RESUMEN
Compound eyes formation in decapod crustaceans occurs after the nauplius stage. However, the key genes and regulatory mechanisms of compound eye development during crustacean embryonic development have not yet been clarified. In this study, RNA-seq was used to investigate the gene expression profiles of Neocaridina denticulata sinensis from nauplius to zoea stage. Based on RNA-seq data analysis, the phototransduction and insect hormone biosynthesis pathways were enriched, and molting-related neuropeptides were highly expressed. There was strong cell proliferation in the embryo prior to compound eye development. The formation of the visual system and the hormonal regulation of hatching were the dominant biological events during compound eye development. The functional analysis of DEGs across all four developmental stages showed that cuticle formation, muscle growth and the establishment of immune system occurred from nauplius to zoea stage. Key genes related to eye development were discovered, including those involved in the determination and differentiation of the eye field, eye-color formation, and visual signal transduction. In conclusion, the results increase the understanding of the molecular mechanism of eye formation in crustacean embryonic stage.
Asunto(s)
Ojo Compuesto de los Artrópodos , Perfilación de la Expresión Génica , Animales , Ojo Compuesto de los Artrópodos/metabolismo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Transcriptoma , Regulación del Desarrollo de la Expresión Génica , Decápodos/genética , Decápodos/crecimiento & desarrollo , Ojo/metabolismo , Ojo/embriología , Ojo/crecimiento & desarrolloRESUMEN
Hippo signaling is a conserved mechanism for controlling organ growth. Increasing evidence suggests that Hippo signaling is modulated by various cellular factors for normal development and tumorigenesis. Hence, identification of these factors is pivotal for understanding the mechanism for the regulation of Hippo signaling. Drosophila Mnat9 is a putative N-acetyltransferase that is required for cell survival by affecting JNK signaling. Here we show that Mnat9 is involved in the negative regulation of Hippo signaling. RNAi knockdown of Mnat9 in the eye disc suppresses the rough eye phenotype of overexpressing Crumbs (Crb), an upstream factor of the Hippo pathway. Conversely, Mnat9 RNAi enhances the eye phenotype caused by overexpressing Expanded (Ex) or Warts (Wts) that acts downstream to Crb. Similar genetic interactions between Mnat9 and Hippo pathway genes are found in the wing. The reduced wing phenotype of Mnat9 RNAi is suppressed by overexpression of Yorkie (Yki), while it is suppressed by knockdown of Hippo upstream factors like Ex, Merlin, or Kibra. Mnat9 co-immunoprecipitates with Mer, implying their function in a protein complex. Furthermore, Mnat9 overexpression together with Hpo knockdown causes tumorous overgrowth in the abdomen. Our data suggest that Mnat9 is required for organ growth and can induce tumorous growth by negatively regulating the Hippo signaling pathway.
Asunto(s)
Carcinogénesis/metabolismo , Drosophila melanogaster/metabolismo , Vía de Señalización Hippo , Acetiltransferasas N-Terminal/metabolismo , Animales , Carcinogénesis/patología , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Acetiltransferasas N-Terminal/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño , Alas de Animales/metabolismoRESUMEN
The eye-antennal disc of Drosophila is composed of three cell layers: a columnar epithelium called the disc proper (DP); an overlying sheet of squamous cells called the peripodial epithelium (PE); and a strip of cuboidal cells that joins the other two cellular sheets to each other and comprises the outer margin (M) of the disc. The M cells play an important role in patterning the eye because it is here that the Hedgehog (Hh), Decapentaplegic (Dpp) and JAK/STAT pathways function to initiate pattern formation. Dpp signaling is lost from the margin of eyes absent (eya) mutant discs and, as a result, the initiation of retinal patterning is blocked. Based on these observations, Eya has been proposed to control the initiation of the morphogenetic furrow via regulation of Dpp signaling within the M. We show that the failure in pattern formation surprisingly results from M cells prematurely adopting a head epidermis fate. This switch in fate normally takes place during pupal development after the eye has been patterned. Our results suggest that the timing of cell fate decisions is essential for correct eye development.
