RESUMEN
Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.
Asunto(s)
Evolución Biológica , Genoma , Ornitorrinco/genética , Tachyglossidae/genética , Animales , Femenino , Masculino , Mamíferos/genética , Filogenia , Cromosomas Sexuales/genéticaRESUMEN
Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.
Asunto(s)
Metilación de ADN , Proteína 2 de Unión a Metil-CpG , Animales , Encéfalo/metabolismo , Genoma , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Vertebrados/genéticaRESUMEN
The evolution of heteromorphic sex chromosomes has occurred independently many times in different lineages. The differentiation of sex chromosomes leads to dramatic changes in sequence composition and function and guides the evolutionary trajectory and utilization of genes in pivotal sex determination and reproduction roles. In addition, meiotic recombination and pairing mechanisms are key in orchestrating the resultant impact, retention and maintenance of heteromorphic sex chromosomes, as the resulting exposure of unpaired DNA at meiosis triggers ancient repair and checkpoint pathways. In this review, we summarize the different ways in which sex chromosome systems are organized at meiosis, how pairing is affected, and differences in unpaired DNA responses. We hypothesize that lineage specific differences in meiotic organization is not only a consequence of sex chromosome evolution, but that the establishment of epigenetic changes on sex chromosomes contributes toward their evolutionary conservation.
Asunto(s)
Evolución Biológica , Linaje de la Célula , Cromosomas/genética , Epigénesis Genética , Meiosis/genética , Cromosomas Sexuales/genética , Animales , Silenciador del Gen , HumanosRESUMEN
The platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly improved reference genome, we called over 6.7 M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative among other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated the de novo mutation rate in the platypus at 7.0 × 10-9/bp/generation (95% CI 4.1 × 10-9-1.2 × 10-8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.
Asunto(s)
Distribución Animal , Ornitorrinco/genética , Animales , Australia , Femenino , Variación Genética , Endogamia , Masculino , Tasa de Mutación , Dinámica Poblacional , Secuenciación Completa del GenomaRESUMEN
Cohesins are vital for chromosome organisation during meiosis and mitosis. In addition to the important function in sister chromatid cohesion, these complexes play key roles in meiotic recombination, DSB repair, homologous chromosome pairing and segregation. Egg-laying mammals (monotremes) feature an unusually complex sex chromosome system, which raises fundamental questions about organisation and segregation during meiosis. We discovered a dynamic and differential accumulation of cohesins on sex chromosomes during platypus prophase I and specific reorganisation of the sex chromosome complex around a large nucleolar body. Detailed analysis revealed a differential loading of SMC3 on the chromatin and chromosomal axis of XY shared regions compared with the chromatin and chromosomal axes of asynapsed X and Y regions during prophase I. At late prophase I, SMC3 accumulation is lost from both the chromatin and chromosome axes of the asynaptic regions of the chain and resolves into subnuclear compartments. This is the first report detailing unpaired DNA specific SMC3 accumulation during meiosis in any species and allows speculation on roles for cohesin in monotreme sex chromosome organisation and segregation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Emparejamiento Cromosómico , Profase Meiótica I/genética , Ornitorrinco/genética , Cromosomas Sexuales/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Hibridación Fluorescente in Situ , Masculino , Ornitorrinco/metabolismo , CohesinasRESUMEN
SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.
RESUMEN
Natural antisense transcripts (NATs) are involved in cellular development and regulatory processes. Multiple NATs at the Sox4 gene locus are spatiotemporally regulated throughout murine cerebral corticogenesis. In the study, we evaluated the potential functional role of Sox4 NATs at Sox4 gene locus. We demonstrated Sox4 sense and NATs formed dsRNA aggregates in the cytoplasm of brain cells. Over expression of Sox4 NATs in NIH/3T3 cells generally did not alter the level of Sox4 mRNA expression or protein translation. Upregulation of a Sox4 NAT known as Sox4ot1 led to the production of a novel small RNA, Sox4_sir3. Its biogenesis is Dicer1-dependent and has characteristics resemble piRNA. Expression of Sox4_sir3 was observed in the marginal and germinative zones of the developing and postnatal brains suggesting a potential role in regulating neurogenesis. We proposed that Sox4 sense-NATs serve as Dicer1-dependent templates to produce a novel endo-siRNA- or piRNA-like Sox4_sir3.
