RESUMO
The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.
Assuntos
Comportamento Animal , Encéfalo , Dano ao DNA , Reparo do DNA , Neurônios , Poli(ADP-Ribose) Polimerase-1 , Sono , Proteínas de Peixe-Zebra , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Brain nuclei are traditionally defined by their anatomy, activity, and expression of specific markers. The hypothalamus contains discrete neuronal populations that coordinate fundamental behavioral functions, including sleep and wakefulness, in all vertebrates. Particularly, the diverse roles of hypocretin/orexin (Hcrt)-releasing neurons suggest functional heterogeneity among Hcrt neurons. Using single-cell RNA sequencing (scRNA-seq) and high-resolution imaging of the adult male and female zebrafish hypothalamic periventricular zone (PVZ), we identified 21 glutamatergic and 28 GABAergic cell types. Integration of zebrafish and mouse scRNA-seq revealed evolutionary conserved and divergent hypothalamic cell types. The expression of specific genes, including npvf, which encodes a sleep-regulating neuropeptide, was enriched in subsets of glutamatergic Hcrt neurons in both larval and adult zebrafish. The genetic profile, activity, and neurite processing of the neuronal subpopulation that co-expresses both Hcrt and Npvf (Hcrt+Npvf+) differ from other Hcrt neurons. These inter-species findings provide a unified annotation of hypothalamic cell types, and suggest that the heterogeneity of Hcrt neurons enables multi-functionality, such as consolidation of both wake and sleep by the Hcrt- and Npvf-releasing neuronal subpopulation.Significance Statement The study reveals the intricate heterogeneity within the hypothalamic periventricular zone (PVZ) of zebrafish, identifying 21 glutamatergic and 28 GABAergic cell types through single-cell RNA sequencing (scRNA-seq) and high-resolution imaging. Comparative analysis with mouse scRNA-seq data revealed conserved and divergent cell types, transcriptional regulatory mechanisms, and neuropeptide localization. Notably, we identified a unique neuronal subpopulation co-expressing both hypocretin/orexin (Hcrt) and neuropeptide VF (Npvf) neuropeptides in zebrafish. The distinct genetic profiles, activity patterns, and neurite processing of this subpopulation suggest a role in regulating both sleep and wakefulness.
RESUMO
Thyroid hormones (THs), thyroxine (T4), and triiodothyronine (T3), regulate growth, metabolism, and neurodevelopment. THs secretion is controlled by the pituitary thyroid-stimulating hormone (TSH) and the hypothalamic-pituitary-thyroid (HPT) axis. The organic anion-transporting polypeptide 1C1 (OATP1C1/SLCO1C1) and the monocarboxylate transporter 8 (MCT8/SLC16A2) actively transport THs, which bind to their nuclear receptors and induce gene expression. A mutation in OATP1C1 is associated with brain hypometabolism, gradual neurodegeneration, and impaired cognitive and motor functioning in adolescent patients. To understand the role of Oatp1c1 and the mechanisms of the disease, we profiled the transcriptome of oatp1c1 mutant (oatp1c1 -/-) and mct8 -/- xoatp1c1 -/- adult male and female zebrafish brains. Among dozens of differentially expressed genes, agouti-related neuropeptide 1 (agrp1) expression increased in oatp1c1 -/- adult brains. Imaging in the hypothalamus revealed enhanced proliferation of Agrp1 neurons in oatp1c1 -/- larvae and adults, and increased food consumption in oatp1c1 -/- larvae. Similarly, feeding and the number of Agrp1 neurons increased in thyroid gland-ablated zebrafish. Pharmacological treatments showed that the T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid), but not T4, normalized the number of Agrp1 neurons in oatp1c1 -/- zebrafish. Since the HPT axis is hyperactive in the oatp1c1 -/- brain, we used the CRISPR-Cas9 system to knockdown tsh in oatp1c1 -/- larvae, and inducibly enhanced the HPT axis in wild-type larvae. These manipulations showed that Tsh promotes proliferation of Agrp1 neurons and increases food consumption in zebrafish. The results revealed upregulation of both the HPT axis-Agrp1 circuitry and feeding in a zebrafish model for OATP1C1 deficiency.SIGNIFICANCE STATEMENT Mutation in the thyroid hormone (TH) transporter OATP1C1 is associated with cognitive and motor functioning disturbances in humans. Here, we used an oatp1c1 -/- zebrafish to understand the role of organic anion-transporting polypeptide 1C1 (Oatp1c1), and the characteristics of OATP1C1 deficiency. Transcriptome profiling identified upregulation of agrp1 expression in the oatp1c1 -/- brain. The oatp1c1 -/- larvae showed increased thyroid-stimulating hormone (tsh) levels, proliferation of Agrp1 neurons and food consumption. Genetic manipulations of the hypothalamic-pituitary-thyroid (HPT) axis showed that Tsh increases the number of Agrp1 neurons and food consumption. The T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid) normalizes the number of Agrp1 neurons and may have potential for the treatment of Oatp1c1 deficiency. The findings demonstrate a functional interaction between the thyroid and feeding systems in the brain of zebrafish and suggest a neuroendocrinological mechanism for OATP1C1 deficiency.