Asunto(s)
Ojo Compuesto de los Artrópodos/citología , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Animales , Diferenciación Celular , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Ojo/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Quinasas Janus/metabolismo , Morfogénesis , Mutación , Factores de Transcripción STAT/metabolismoRESUMEN
The arthropod compound eye represents one of two major eye types in the animal kingdom and has served as an essential experimental paradigm for defining fundamental mechanisms underlying sensory organ formation, function, and maintenance. One of the most distinguishing features of the compound eye is the highly regular array of lens facets that define individual eye (ommatidial) units. These lens facets are produced by a deeply conserved quartet of cuticle-secreting cells, called Semper cells (SCs). Also widely known as cone cells, SCs were originally identified for their secretion of the dioptric system, i.e. the corneal lens and underlying crystalline cones. Additionally, SCs are now known to execute a diversity of patterning and glial functions in compound eye development and maintenance. Here, we present an integrated account of our current knowledge of SC multifunctionality in the Drosophila compound eye, highlighting emerging gene regulatory modules that may drive the diverse roles for these cells. Drawing comparisons with other deeply conserved retinal glia in the vertebrate single lens eye, this discussion speaks to glial cell origins and opens new avenues for understanding sensory system support programs.
Asunto(s)
Ojo Compuesto de los Artrópodos/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Animales , Ojo Compuesto de los Artrópodos/metabolismo , Córnea/metabolismo , Córnea/fisiología , Drosophila/genética , Proteínas de Drosophila/genética , Ojo/metabolismo , Proteínas del Ojo/genética , Cristalino/metabolismo , Cristalino/fisiología , Neuroglía/fisiología , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Relación Estructura-ActividadRESUMEN
In the last larval instar, uncommitted progenitor cells in the Drosophila eye primordium start to adopt individual retinal cell fates, arrest their growth and proliferation, and initiate terminal differentiation into photoreceptor neurons and other retinal cell types. To explore the regulation of these processes, we have performed mRNA-Seq studies of the larval eye and antennal primordial at multiple developmental stages. A total of 10,893 fly genes were expressed during these stages and could be adaptively clustered into gene groups, some of whose expression increases or decreases in parallel with the cessation of proliferation and onset of differentiation. Using in situ hybridization of a sample of 98 genes to verify spatial and temporal expression patterns, we estimate that 534 genes or more are transcriptionally upregulated during retinal differentiation, and 1367 or more downregulated as progenitor cells differentiate. Each group of co-expressed genes is enriched for regulatory motifs recognized by co-expressed transcription factors, suggesting that they represent coherent transcriptional regulatory programs. Using available mutant strains, we describe novel roles for the transcription factors SoxNeuro (SoxN), H6-like homeobox (Hmx), CG10253, without children (woc), Structure specific recognition protein (Ssrp), and multisex combs (mxc).
Asunto(s)
Ojo Compuesto de los Artrópodos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Transcriptoma , Animales , Diferenciación Celular , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Using a gain-of-function screen in Drosophila, we identified the Krüppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and our data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells.
Asunto(s)
Ciclo Celular/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Animales , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Mutación con Ganancia de Función , Prueba de Complementación Genética , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Factores de Transcripción/genéticaRESUMEN
Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a Drosophila 'HPV 18 E6' model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKK[Formula: see text]-a regulator of NF-κB-as an enhancer of E6-induced cellular defects. We demonstrate that inhibition of IKK[Formula: see text] reduces Magi degradation and that this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKK[Formula: see text] suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKK[Formula: see text] and E6 is conserved in human cells: inhibition of IKK[Formula: see text] blocked the growth of cervical cancer cells, suggesting that IKK[Formula: see text] may serve as a novel therapeutic target for HPV-mediated cancers.
