RESUMO
Metabolic homeostasis is coordinated through a robust network of signaling pathways acting across all tissues. A key part of this network is insulin-like signaling, which is fundamental for surviving glucose stress. Here, we show that Caenorhabditis elegans fed excess dietary glucose reduce insulin-1 (INS-1) expression specifically in the BAG glutamatergic sensory neurons. We demonstrate that INS-1 expression in the BAG neurons is directly controlled by the transcription factor ETS-5, which is also down-regulated by glucose. We further find that INS-1 acts exclusively from the BAG neurons, and not other INS-1-expressing neurons, to systemically inhibit fat storage via the insulin-like receptor DAF-2. Together, these findings reveal an intertissue regulatory pathway where regulation of insulin expression in a specific neuron controls systemic metabolism in response to excess dietary glucose.
Assuntos
Proteínas de Caenorhabditis elegans , Insulina , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dieta , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Neurônios/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Neuropeptide release from dense-core vesicles in Caenorhabditis elegans is promoted by UNC-31, ortholog of the calcium-dependent activator protein for secretion (CAPS). Loss of UNC-31 causes multiple phenotypes in C. elegans including reduced motility, retention of late-stage eggs, and reduction in evoked synaptic release. However, the ability to analyze UNC-31 function over discrete timescales and in specific neurons is lacking. Here, we generated and validated a tool to enable UNC-31 expression and spatiotemporal functional analysis. We show that endogenously tagged UNC-31 is expressed in major ganglia and nerve cords from late embryonic stages through to adult. Using the auxin-inducible degradation system, we depleted UNC-31 postembryonically from the hermaphrodite nervous system and revealed defects in egg laying, locomotion, and vesicle release that were comparable to those in unc-31 null mutant animals. In addition, we found that depleting UNC-31 specifically from the BAG sensory neurons causes increased intestinal fat storage, highlighting the spatial sensitivity of this system. Together, this protein degradation tool may be used to facilitate studies of neuropeptide function at precise cellular and temporal scales.SIGNIFICANCE STATEMENT Animal behavior and physiology is controlled by neuropeptides that are released from specific neuronal sources. The ability to dissect discrete neuropeptide functions requires precise manipulation of neuropeptide release. We have developed and validated a tool that enables precise spatiotemporal regulation of neuropeptide release that will enable researchers to examine neuropeptide function at exceptional resolution.
Assuntos
Proteínas de Caenorhabditis elegans , Neuropeptídeos , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neurônios/metabolismo , Análise Espaço-Temporal , MutaçãoRESUMO
Brain development requires precise regulation of axon outgrowth, guidance and termination by multiple signaling and adhesion molecules. How the expression of these neurodevelopmental regulators is transcriptionally controlled is poorly understood. The Caenorhabditis elegans SMD motor neurons terminate axon outgrowth upon sexual maturity and partially retract their axons during early adulthood. Here we show that C-terminal binding protein 1 (CTBP-1), a transcriptional corepressor, is required for correct SMD axonal development. Loss of CTBP-1 causes multiple defects in SMD axon development: premature outgrowth, defective guidance, delayed termination and absence of retraction. CTBP-1 controls SMD axon guidance by repressing the expression of SAX-7, an L1 cell adhesion molecule (L1CAM). CTBP-1-regulated repression is crucial because deregulated SAX-7/L1CAM causes severely aberrant SMD axons. We found that axonal defects caused by deregulated SAX-7/L1CAM are dependent on a distinct L1CAM, called LAD-2, which itself plays a parallel role in SMD axon guidance. Our results reveal that harmonization of L1CAM expression controls the development and maturation of a single neuron.
Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Motores/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Crescimento Neuronal , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Crescimento Neuronal/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Animals constantly encounter environmental and physiological stressors that threaten survival and fertility. Somatic stress responses and germ cell arrest/repair mechanisms are employed to withstand such challenges. The Caenorhabditis elegans germline combats stress by initiating mitotic germ cell quiescence to preserve genome integrity, and by removing meiotic germ cells to prevent inheritance of damaged DNA or to tolerate lack of germline nutrient supply. Here, we review examples of germline recovery from distinct stressors - acute starvation and defective splicing - where quiescent mitotic germ cells resume proliferation to repopulate a germ line following apoptotic removal of meiotic germ cells. These protective mechanisms reveal the plastic nature of germline stem cells.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Sobrevivência Celular , Células Germinativas/metabolismo , Divisão Celular , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
Rac GTPases act as master switches to coordinate multiple interweaved signaling pathways. A major function for Rac GTPases is to control neurite development by influencing downstream effector molecules and pathways. In Caenorhabditis elegans, the Rac proteins CED-10, RAC-2 and MIG-2 act in parallel to control axon outgrowth and guidance. Here, we have identified a single glycine residue in the CED-10/Rac1 Switch 1 region that confers a non-redundant function in axon outgrowth but not guidance. Mutation of this glycine to glutamic acid (G30E) reduces GTP binding and inhibits axon outgrowth but does not affect other canonical CED-10 functions. This demonstrates previously unappreciated domain-specific functions within the CED-10 protein. Further, we reveal that when CED-10 function is diminished, the adaptor protein NAB-1 (Neurabin) and its interacting partner SYD-1 (Rho-GAP-like protein) can act as inhibitors of axon outgrowth. Together, we reveal that specific domains and residues within Rac GTPases can confer context-dependent functions during animal development.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Crescimento Neuronal/genética , Domínios Proteicos/fisiologia , Proteínas rac de Ligação ao GTP/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Feminino , Ácido Glutâmico/genética , Glicina/genética , Masculino , Mutagênese , Domínios Proteicos/genética , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract in vertebrates. Many disorders of female reproduction can be attributed to anomalies of Müllerian duct development. However, the molecular genetics of Müllerian duct formation is poorly understood and most disorders of duct development have unknown etiology. In this study, we describe for the first time the transcriptional landscape of the embryonic Müllerian duct, using the chicken embryo as a model system. RNA sequencing was conducted at 1 day intervals during duct formation to identify developmentally-regulated genes, validated by in situ hybridization. RESULTS: This analysis detected hundreds of genes specifically up-regulated during duct morphogenesis. Gene ontology and pathway analysis revealed enrichment for developmental pathways associated with cell adhesion, cell migration and proliferation, ERK and WNT signaling, and, interestingly, axonal guidance. The latter included factors linked to neuronal cell migration or axonal outgrowth, such as Ephrin B2, netrin receptor, SLIT1 and class A semaphorins. A number of transcriptional modules were identified that centred around key hub genes specifying matrix-associated signaling factors; SPOCK1, HTRA3 and ADGRD1. Several novel regulators of the WNT and TFG-ß signaling pathway were identified in Müllerian ducts, including APCDD1 and DKK1, BMP3 and TGFBI. A number of novel transcription factors were also identified, including OSR1, FOXE1, PRICKLE1, TSHZ3 and SMARCA2. In addition, over 100 long non-coding RNAs (lncRNAs) were expressed during duct formation. CONCLUSIONS: This study provides a rich resource of new candidate genes for Müllerian duct development and its disorders. It also sheds light on the molecular pathways engaged during tubulogenesis, a fundamental process in embryonic development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Ductos Paramesonéfricos/metabolismo , Transcriptoma , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Embrião de Galinha , Feminino , Ductos Paramesonéfricos/embriologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
miR-137 is a highly conserved microRNA (miRNA) that is associated with the control of brain function and the etiology of psychiatric disorders including schizophrenia and bipolar disorder. The Caenorhabditis elegans genome encodes a single miR-137 ortholog called mir-234, the function of which is unknown. Here we show that mir-234 is expressed in a subset of sensory, motor and interneurons in C. elegans. Using a mir-234 deletion strain, we systematically examined the development and function of these neurons in addition to global C. elegans behaviors. We were however unable to detect phenotypes associated with loss of mir-234, possibly due to genetic redundancy. To circumvent this issue, we overexpressed mir-234 in mir-234-expressing neurons to uncover possible phenotypes. We found that mir-234-overexpression endows resistance to the acetylcholinesterase inhibitor aldicarb, suggesting modification of neuromuscular junction (NMJ) function. Further analysis revealed that mir-234 controls neuropeptide levels, therefore positing a cause of NMJ dysfunction. Together, our data suggest that mir-234 functions to control the expression of target genes that are important for neuropeptide maturation and/or transport in C. elegans. SIGNIFICANCE STATEMENT: The miR-137 family of miRNAs is linked to the control of brain function in humans. Defective regulation of miR-137 is associated with psychiatric disorders that include schizophrenia and bipolar disorder. Previous studies have revealed that miR-137 is required for the development of dendrites and for controlling the release of fast-acting neurotransmitters. Here, we analyzed the function a miR-137 family member (called mir-234) in the nematode animal model using anatomical, behavioral, electrophysiological and neuropeptide analysis. We reveal for the first time that mir-234/miR-137 is required for the release of slow-acting neuropeptides, which may also be of relevance for controlling human brain function.