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Transportadores de Ácidos Monocarboxílicos , Transportadores de Ânions Orgânicos , Simportadores , Adolescente , Animais , Feminino , Humanos , Masculino , Ânions , Proliferação de Células , Larva/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Hormônios Tireóideos , Tireotropina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
A-to-I RNA editing is a common post transcriptional mechanism, mediated by the Adenosine deaminase that acts on RNA (ADAR) enzymes, that increases transcript and protein diversity. The study of RNA editing is limited by the absence of editing maps for most model organisms, hindering the understanding of its impact on various physiological conditions. Here, we mapped the vertebrate developmental landscape of A-to-I RNA editing, and generated the first comprehensive atlas of editing sites in zebrafish. Tens of thousands unique editing events and 149 coding sites were identified with high-accuracy. Some of these edited sites are conserved between zebrafish and humans. Sequence analysis of RNA over seven developmental stages revealed high levels of editing activity in early stages of embryogenesis, when embryos rely on maternal mRNAs and proteins. In contrast to the other organisms studied so far, the highest levels of editing were detected in the zebrafish ovary and testes. This resource can serve as the basis for understanding of the role of editing during zebrafish development and maturity.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Edição de RNA , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Adenosina/genética , Animais , Código Genético , Inosina/genéticaRESUMO
Thyroid hormones (THs; T3 and T4) enter cells using specific transporters and regulate development and metabolism. Mutation in the TH transporter monocarboxylate transporter 8 (MCT8, SLC16A2) is associated with brain hypothyroidism and neurological impairment. We established mct8 mutant (mct8-/-) zebrafish as a model for MCT8 deficiency, which causes endocrinological, neurological, and behavioral alterations. Here, we profiled the transcriptome of mct8-/- larvae. Among hundreds of differentially expressed genes, the expression of a cluster of vision-related genes was distinct. Specifically, the expression of the opsin 1 medium wave sensitive 2 (opn1mw2) decreased in two mct8 mutants: mct8-/- and mct8-25bp-/- larvae, and under pharmacological inhibition of TH production. Optokinetic reflex (OKR) assays showed a reduction in the number of conjugated eye movements, and live imaging of genetically encoded Ca2+ indicator revealed altered neuronal activity in the pretectum area of mct8-25bp-/- larvae. These results imply that MCT8 and THs regulate the development of the visual system and suggest a mechanism to the deficiencies observed in the visual system of MCT8-deficiency patients.
Assuntos
Hipotireoidismo , Simportadores , Animais , Encéfalo/metabolismo , Humanos , Hipotireoidismo/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day-night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator's period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep-wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.
Assuntos
Relógios Circadianos/genética , Proteínas F-Box/genética , Doenças Genéticas Inatas/genética , Doenças Raras/genética , Peixe-Zebra/genética , Animais , Ritmo Circadiano/genética , Humanos , Deficiência Intelectual/genética , Mamíferos/genética , Modelos Animais , Mutação/genéticaRESUMO
BACKGROUND: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. MATERIALS AND METHODS: In order to study the role of Nts, nts mutant (nts-/-), and overexpressing zebrafish were generated. RESULTS: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts-/- larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts-/- larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts-/- larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts-/- larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts-/- larvae. CONCLUSION: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.
Assuntos
Nível de Alerta/fisiologia , Hormônios Hipotalâmicos/metabolismo , Locomoção/fisiologia , Melaninas/metabolismo , Neurotensina/fisiologia , Hormônios Hipofisários/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Neurotensina/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the fmr1 zebrafish mutant (fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS.
