variety of developmental processes including gonad function. We determined that reduced progeny production or complete sterility enhanced anoxia survival except in the case of sterile hermaphrodites [spe-9(hc52ts), fer-15(hc15ts)] undergoing oocyte maturation and ovulation as exhibited by the presence of laid unfertilized oocytes. Furthermore, the fog-2(q71) long-term anoxia survival phenotype was suppressed when oocyte maturation and ovulation were induced by mating with males that have functional or nonfunctional sperm. The mutants with a reduced progeny production survive long-term anoxia in a daf-16- and hif-1-independent manner. Finally, we determined that wild-type males were able to survive long-term anoxia in a daf-16-independent manner. Together, these results suggest that the insulin signaling pathway is not the only mechanism to survive oxygen deprivation and that altering gonad function, in particular oocyte maturation and ovulation, leads to a physiological state conducive for oxygen deprivation survival.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Ovulación/fisiología , Factores de Transcripción/fisiología , Animales , Antimetabolitos Antineoplásicos/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Floxuridina/farmacología , Factores de Transcripción Forkhead , Hipoxia/fisiopatología , Masculino , Mutación , Fenotipo , Interferencia de ARN , Reproducción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.
Asunto(s)
Caenorhabditis elegans/fisiología , Calor , Hipoxia , Oocitos/fisiología , Ovulación/fisiología , Estrés Oxidativo , Ribonucleoproteínas/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Femenino , Fertilización/fisiología , Masculino , Oocitos/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
BACKGROUND: The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant. RESULTS: The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. CONCLUSION: Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.
RESUMEN
Oxygen deprivation has a role in the pathology of many human diseases. Thus it is of interest in understanding the genetic and cellular responses to hypoxia or anoxia in oxygen-deprivation-tolerant organisms such as Caenorhabditis elegans. In C. elegans the DAF-2/DAF-16 pathway, an IGF-1/insulin-like signaling pathway, is involved with dauer formation, longevity, and stress resistance. In this report we compared the response of wild-type and daf-2(e1370) animals to anoxia. Unlike wild-type animals, the daf-2(e1370) animals have an enhanced anoxia-survival phenotype in that they survive long-term anoxia and high-temperature anoxia, do not accumulate significant tissue damage in either of these conditions, and are motile after 24 hr of anoxia. RNA interference was used to screen DAF-16-regulated genes that suppress the daf-2(e1370)-enhanced anoxia-survival phenotype. We identified gpd-2 and gpd-3, two nearly identical genes in an operon that encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. We found that not only is the daf-2(e1370)-enhanced anoxia phenotype dependent upon gpd-2 and gpd-3, but also the motility of animals exposed to brief periods of anoxia is prematurely arrested in gpd-2/3(RNAi) and daf-2(e1370);gpd-2/3(RNAi) animals. These data suggest that gpd-2 and gpd-3 may serve a protective role in tissue exposed to oxygen deprivation.