Asunto(s)
Encéfalo/crecimiento & desarrollo , ARN sin Sentido/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXC/genética , Animales , Encéfalo/metabolismo , Citoplasma/metabolismo , ARN Helicasas DEAD-box/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Células 3T3 NIH , Neurogénesis , ARN sin Sentido/metabolismo , ARN Bicatenario/genética , Ribonucleasa III/metabolismo , Factores de Transcripción SOXC/metabolismoRESUMEN
BACKGROUND: In therian mammals heteromorphic sex chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during meiotic prophase I while the autosomes maintain transcriptional activity. The evolution of this sex chromosome silencing is thought to result in retroposition of genes required in spermatogenesis from the sex chromosomes to autosomes. In birds sex chromosome specific silencing appears to be absent and global transcriptional reductions occur through pachytene and sex chromosome-derived autosomal retrogenes are lacking. Egg laying monotremes are the most basal mammalian lineage, feature a complex and highly differentiated XY sex chromosome system with homology to the avian sex chromosomes, and also lack autosomal retrogenes. In order to delineate the point of origin of sex chromosome specific silencing in mammals we investigated whether MSCI exists in platypus. RESULTS: Our results show that platypus sex chromosomes display only partial or transient colocalisation with a repressive histone variant linked to therian sex chromosome silencing and surprisingly lack a hallmark MSCI epigenetic signature present in other mammals. Remarkably, platypus instead feature an avian like period of general low level transcription through prophase I with the sex chromosomes and the future mammalian X maintaining association with a nucleolus-like structure. CONCLUSIONS: Our work demonstrates for the first time that in mammals meiotic silencing of sex chromosomes evolved after the divergence of monotremes presumably as a result of the differentiation of the therian XY sex chromosomes. We provide a novel evolutionary scenario on how the future therian X chromosome commenced the trajectory toward MSCI.
Asunto(s)
Epigénesis Genética , Silenciador del Gen , Meiosis , Ornitorrinco/genética , Cromosomas Sexuales/genética , Animales , Evolución Molecular , Perfilación de la Expresión Génica , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Profase Meiótica IRESUMEN
The platypus and echidna are the only extant species belonging to the clade of monotremata, the most basal mammalian lineage. The platypus is particularly well known for its mix of mammalian and reptilian characteristics and work in recent years has revealed this also extends to the genetic level. Amongst the monotreme specific features is the unique multiple sex chromosome system (5X4Y in the echidna and 5X5Y in the platypus), which forms a chain in meiosis. This raises questions about sex chromosome organisation at meiosis, including whether there has been changes in genes coding for synaptonemal complex proteins which are involved in homologous synapsis. Here we investigate the key structural components of the synaptonemal complex in platypus and echidna, synaptonemal complex proteins 1, 2 and 3 (SYCP1, SYCP2 and SYCP3). SYCP1 and SYCP2 orthologues are present, conserved and expressed in platypus testis. SYCP3 in contrast is highly diverged, but key residues required for self-association are conserved, while those required for tetramer stabilisation and DNA binding are missing. We also discovered a second SYCP3-like gene (SYCP3-like) in the same region. Comparison with the recently published Y-borne SYCP3 amino acid sequences revealed that SYCP3Y is more similar to SYCP3 in other mammals than the monotreme autosomal SYCP3. It is currently unclear if these changes in the SYCP3 gene repertoire are related to meiotic organisation of the extraordinary monotreme sex chromosome system.
Asunto(s)
Proteínas Nucleares/genética , Ornitorrinco/genética , Complejo Sinaptonémico/genética , Tachyglossidae/genética , Secuencia de Aminoácidos , Animales , Cromosomas de los Mamíferos/genética , Evolución Molecular , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Filogenia , Homología de Secuencia de Aminoácido , Cromosomas Sexuales/genética , Complejo Sinaptonémico/metabolismo , Testículo/metabolismoRESUMEN
Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production.
Asunto(s)
Evolución Molecular , ARN Largo no Codificante/genética , Animales , Anuros/genética , Pollos/genética , Secuencia Conservada/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genómica , Humanos , Ratones , MicroARNs/genética , Familia de Multigenes , Primates/genética , Proteínas/genética , Precursores del ARN/genética , TranscriptomaRESUMEN
As a result of sex chromosome differentiation from ancestral autosomes, male mammalian cells only contain one X chromosome. It has long been hypothesized that X-linked gene expression levels have become doubled in males to restore the original transcriptional output, and that the resulting X overexpression in females then drove the evolution of X inactivation (XCI). However, this model has never been directly tested and patterns and mechanisms of dosage compensation across different mammals and birds generally remain little understood. Here we trace the evolution of dosage compensation using extensive transcriptome data from males and females representing all major mammalian lineages and birds. Our analyses suggest that the X has become globally upregulated in marsupials, whereas we do not detect a global upregulation of this chromosome in placental mammals. However, we find that a subset of autosomal genes interacting with X-linked genes have become downregulated in placentals upon the emergence of sex chromosomes. Thus, different driving forces may underlie the evolution of XCI and the highly efficient equilibration of X expression levels between the sexes observed for both of these lineages. In the egg-laying monotremes and birds, which have partially homologous sex chromosome systems, partial upregulation of the X (Z in birds) evolved but is largely restricted to the heterogametic sex, which provides an explanation for the partially sex-biased X (Z) expression and lack of global inactivation mechanisms in these lineages. Our findings suggest that dosage reductions imposed by sex chromosome differentiation events in amniotes were resolved in strikingly different ways.
