RESUMO
Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.
Assuntos
Sinalização do Cálcio/fisiologia , Cerebelo/metabolismo , Cerebelo/fisiologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Cálcio/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
BACKGROUND: Reproductive containment provides an opportunity to implement a staged-release strategy for genetic control of malaria vectors, in particular allowing predictions about the spread and persistence of (self-limiting) sterile and male-biased strains to be compared to outcomes before moving to (self-sustaining) gene-drive strains. METHODS: In this study, we: (i) describe a diffusion-advection-reaction model of the spread and persistence of a single cohort of male mosquitoes; (ii) elicit informative prior distributions for model parameters, for wild-type (WT) and genetically modified dominant sterile strains (DSM); (iii) estimate posterior distributions for WT strains using data from published mark-recapture-release (MRR) experiments, with inference performed through the Delayed-Rejection Adaptive Metropolis algorithm; and (iv) weight prior distributions, in order to make predictions about genetically modified strains using Bayes factors calculated for the WT strains. RESULTS: If a single cohort of 5000 genetically modified dominant sterile male mosquitoes are released at the same location as previous MRR experiments with their WT counterparts, there is a 90% probability that the expected number of released mosquitoes will fall to < 1 in 10 days, and that by 12 days there will be a 99% probability that no mosquitoes will be found more than 150 m from the release location. CONCLUSIONS: Spread and persistence models should form a key component of risk assessments of novel genetic control strategies for malaria vectors. Our predictions, used in an independent risk assessment, suggest that genetically modified sterile male mosquitoes will remain within the locality of the release site, and that they will persist for a very limited amount of time. Data gathered following the release of these mosquitoes will enable us to test the accuracy of these predictions and also provide a means to update parameter distributions for genetic strains in a coherent (Bayesian) framework. We anticipate this will provide additional insights about how to conduct probabilistic risk assessments of stage-released genetically modified mosquitoes.
Assuntos
Animais Geneticamente Modificados/fisiologia , Infertilidade Masculina , Controle de Mosquitos/métodos , Mosquitos Vetores/genética , Animais , Anopheles/classificação , Anopheles/genética , Anopheles/fisiologia , Teorema de Bayes , Estudos de Coortes , Tecnologia de Impulso Genético , Malária/prevenção & controle , Malária/transmissão , Masculino , Mosquitos Vetores/fisiologiaRESUMO
BACKGROUND: Whole transgenic or non-transgenic organism model systems allow the screening of pharmacological compounds for protective actions in Alzheimer's disease (AD). AIM: In this study, a plant parasitic nematode, Globodera pallida, which assimilates intact peptides from the external environment, was investigated as a new potential non-transgenic model system of AD. Methods: Fresh second-stage juveniles of G. pallida were used to measure their chemosensory, perform immunocytochemistry on their neurological structures, evaluate their survival rate, measure reactive oxygen species, and determine total oxidized glutathione to reduced glutathione ratio (GSSG/GSH) levels, before and after treatment with 100 µM of various amyloid beta (Aß) peptides (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16). Wild-type N2 C. elegans (strain N2) was cultured on Nematode Growth Medium and directly used, as control, for chemosensory assays. RESULTS: We demonstrated that: (i) G. pallida (unlike Caenorhabditis elegans) assimilates amyloid-ß (Aß) peptides which co-localise with its neurological structures; (ii) pre-treatment with various Aß isoforms (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16) impairs G. pallida's chemotaxis to differing extents; (iii) Aß peptides reduced survival, increased the production of ROS, and increased GSSG/GSH levels in this model; (iv) this unique model can distinguish differences between different treatment concentrations, durations, and modalities, displaying good sensitivity; (v) clinically approved neuroprotective agents were effective in protecting G. pallida from Aß (1-42) exposure. Taken together, the data indicate that G. pallida is an interesting in vivo model with strong potential for discovery of novel bioactive compounds with anti-AD activity.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais Geneticamente Modificados/fisiologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tylenchoidea/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Quimiotaxia , Tylenchoidea/efeitos dos fármacosRESUMO
Regeneration after peripheral nerve damage requires that axons re-grow to the correct target tissues in a process called target-specific regeneration. Although much is known about the mechanisms that promote axon re-growth, re-growing axons often fail to reach the correct targets, resulting in impaired nerve function. We know very little about how axons achieve target-specific regeneration, particularly in branched nerves that require distinct targeting decisions at branch points. The zebrafish vagus motor nerve is a branched nerve with a well-defined topographic organization. Here, we track regeneration of individual vagus axons after whole-nerve laser severing and find a robust capacity for target-specific, functional re-growth. We then develop a new single-cell chimera injury model for precise manipulation of axon-environment interactions and find that (1) the guidance mechanism used during regeneration is distinct from the nerve's developmental guidance mechanism, (2) target selection is specified by neurons' intrinsic memory of their position within the brain, and (3) targeting to a branch requires its pre-existing innervation. This work establishes the zebrafish vagus nerve as a tractable regeneration model and reveals the mechanistic basis of target-specific regeneration.
Assuntos
Axônios/fisiologia , Regeneração Nervosa/fisiologia , Nervo Vago/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologiaRESUMO
Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.