Asunto(s)
Ojo Compuesto de los Artrópodos/anomalías , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Neoplasias del Cuello Uterino/patología , Animales , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Viral , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Drosophila , Femenino , Humanos , Nucleósido-Fosfato Quinasa/metabolismo , Dominios PDZ , Fosforilación , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Drosophila melanogaster has been a model for multiple human disease conditions, including cancer. Among Drosophila tissues, the eye development is particularly sensitive to perturbations of the embryonic signaling pathways, whose improper activation in humans underlies various forms of cancer. We have launched the HumanaFly project, whereas human genes expressed in breast cancer patients are screened for their ability to aberrate development of the Drosophila eye, hoping to thus identify novel oncogenes. Here we report identification of a breast cancer transgene, which upon expression in Drosophila produces eye malformation similar to the famous Glazed phenotype discovered by Thomas Morgan and decades later dissected to originate from mis-expression of Wingless (Wg). Wg is the ortholog of human Wnt proteins serving as ligands to initiate the developmental/oncogenic Wnt signaling pathway. Through genetic experiments we identified that this transgene interacted with the Wg production machinery, rather than with Wg signal transduction. In Drosophila imaginal discs, we directly show that the transgene promoted long-range diffusion of Wg, affecting expression of the Wg target genes. The transgene emerged to encode RPS12-a protein of the small ribosomal subunit overexpressed in several cancer types and known to also possess extra-ribosomal functions. Our work identifies RPS12 as an unexpected regulator of secretion and activity of Wnts. As Wnt signaling is particularly important in the context of breast cancer initiation and progression, RPS12 might be implicated in tumorigenesis in this and other Wnt-dependent cancers. Continuation of our HumanaFly project may bring further discoveries on oncogenic mechanisms.
Asunto(s)
Neoplasias de la Mama/genética , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Ribosómicas/genética , Transgenes , Vía de Señalización Wnt , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Femenino , Humanos , Discos Imaginales/metabolismo , Proteínas Mitocondriales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.
Asunto(s)
Ojo Compuesto de los Artrópodos/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Neurogénesis , Animales , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Mutación con Pérdida de Función , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Vía de Señalización WntRESUMEN
Drosophila Lobe (L) alleles were first discovered â¼100 years ago as spontaneous dominant mutants with characteristic developmental eye defects. However, the molecular basis for L dominant eye phenotypes has not been clearly understood. A previous work reported identification of CG10109/PRAS40 as the L gene, but subsequent analyses suggested that PRAS40 may not be related to L Here, we revisited the L gene to clarify this discrepancy and understand the basis for the dominance of L mutations. Genetic analysis localized the L gene to Oaz, which encodes a homolog of the vertebrate zinc finger protein 423 (Zfp423) family transcriptional regulators. We demonstrate that RNAi knockdown of Oaz almost completely restores all L dominant alleles tested. Lrev6-3 , a revertant allele of the L2 dominant eye phenotype, has an inframe deletion in the Oaz coding sequence. Molecular analysis of L dominant mutants identified allele-specific insertions of natural transposons (roo[ ]L1 , hopper[ ]L5 , and roo[ ]Lr ) or alterations of a preexisting transposon (L2 -specific mutations in roo[ ]Mohr) in the Oaz region. In addition, we generated additional L2 -reversion alleles by CRISPR targeting at Oaz These new loss-of-function Oaz mutations suppress the dominant L eye phenotype. Oaz protein is not expressed in wild-type eye disc but is expressed ectopically in L2/+ mutant eye disc. We induced male recombination between Oaz-GAL4 insertions and the L2 mutation through homologous recombination. By using the L2 -recombined GAL4 reporters, we show that Oaz-GAL4 is expressed ectopically in L2 eye imaginal disc. Taken together, our data suggest that neomorphic L eye phenotypes are likely due to misregulation of Oaz by spontaneous transposon insertions.
Asunto(s)
Ojo Compuesto de los Artrópodos/metabolismo , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Factores de Transcripción/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Ojo/metabolismo , Genes Dominantes , Recombinación Homóloga , Mutación con Pérdida de Función , Fenotipo , Factores de Transcripción/metabolismoRESUMEN
While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.