Asunto(s)
Aves/genética , Compensación de Dosificación (Genética) , Evolución Molecular , Mamíferos/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Simulación por Computador , Femenino , Duplicación de Gen , Regulación de la Expresión Génica , Genes Ligados a X , Masculino , Análisis de Secuencia de ARN , Cromosomas Sexuales , Testículo/citología , TranscriptomaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that can exert multilevel inhibition/repression at a post-transcriptional or protein synthesis level during disease or development. Characterisation of miRNAs in adult mammalian brains by deep sequencing has been reported previously. However, to date, no small RNA profiling of the developing brain has been undertaken using this method. We have performed deep sequencing and small RNA analysis of a developing (E15.5) mouse brain. RESULTS: We identified the expression of 294 known miRNAs in the E15.5 developing mouse brain, which were mostly represented by let-7 family and other brain-specific miRNAs such as miR-9 and miR-124. We also discovered 4 putative 22-23 nt miRNAs: mm_br_e15_1181, mm_br_e15_279920, mm_br_e15_96719 and mm_br_e15_294354 each with a 70-76 nt predicted pre-miRNA. We validated the 4 putative miRNAs and further characterised one of them, mm_br_e15_1181, throughout embryogenesis. Mm_br_e15_1181 biogenesis was Dicer1-dependent and was expressed in E3.5 blastocysts and E7 whole embryos. Embryo-wide expression patterns were observed at E9.5 and E11.5 followed by a near complete loss of expression by E13.5, with expression restricted to a specialised layer of cells within the developing and early postnatal brain. Mm_br_e15_1181 was upregulated during neurodifferentiation of P19 teratocarcinoma cells. This novel miRNA has been identified as miR-3099. CONCLUSIONS: We have generated and analysed the first deep sequencing dataset of small RNA sequences of the developing mouse brain. The analysis revealed a novel miRNA, miR-3099, with potential regulatory effects on early embryogenesis, and involvement in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development.
Asunto(s)
Encéfalo/metabolismo , MicroARNs/genética , Animales , Encéfalo/crecimiento & desarrollo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , MicroARNs/metabolismoRESUMEN
Monotremes are phylogenetically and phenotypically unique animals with an unusually complex sex chromosome system that is composed of ten chromosomes in platypus and nine in echidna. These chromosomes are alternately linked (X1Y1, X2Y2, ...) at meiosis via pseudoautosomal regions and segregate to form spermatozoa containing either X or Y chromosomes. The physical and epigenetic mechanisms involved in pairing and assembly of the complex sex chromosome chain in early meiotic prophase I are completely unknown. We have analysed the pairing dynamics of specific sex chromosome pseudoautosomal regions in platypus spermatocytes during prophase of meiosis I. Our data show a highly coordinated pairing process that begins at the terminal Y5 chromosome and completes with the union of sex chromosomes X1Y1. The consistency of this ordered assembly of the chain is remarkable and raises questions about the mechanisms and factors that regulate the differential pairing of sex chromosomes and how this relates to potential meiotic silencing mechanisms and alternate segregation.
Asunto(s)
Emparejamiento Cromosómico/fisiología , Meiosis/genética , Ornitorrinco/genética , Cromosomas Sexuales/genética , Cromosomas Sexuales/metabolismo , Animales , Cromosomas Artificiales Bacterianos , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Momento de Replicación del ADN/fisiología , Hibridación Fluorescente in Situ , Masculino , Modelos Biológicos , Telómero/metabolismoRESUMEN
Platypus and echidnas are the only living representative of the egg-laying mammals that diverged 166 million years ago from the mammalian lineage. Despite occupying a key spot in mammalian phylogeny, research on monotremes has been limited by access to material and lack of molecular genetic resources. This has changed recently, and the sequencing of the platypus genome has promoted monotremes into a generally accessible tool in comparative genomics. The most extraordinary aspect of the monotreme genome is an amazingly complex sex chromosomes system that shares extensive homology with bird sex chromosomes and no homology with sex chromosomes of other mammals. This raises important questions about dosage compensation of the five pairs of sex chromosomes in females and meiotic silencing in males, and we are only beginning to unravel possible mechanisms and pathways that may be involved. The homology between monotreme and bird sex chromosomes makes comparison between those species worthwhile, also as they provide a well-defined example where the same sex chromosomes changed from female heterogamety (chicken) to male heterogamety (monotremes). We summarize recent research on monotreme and chicken sex chromosomes and discuss possible mechanisms that may contribute to sex chromosome silencing in monotremes.