Assuntos
Adenosina Desaminase/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Adenosina Desaminase/biossíntese , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Síndrome do Cromossomo X Frágil/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Atividade Motora/genética , Neurônios/metabolismo , Neurônios/patologia , Edição de RNA/genética , Proteínas de Ligação a RNA/biossíntese , Sinapses/metabolismo , Sinapses/patologia , Transcriptoma/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8-/-) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8-/- larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8-/- larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8-/- larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8-/- larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Hipotonia Muscular/diagnóstico , Hipotonia Muscular/etiologia , Atrofia Muscular/diagnóstico , Atrofia Muscular/etiologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Sistema Hipotálamo-Hipofisário , Fatores de Transcrição Kruppel-Like/genética , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Transportadores de Ácidos Monocarboxílicos/genética , Atividade Motora/genética , Hipotonia Muscular/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Mutação , Bainha de Mielina/metabolismo , Neurogranina/genética , Neurônios/metabolismo , Fenótipo , Fotoperíodo , Pseudópodes/genética , Pseudópodes/metabolismo , Glândula Tireoide , Receptores alfa dos Hormônios Tireóideos/genética , Hormônios Tireóideos/farmacologia , Peixe-Zebra , Dedos de ZincoRESUMO
Neuronal-activity-regulated pentraxin (NARP/NPTX2/NP2) is a secreted synaptic protein that regulates the trafficking of glutamate receptors and mediates learning, memory, and drug addiction. The role of NPTX2 in regulating structural synaptic plasticity and behavior in a developing vertebrate is indefinite. We characterized the expression of nptx2a in larvae and adult zebrafish and established a transcription activator-like effector nuclease (TALEN)-mediated nptx2a mutant (nptx2a(-/-)) to study the role of Nptx2a in regulating structural synaptic plasticity and behavior. Similar to mammals, the zebrafish nptx2a was expressed in excitatory neurons in the brain and spinal cord. Its expression was induced in response to a mechanosensory stimulus but did not change during day and night. Behavioral assays showed that loss of Nptx2a results in reduced locomotor response to light-to-dark transition states and to a sound stimulus. Live imaging of synapses using the transgenic nptx2a:GAL4VP16 zebrafish and a fluorescent presynaptic synaptophysin (SYP) marker revealed reduced synaptic density in the axons of the spinal motor neurons and the anterodorsal lateral-line ganglion (gAD), which regulate locomotor activity and locomotor response to mechanosensory stimuli, respectively. These results suggest that Nptx2a affects locomotor response to external stimuli by mediating structural synaptic plasticity in excitatory neuronal circuits.
Assuntos
Proteína C-Reativa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Proteína C-Reativa/deficiência , Proteína C-Reativa/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Larva/citologia , Larva/fisiologia , Mecanotransdução Celular , Atividade Motora , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Estimulação Luminosa , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
Assuntos
Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Deficiência Intelectual Ligada ao Cromossomo X/genética , Hipotonia Muscular/genética , Atrofia Muscular/genética , Mutação , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Modelos Genéticos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/patologia , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Simportadores , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Peixe-ZebraRESUMO
The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Ritmo Circadiano/genética , Relógios Circadianos/genética , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/fisiologia , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Fotoperíodo , Cnidários/fisiologia , Cnidários/genéticaRESUMO
The ability of an animal to effectively capture prey and defend against predators is pivotal for survival. Venom is often a mixture of many components including toxin proteins that shape predator-prey interactions. Here, we used the sea anemone Nematostella vectensis to test the impact of toxin genotypes on predator-prey interactions. We developed a genetic manipulation technique to demonstrate that both transgenically deficient and a native Nematostella strain lacking a major neurotoxin (Nv1) have a reduced ability to defend themselves against grass shrimp, a native predator. In addition, secreted Nv1 can act indirectly in defense by attracting mummichog fish, which prey on grass shrimp. Here, we provide evidence at the molecular level of an animal-specific tritrophic interaction between a prey, its antagonist, and a predator. Last, this study reveals an evolutionary trade-off, as the reduction of Nv1 levels allows for faster growth and increased reproductive rates.
Assuntos
Anêmonas-do-Mar , Peçonhas , Animais , Reprodução , Evolução Biológica , Neurotoxinas/genética , Anêmonas-do-Mar/genética , Comportamento Predatório/fisiologiaRESUMO
Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and cellular stress, we compared the DNA Damage Response (DDR) and oxidative stress levels between A. mexicanus populations. We measured markers of chronic sleep loss and discovered elevated levels of the DNA damage marker γH2AX in the brain, and increased oxidative stress in the gut of cavefish, consistent with chronic sleep deprivation. Notably, we found that acute UV-induced DNA damage elicited an increase in sleep in surface fish but not in cavefish. On a transcriptional level, only the surface fish activated the photoreactivation repair pathway following UV damage. These findings suggest a reduction of the DDR in cavefish compared to surface fish that coincides with elevated DNA damage in cavefish. To examine DDR pathways at a cellular level, we created an embryonic fibroblast cell line from the two populations of A. mexicanus. We observed that both the DDR and DNA repair were diminished in the cavefish cells, corroborating the in vivo findings and suggesting that the acute response to DNA damage is lost in cavefish. To investigate the long-term impact of these changes, we compared the transcriptome in the brain and gut of aged surface fish and cavefish. Strikingly, many genes that are differentially expressed between young and old surface fish do not transcriptionally vary by age in cavefish. Taken together, these findings suggest that have developed resilience to sleep loss, despite possessing cellular hallmarks of chronic sleep deprivation.