Assuntos
MicroRNAs/metabolismo , Junção Neuromuscular/metabolismo , Transmissão Sináptica , Animais , Caenorhabditis elegans , MicroRNAs/genética , Movimento , Junção Neuromuscular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in Caenorhabditis elegans Here we report that the C. elegans transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety-induced quiescence. Nutritional status has a major influence on C. elegans behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food, C. elegans become sated and enter quiescence. We show that ETS-5 acts to promote roaming and inhibit quiescence by setting the internal "satiety quotient" through fat regulation. Acting from the ASG and BAG sensory neurons, we show that ETS-5 functions in a complex network with serotonergic and neuropeptide signaling pathways to control food-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in C. elegans, and establish a paradigm for the elucidation of obesity-relevant mechanisms.
Assuntos
Comportamento Animal/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Animais , Neuropeptídeos/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Neurônios Serotoninérgicos/metabolismo , Neurônios Serotoninérgicos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Studies of nervous system connectivity, in a wide variety of species and at different scales of resolution, have identified several highly conserved motifs of network organization. One such motif is a heterogeneous distribution of connectivity across neural elements, such that some elements act as highly connected and functionally important network hubs. These brain network hubs are also densely interconnected, forming a so-called rich club. Recent work in mouse has identified a distinctive transcriptional signature of neural hubs, characterized by tightly coupled expression of oxidative metabolism genes, with similar genes characterizing macroscale inter-modular hub regions of the human cortex. Here, we sought to determine whether hubs of the neuronal C. elegans connectome also show tightly coupled gene expression. Using open data on the chemical and electrical connectivity of 279 C. elegans neurons, and binary gene expression data for each neuron across 948 genes, we computed a correlated gene expression score for each pair of neurons, providing a measure of their gene expression similarity. We demonstrate that connections between hub neurons are the most similar in their gene expression while connections between nonhubs are the least similar. Genes with the greatest contribution to this effect are involved in glutamatergic and cholinergic signaling, and other communication processes. We further show that coupled expression between hub neurons cannot be explained by their neuronal subtype (i.e., sensory, motor, or interneuron), separation distance, chemically secreted neurotransmitter, birth time, pairwise lineage distance, or their topological module affiliation. Instead, this coupling is intrinsically linked to the identity of most hubs as command interneurons, a specific class of interneurons that regulates locomotion. Our results suggest that neural hubs may possess a distinctive transcriptional signature, preserved across scales and species, that is related to the involvement of hubs in regulating the higher-order behaviors of a given organism.
Assuntos
Caenorhabditis elegans/fisiologia , Conectoma , Neurônios/fisiologia , Animais , Encéfalo/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Córtex Cerebral/fisiologia , Simulação por Computador , Junções Comunicantes , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interneurônios/fisiologia , Camundongos , Modelos Neurológicos , Vias Neurais/fisiologia , Oxigênio/químicaRESUMO
Neurodegenerative diseases (NDs) affect millions of people worldwide. Characterized by the functional loss and death of neurons, NDs lead to symptoms (dementia and seizures) that affect the daily lives of patients. In spite of extensive research into NDs, the number of approved drugs for their treatment remains limited. There is therefore an urgent need to develop new approaches for the prevention and treatment of NDs. Glycans (carbohydrate chains) are ubiquitous, abundant, and structural complex natural biopolymers. Glycans often covalently attach to proteins and lipids to regulate cellular recognition, adhesion, and signaling. The importance of glycans in both the developing and mature nervous system is well characterized. Moreover, glycan dysregulation has been observed in NDs such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). Therefore, glycans are promising but underexploited therapeutic targets. In this review, we summarize the current understanding of glycans in NDs. We also discuss a number of natural products that functionally mimic glycans to protect neurons, which therefore represent promising new therapeutic approaches for patients with NDs.