Asunto(s)
Variaciones en el Número de Copia de ADN , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Trastornos del Neurodesarrollo/genética , Animales , Ojo Compuesto de los Artrópodos/embriología , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neuronas/citología , Neuronas/metabolismo , Especificidad de Órganos , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal , Alas de Animales/embriología , Alas de Animales/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
The objective of this study was screening of ommochromes from the compound eyes of insects and comparison of their antioxidant properties. Ommochromes were isolated in preparative quantities from insects of five different families: Stratiomyidae, Sphingidae, Blaberidae, Acrididae, and Tenebrionidae. The yield of ommochromes (dry pigment weight) was 0.9-5.4% of tissue wet weight depending on the insect species. Isolated pigments were analyzed by high-performance liquid chromatography and represented a mixture of several ommochromes of the ommatin series. The isolated ommochromes displayed a pronounced fluorescence with the emission maxima at 435-450 nm and 520-535 nm; furthermore, the emission intensity increased significantly upon ommochrome oxidation with hydrogen peroxide. The ommochromes produced a stable EPR signal consisting of a singlet line with g = 2.0045-2.0048, width of 1.20-1.27 mT, and high concentration of paramagnetic centers (> 1017 spin/g dry weight). All the investigated ommochromes demonstrated high antiradical activity measured from the degree of chemiluminescence quenching in a model system containing luminol, hemoglobin, and hydrogen peroxide. The ommochromes strongly inhibited peroxidation of the photoreceptor cell outer segments induced by visible light in the presence of lipofuscin granules from the human retinal pigment epithelium, as well as suppressed iron/ascorbate-mediated lipid peroxidation. The obtained results are important for understanding the biological functions of ommochromes in invertebrates and identifying invertebrate species that could be used as efficient sources of ommochromes for pharmacological preparations to prevent and treat pathologies associated with the oxidative stress development.
Asunto(s)
Antioxidantes/farmacología , Fenómenos Químicos , Ojo Compuesto de los Artrópodos/química , Insectos/metabolismo , Fenotiazinas/farmacología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Ojo Compuesto de los Artrópodos/metabolismo , Peróxido de Hidrógeno , Insectos/efectos de los fármacos , Luz , Peroxidación de Lípido , Oxidación-Reducción , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
The Hippo pathway is an evolutionarily conserved signaling network that regulates organ size, cell fate, and tumorigenesis. In the context of organ size control, the pathway incorporates a large variety of cellular cues, such as cell polarity and adhesion, into an integrated transcriptional response. The central Hippo signaling effector is the transcriptional coactivator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood, and therefore we sought to identify proteins that potentially operate together with Tgi. Using an affinity purification and mass-spectrometry approach we identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologs of Tgi, CtBP, and Pits (VGLL4, CTBP2, and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Portadoras/metabolismo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Oxidorreductasas de Alcohol/genética , Animales , Proteínas Portadoras/genética , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Nucleares/genética , Unión Proteica , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The expression of multiple growth-promoting genes is coordinated by the transcriptional co-activator Yorkie with its major regulatory input provided by the Hippo-Warts kinase cascade. Here, we identify Atg1/ULK1-mediated phosphorylation of Yorkie as an additional inhibitory input independent of the Hippo-Warts pathway. Two serine residues in Yorkie, S74 and S97, are Atg1/ULK1 consensus target sites and are phosphorylated by ULK1 in vitro, thereby preventing its binding to Scalloped. In vivo, gain of function of Atg1, or its activator Acinus, caused elevated Yorkie phosphorylation and inhibited Yorkie's growth-promoting activity. Loss of function of Atg1 or Acinus raised expression of Yorkie target genes and increased tissue size. Unlike Atg1's role in autophagy, Atg1-mediated phosphorylation of Yorkie does not require Atg13. Atg1 is activated by starvation and other cellular stressors and therefore can impose temporary stress-induced constraints on the growth-promoting gene networks under the control of Hippo-Yorkie signaling.