Asunto(s)
Compensación de Dosificación (Genética) , Evolución Molecular , Monotremata/genética , Cromosomas Sexuales/genética , Animales , Femenino , Humanos , Masculino , Mamíferos/genética , Meiosis/genéticaRESUMEN
The sequential modifications of histones form the basis of the histone code that translates into either gene activation or repression. Nuclear receptors recruit a cohort of histone-modifying enzymes in response to ligand binding and regulate proliferation, differentiation, and cell death. In Drosophila melanogaster, the steroid hormone ecdysone binds its heterodimeric receptor ecdysone receptor/ultraspiracle to spatiotemporally regulate the transcription of several genes. In this study, we identify a novel cofactor, Drosophila lysine ketoglutarate reductase (dLKR)/saccharopine dehydrogenase (SDH), that is involved in ecdysone-mediated transcription. dLKR/SDH binds histones H3 and H4 and suppresses ecdysone-mediated transcription of cell death genes by inhibiting histone H3R17me2 mediated by the Drosophila arginine methyl transferase CARMER. Our data suggest that the dynamic recruitment of dLKR/SDH to ecdysone-regulated gene promoters controls the timing of hormone-induced gene expression. In the absence of dLKR/SDH, histone methylation occurs prematurely, resulting in enhanced gene activation. Consistent with these observations, the loss of dLKR/SDH in Drosophila enhances hormone-regulated gene expression, affecting the developmental timing of gene activation.
Asunto(s)
Arginina/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Ecdisona/farmacología , Histonas/metabolismo , Sacaropina Deshidrogenasas/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes de Insecto , Cinética , Metilación/efectos de los fármacos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Receptores de Esteroides/metabolismo , Activación TranscripcionalRESUMEN
The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.
Asunto(s)
Apoptosis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Animales , Apoptosis/efectos de la radiación , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero/anomalías , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Genes Letales/genética , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/genética , Mutación/genética , Mutación/efectos de la radiación , Radiación Ionizante , Glándulas Salivales/citología , Glándulas Salivales/crecimiento & desarrollo , Glándulas Salivales/metabolismo , Transducción de Señal/genéticaRESUMEN
dronc, the only apical caspase in Drosophila, is thought to be essential and non-redundant for apoptosis. Recent analyses of several independently derived dronc mutants have demonstrated that DRONC is required for normal development. Interestingly, analysis of these mutants show that DRONC is not essential for cell death in all tissues and that in some cases, DRONC-independent effector caspase activation and apoptosis can occur. These mutants provide a valuable resource to investigate the recently reported roles of DRONC in nonapoptotic pathways. Insights gained from the dronc mutants will help in advancing our understanding of caspase function in various developmental pathways.
Asunto(s)
Caspasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Animales , Muerte Celular , Modelos BiológicosRESUMEN
Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis.
Asunto(s)
Apoptosis , Caspasas/fisiología , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Alelos , Animales , Tipificación del Cuerpo , Caspasas/metabolismo , Muerte Celular , Drosophila melanogaster , Ojo/embriología , Ojo/metabolismo , Prueba de Complementación Genética , Genoma , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Hemocitos/metabolismo , Homocigoto , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Modelos Genéticos , Mutagénesis , Mutación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.
Asunto(s)
Apoptosis , Caspasas/fisiología , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Animales , Animales Modificados Genéticamente , Caspasas/metabolismo , Muerte Celular , Línea Celular , Cruzamientos Genéticos , Daño del ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Larva/metabolismo , Modelos Biológicos , Mutación , Fenotipo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Glándulas Salivales/metabolismo , Factores de Tiempo , TransgenesRESUMEN
The steroid hormone ecdysone regulates moulting, cell death, and differentiation during insect development. Ecdysone mediates its biological effects by either direct activation of gene transcription after binding to its receptor EcR-Usp or via hierarchical transcriptional regulation of several primary transcription factors. In turn, these transcription factors regulate the expression of several downstream genes responsible for specific biological outcomes. DRONC, the Drosophila initiator caspase, is transcriptionally regulated by ecdysone during development. We demonstrate here that the dronc promoter directly binds EcR-Usp. We further show that mutation of the EcR-Usp binding element (EcRBE) reduces transcription of a reporter and abolishes transactivation by an EcR isoform. We demonstrate that EcRBE is required for temporal regulation of dronc expression in response to ecdysone in specific tissues. We also uncover the participation of a putative repressor whose function appears to be coupled with EcR-Usp. These results indicate that direct binding of EcR-Usp is crucial for controlling the timing of dronc expression in specific tissues.