RESUMO
Sleep is an essential biological need of all animals studied to date. The sleep disorder narcolepsy is characterized by excessive daytime sleepiness, fragmentation of nighttime sleep, and cataplexy. Narcolepsy is caused by selective degeneration of hypothalamic hypocretin/orexin (HCRT) neurons. In mammals, HCRT neurons primarily regulate the sleep/wake cycle, feeding, reward-seeking, and addiction. The role of HCRT neurons in zebrafish is implicated in both sleep and wake regulation. We established a transgenic zebrafish model enabling inducible ablation of HCRT neurons and used these animals to understand the function of HCRT neurons and narcolepsy. Loss of HCRT neurons increased the expression of the HCRT receptor (hcrtr). Behavioral assays revealed that HCRT neuron-ablated larvae had normal locomotor activity, but demonstrated an increase in sleep time during the day and an increased number of sleep/wake transitions during both day and night. Mild sleep disturbance reduced sleep and increased c-fos expression in HCRT neuron-ablated larvae. Furthermore, ablation of HCRT neurons altered the behavioral response to external stimuli. Exposure to light during the night decreased locomotor activity of wild-type siblings, but induced an opposite response in HCRT neuron-ablated larvae. Sound stimulus during the day reduced the locomotor activity of wild-type sibling larvae, while HCRT neuron-ablated larvae demonstrated a hyposensitive response. This study establishes zebrafish as a model for narcolepsy, and indicating a role of HCRT neurons in regulation of sleep/wake transitions during both day and night. Our results further suggest a key role of HCRT neurons in mediating behavioral state transitions in response to external stimuli.
Assuntos
Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Locomoção/fisiologia , Narcolepsia/fisiopatologia , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Fases do Sono/fisiologia , Peixe-Zebra/fisiologia , Animais , Comportamento Animal/fisiologia , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neuropeptídeos/genética , OrexinasRESUMO
Glial cells support the function of neurons. Recent evidence shows that astrocytes are also involved in brain computations. To explore whether and how their excitable nature affects brain computations and motor behaviors, we used two-photon Ca2+ imaging of zebrafish larvae expressing GCaMP in both neurons and radial astrocytes (RAs). We found that in the optic tectum, RAs synchronize their Ca2+ transients immediately after the end of an escape behavior. Using optogenetics, ablations, and a genetically encoded norepinephrine sensor, we observed that RA synchronous Ca2+ events are mediated by the locus coeruleus (LC)-norepinephrine circuit. RA synchronization did not induce direct excitation or inhibition of tectal neurons. Nevertheless, it modulated the direction selectivity and the long-distance functional correlations among neurons. This mechanism supports freezing behavior following a switch to an alerted state. These results show that LC-mediated neuro-glial interactions modulate the visual system during transitions between behavioral states.
Assuntos
Astrócitos , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Neurônios/fisiologia , Colículos Superiores/fisiologia , NorepinefrinaRESUMO
For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.
Assuntos
Elementos E-Box , Regulação da Expressão Gênica , Luz , Proteínas Circadianas Period , Peixe-Zebra/genética , Animais , Sequência de Bases , Ritmo Circadiano/genética , Sequência Conservada , DNA/genética , DNA/metabolismo , Humanos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Glândula Pineal/fisiologia , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr-/-). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr-/- pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr-/- fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr-/- fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Melatonina/fisiologia , Neuropeptídeos/fisiologia , Sono , Vigília , Peixe-Zebra/fisiologia , Animais , Melatonina/biossíntese , Receptores de Orexina , Orexinas , Glândula Pineal/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/fisiologiaRESUMO
The multifunctional, hypothalamic hypocretin/orexin (HCRT)-producing neurons regulate an array of physiological and behavioral states including arousal, sleep, feeding, emotions, stress, and reward. How a presumably uniform HCRT neuron population regulates such a diverse set of functions is not clear. The role of the HCRT neuropeptides may vary depending on the timing and localization of secretion and neuronal activity. Moreover, HCRT neuropeptides may not mediate all functions ascribed to HCRT neurons. Some could be orchestrated by additional neurotransmitters and neuropeptides that are expressed in HCRT neurons. We hypothesize that HCRT neurons are segregated into genetically, anatomically and functionally distinct subpopulations. We discuss accumulating data that suggest the existence of such HCRT neuron subpopulations that may effectuate the diverse functions of these neurons in mammals and fish.