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Sitios de Unión , Núcleo Celular/metabolismo , Ojo Compuesto de los Artrópodos/metabolismo , Secuencia de Consenso , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The establishment of the functional nervous system requires coordinated development of neurons and glia in the embryo. Our understanding of underlying molecular and cellular mechanisms, however, remains limited. The developing Drosophila visual system is an excellent model for understanding the developmental control of the nervous system. By performing a systematic transgenic RNAi screen, we investigated the requirements of secreted proteins and cell-surface receptors for the development of photoreceptor neurons (R cells) and wrapping glia (WG) in the Drosophila visual system. From the screen, we identified seven genes whose knockdown disrupted the development of R cells and/or WG, including amalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific analysis revealed that ama is required in the developing eye disc for promoting cell proliferation and differentiation, which is essential for the migration of glia in the optic stalk. Our results also suggest that nrg functions in both eye disc and WG for coordinating R-cell and WG development.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Discos Imaginales/metabolismo , Inmunoglobulinas/fisiología , Neurogénesis/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Moléculas de Adhesión Celular Neuronal/genética , Linaje de la Célula , Movimiento Celular , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Técnicas de Silenciamiento del Gen , Discos Imaginales/citología , Inmunoglobulinas/genética , Larva , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismoRESUMEN
We previously identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-coloured compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the Amyloid precursor protein-like protein, which is an orthologue of Amyloid precursor protein, which is a causative molecule of Alzheimer's disease. In this study, we examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. We found that abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies, but were observed in very aged red-eyed wild-type flies. Thus, our data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Ojo Compuesto de los Artrópodos/metabolismo , Ojo Compuesto de los Artrópodos/ultraestructura , Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/ultraestructura , Proteínas del Ojo/genética , Mutación , Proteínas Quinasas/genética , Factores de Edad , Animales , Estudios de Asociación Genética , Longevidad/genética , FenotipoRESUMEN
Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.
Asunto(s)
Procesos de Crecimiento Celular/genética , Fosfatidilinositoles/genética , Transducción de Señal , Animales , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Light is the most important Zeitgeber for entraining animal activity rhythms to the 24-h day. In all animals, the eyes are the main visual organs that are not only responsible for motion and colour (image) vision, but also transfer light information to the circadian clock in the brain. The way in which light entrains the circadian clock appears, however, variable in different species. As do vertebrates, insects possess extraretinal photoreceptors in addition to their eyes (and ocelli) that are sometimes located close to (underneath) the eyes, but sometimes even in the central brain. These extraretinal photoreceptors contribute to entrainment of their circadian clocks to different degrees. The fruit fly Drosophila melanogaster is special, because it expresses the blue light-sensitive cryptochrome (CRY) directly in its circadian clock neurons, and CRY is usually regarded as the fly's main circadian photoreceptor. Nevertheless, recent studies show that the retinal and extraretinal eyes transfer light information to almost every clock neuron and that the eyes are similarly important for entraining the fly's activity rhythm as in other insects, or more generally spoken in other animals. Here, I compare the light input pathways between selected insect species with a focus on Drosophila's special case.
Asunto(s)
Ritmo Circadiano , Ojo Compuesto de los Artrópodos/fisiología , Drosophila melanogaster/fisiología , Fotoperiodo , Células Fotorreceptoras de Invertebrados/fisiología , Visión Ocular , Percepción Visual , Animales , Ojo Compuesto de los Artrópodos/citología , Ojo Compuesto de los Artrópodos/metabolismo , Criptocromos/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Luz , Estimulación Luminosa , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentos Retinianos/metabolismo , Especificidad de la Especie , Vías Visuales/fisiologíaRESUMEN
Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.
Asunto(s)
Autofagia , Proteínas de Drosophila/genética , Heparitina Sulfato/biosíntesis , Longevidad , Estrés Oxidativo , Ubiquitina-Proteína Ligasas/genética , Animales , Encéfalo/metabolismo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Heparitina Sulfato/genética , Músculos/metabolismo , Mutación , Presenilinas/genética , Presenilinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. We previously performed modifier screening on the dFIG4 knockdown-induced rough eye phenotype and identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA) as genetic interactants with dFIG4. In the present study, in more extensive genetic screening, we found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, we established a CR43467-specific knockdown line using the CRISPR-dCas9 system. By using this system, we demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. The knockdown of CR43467 also suppressed the rough eye phenotype induced by other peripheral neuropathy-related genes, such as dCOA7, dHADHB, and dPDHB. We herein identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.