Assuntos
Mimetismo Biológico , Produtos Biológicos/química , Biomimética , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Biomimética/métodos , Descoberta de Drogas/métodos , Glicosilação , Humanos , Estrutura Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Polissacarídeos/biossíntese , Polissacarídeos/uso terapêuticoRESUMO
The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1-L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage-specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system-wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the primary accession identifier PXD006676.
Assuntos
Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/química , Caenorhabditis elegans/crescimento & desenvolvimento , Animais , Larva/química , Proteômica , Espectrometria de Massas em TandemRESUMO
The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analyzed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analyzing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organization and cytoskeletal structures that will have a major impact on the characterization of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.
Assuntos
Células Germinativas/metabolismo , Imageamento Tridimensional/métodos , Reconhecimento Automatizado de Padrão/métodos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação CelularRESUMO
Animals harbor specialized neuronal systems that are used for sensing and coordinating responses to changes in oxygen (O2) and carbon dioxide (CO2). In Caenorhabditis elegans, the O2/CO2 sensory system comprises functionally and morphologically distinct sensory neurons that mediate rapid behavioral responses to exquisite changes in O2 or CO2 levels via different sensory receptors. How the diversification of the O2- and CO2-sensing neurons is established is poorly understood. We show here that the molecular identity of both the BAG (O2/CO2-sensing) and the URX (O2-sensing) neurons is controlled by the phylogenetically conserved SoxD transcription factor homolog EGL-13. egl-13 mutant animals fail to fully express the distinct terminal gene batteries of the BAG and URX neurons and, as such, are unable to mount behavioral responses to changes in O2 and CO2. We found that the expression of egl-13 is regulated in the BAG and URX neurons by two conserved transcription factors-ETS-5(Ets factor) in the BAG neurons and AHR-1(bHLH factor) in the URX neurons. In addition, we found that EGL-13 acts in partially parallel pathways with both ETS-5 and AHR-1 to direct BAG and URX neuronal fate respectively. Finally, we found that EGL-13 is sufficient to induce O2- and CO2-sensing cell fates in some cellular contexts. Thus, the same core regulatory factor, egl-13, is required and sufficient to specify the distinct fates of O2- and CO2-sensing neurons in C. elegans. These findings extend our understanding of mechanisms of neuronal diversification and the regulation of molecular factors that may be conserved in higher organisms.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Dióxido de Carbono/metabolismo , Dióxido de Carbono/fisiologia , Mutação , Oxigênio/metabolismo , Oxigênio/fisiologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Células Receptoras Sensoriais/citologia , Fatores de Transcrição/metabolismoRESUMO
Neuronal wiring during development requires that the growth cones of axons and dendrites are correctly guided to their appropriate targets. As in other animals, axon growth cones in Caenorhabditis elegans integrate information in their extracellular environment via interactions among transiently expressed cell surface receptors, their ligands, and the extracellular matrix (ECM). Components of the ECM undergo a wide variety of post-translational modifications that may affect efficacy of binding to neuronal guidance molecules. The most common modification of the ECM is prolyl 4-hydroxylation. However, little is known of its importance in the control of axon guidance. In a screen of prolyl 4-hydroxylase (P4H) mutants, we found that genetic removal of a specific P4H subunit, DPY-18, causes dramatic defects in C. elegans neuroanatomy. In dpy-18 mutant animals, the axons of specific ventral nerve cord neurons do not respect the ventral midline boundary and cross over to the contralateral axon fascicle. We found that these defects are independent of the known role of dpy-18 in regulating body size and that dpy-18 acts from multiple tissues to regulate axon guidance. Finally, we found that the neuronal defects in dpy-18 mutant animals are dependent on the expression of muscle-derived basement membrane collagens and motor neuron-derived ephrin ligands. Loss of dpy-18 causes dysregulated ephrin expression and this is at least partially responsible for the neurodevelopmental defects observed. Together, our data suggest that DPY-18 regulates ephrin expression to direct axon guidance, a role for P4Hs that may be conserved in higher organisms.
Assuntos
Axônios/fisiologia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Prolil Hidroxilases/fisiologia , Animais , Axônios/ultraestrutura , Rastreamento de Células , Efrinas/metabolismo , Hidroxilação , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Mutação , Prolil Hidroxilases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologiaRESUMO
BACKGROUND: Quantitative real-time PCR (qPCR) has become the "gold standard" for measuring expression levels of individual miRNAs. However, little is known about the validity of reference miRNAs, the improper use of which can result in misleading interpretation of data. RESULTS: Here we undertook a systematic approach to identify highly stable miRNAs in different stress conditions such as low oxygen (hypoxia), UV-stress and high temperature (heat-stress) in the nematode Caenorhabditis elegans. We conducted genome-wide RNA-seq for small RNAs and selected abundant miRNAs with minimal variation of expression between the different conditions. We further validated the stable expression of a selection of those constitutively expressed candidates in the different stress conditions by SYBR Green qPCR. The selected miRNA candidates were analyzed for stability by applying the widely used geNorm logarithm. With this approach, we were able to successfully identify suitable reference miRNAs for each stress condition. Interestingly, we also found that 3 miRNAs, namely mir-2-5p, mir-46-3p and mir-47-3p, are stable in all the above-mentioned conditions suggesting that they might have general functions independent of stress. CONCLUSIONS: Our analysis offers a comprehensive list of stably expressed miRNAs in different stress conditions that can be confidently used as reference miRNAs for qPCR analysis in C. elegans.
Assuntos
Caenorhabditis elegans/genética , MicroRNAs/metabolismo , Animais , Perfilação da Expressão Gênica/normas , Biblioteca Gênica , Genoma , Hipóxia , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Temperatura , Raios UltravioletaRESUMO
Establishing a functional nervous system is a complex process requiring tightly controlled gene expression programs to achieve the correct differentiation of distinct neuronal subtypes. The molecular programs required for neurons to acquire neuron-type-specific, and core pan-neuronal features mostly rely on sequence-specific transcription factors (TFs), which recognize and bind to cis-regulatory motifs present in the promoters of target genes. Recently, we investigated the role and mode of action of the NF-Y complex, a ubiquitously expressed transcriptional master regulator, in the Caenorhabditis elegans nervous system. We found that NFYA-1 is a pervasive regulator of neuron-specific and pan-neuronal gene batteries that are essential for neuronal development and function. Furthermore, we concluded that NFYA-1 acts cell autonomously by either directly binding to conserved motifs in target gene promoter regions or indirectly by regulating other transcriptional regulators to fine-tune gene expression. However, further studies are required to fully define the impact of the NF-Y complex on nervous system regulatory networks and how NF-Y coordinates with other TFs in this regard.
Assuntos
Fator de Ligação a CCAAT , Caenorhabditis elegans , Regulação da Expressão Gênica , Neurônios , Animais , Neurônios/metabolismo , Fator de Ligação a CCAAT/metabolismo , Fator de Ligação a CCAAT/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regiões Promotoras Genéticas , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Mitochondria and the endoplasmic reticulum (ER) utilise unique unfolded protein response (UPR) mechanisms to maintain cellular proteostasis. Heat shock proteins (HSPs) are UPR chaperones induced by specific stressors to promote protein folding. Previous research has successfully employed transgenic reporters in Caenorhabditis elegans to report HSP induction. However, transgenic reporters are overexpressed and only show promoter regulation and not post-transcriptional regulation. To examine endogenous HSP regulation, we attempted to generate and validate endogenous reporters for mitochondrial ( HSP-60 ) and ER ( HSP-4 ) chaperones. Using CRISPR/Cas9 technology, F2A-GFP-H2B coding DNA was inserted downstream of each HSP gene and stress induction assays conducted to validate these tools. Endogenous reporters were successfully generated for hsp-4 and hsp-60 . However, GFP induction could not be detected with these endogenous reporters upon stress induction, likely due to low level expression.
RESUMO
Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.
Assuntos
Acetilcolina , Caenorhabditis elegans , Mitocôndrias , Transdução de Sinais , Animais , Acetilcolina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ácido gama-Aminobutírico/metabolismo , Intestinos/fisiologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Estresse Fisiológico , Transmissão Sináptica/fisiologia , Resposta a Proteínas não DobradasRESUMO
The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Diferenciação Celular , Matriz Extracelular , Células Germinativas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Matriz Extracelular/metabolismo , Células Germinativas/metabolismo , Células Germinativas/citologia , Diferenciação Celular/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Linhagem da Célula/genética , Oócitos/metabolismo , Oócitos/citologiaRESUMO
Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Nucleic acid binding proteins (NBPs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire NBP repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed NBPs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 NBPs with discrete autonomous germline functions. By identifying NBPs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.