Assuntos
Proliferação de Células/fisiologia , Miócitos Cardíacos/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Calpaína/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , Citocinese/fisiologia , Feminino , Coração/fisiologia , Masculino , Mamíferos/metabolismo , Mamíferos/fisiologia , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Regeneração/fisiologia , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Thermotolerance is a complex trait that can greatly differ between heat-susceptible (HS) and heat-adapted populations of small insects including Drosophila, with short-term effects after a sub-lethal level of heat stress on many physiological functions. Cardiac performance could accordingly be more robust in heat-resistant (HR) than in HS individuals under heat stress. Here, we tested heart performance under heat-stress effects in two recombinant inbred lines (RIL) of Drosophila melanogaster that dramatically differ in heat knockdown resistance. Heart rate did not strongly differ between heat-susceptible and heat-tolerant flies after a sub-lethal heat stress. Instead, heat-susceptible flies showed a much higher arrhythmia incidence, a longer duration of each heartbeat, and a larger amount of bradycardia than heat-tolerant flies. The highly conserved cardiac proteins SERCA, RyR and NCX that participate in the excitation/contraction coupling, did not differ in activity level between HR and HS flies. Available information for both RIL suggests that heart performance under heat stress may be linked, at least partially, to candidate genes of previously identified quantitative trait loci (QTL) for thermotolerance. This study indicates that HR flies can be genetically more robust in their heart performance than HS flies under even sub-lethal levels of heat stress.
Assuntos
Drosophila melanogaster/fisiologia , Coração/fisiologia , Resposta ao Choque Térmico/genética , Adaptação Fisiológica , Animais , Animais Geneticamente Modificados/fisiologia , Termotolerância/genéticaRESUMO
Neurotoxic aggregation of ß-amyloid (Aß) peptides is a hallmark of Alzheimer's disease and increased reactive oxygen species (ROS) is an associated process. In the present study, we report the neuroprotective effects of disulfide-rich, circular peptides from Clitoria ternatea (C. ternatea) (butterfly pea) on Aß-induced toxicity in transgenic Caenorhabditis elegans. Cyclotides (â¼30 amino acids long) are a special class of cyclic cysteine knot peptides. We show that cyclotide-rich fractions from different plant tissues delay Aß-induced paralysis in the transgenic CL4176 strain expressing the human muscle-specific Aß1-42 gene. They also improved Aß-induced chemotaxis defects in CL2355 strain expressing Aß1-42 in the neuronal cells. ROS assay suggests that this protection is likely mediated by the inhibition of Aß oligomerization. Furthermore, Aß deposits were reduced in the CL2006 strain treated with the fractions. The study shows that cyclotides from C. ternatea could be a source of a novel pharmacophore scaffold against neurodegenerative diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Caenorhabditis elegans/metabolismo , Clitoria/metabolismo , Dissulfetos/química , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Caenorhabditis elegans/fisiologia , Quimiotaxia/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/uso terapêutico , Paralisia/tratamento farmacológico , Paralisia/patologia , Fragmentos de Peptídeos/genética , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Synapses exhibit multiple forms of short-term plasticities, which have been attributed to the heterogeneity of neurotransmitter release probability. However, the molecular mechanisms that underlie the differential release states remain to be fully elucidated. The Unc-13 proteins appear to have key roles in synaptic function through multiple regulatory domains. Here, we report that deleting the M domain in Caenorhabditis elegans UNC-13MR leads to a significant increase in release probability, revealing an inhibitory function of this domain. The inhibitory effect of this domain is eliminated when the C1 and C2B domains are absent or activated, suggesting that the M domain inhibits release probability by suppressing the activity of C1 and C2B domains. When fused directly to the MUNC2C fragment of UNC-13, the M domain greatly enhances release probability. Thus, our findings reveal a mechanism by which the UNC-13 M domain regulates synaptic transmission and provides molecular insights into the regulation of release probability.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cálcio/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Genótipo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios Proteicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismoRESUMO
Transgenic zebrafish in which the germline is specifically labeled with enhanced green fluorescent protein (eGFP) can be used for continuous observation of germline development during the lifetime, from the primordial germ cells (PGCs) in the early embryo to the gametes in the mature gonad. In this chapter, we describe a procedure for the generation of transgenic fish Tg(piwil1:egfp-UTRnanos3), the sample preparation for live imaging of PGCs, for high-resolution imaging of germ cells in developing gonads, and quantifying PGC numbers. The methods described in this chapter are not only applicable to the study of germ cells, but also provide general advices for researchers who are willing to generate transgenic zebrafish and do observation on live embryos as well as on fixed tissues.
Assuntos
Animais Geneticamente Modificados/fisiologia , Células Germinativas/fisiologia , Animais , Embrião não Mamífero/fisiologia , Feminino , Gônadas/fisiologia , Masculino , Peixe-Zebra/fisiologiaRESUMO
Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Animais , Linfócitos T/metabolismo , Teratoma/metabolismo , Animais , Animais Geneticamente Modificados/fisiologia , Prótese Vascular , Humanos , Camundongos , Camundongos Nus , Mutação , Coelhos , Imunodeficiência Combinada Severa/genética , Transplante HeterólogoRESUMO
Genome association studies in human and genetic studies in mouse implicated members of the transmembrane protein 132 (TMEM132) family in multiple conditions including panic disorder, hearing loss, limb and kidney malformation. However, the presence of five TMEM132 paralogs in mammalian genomes makes it extremely challenging to reveal the full requirement for these proteins in vivo. In contrast, there is only one TMEM132 homolog, detonator (dtn), in the genome of fruit fly Drosophila melanogaster, enabling straightforward research into its in vivo function. In the current study, we generate multiple loss-of-function dtn mutant fly strains through a polycistronic tRNA-gRNA approach, and show that most embryos lacking both maternal and paternal dtn fail to hatch into larvae, indicating an essential role of dtn in Drosophila reproduction.
Assuntos
Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Edição de Genes , RNA Guia de Cinetoplastídeos/genética , RNA de Transferência/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Fertilidade , Edição de Genes/métodos , Mutação com Perda de Função , Masculino , ReproduçãoRESUMO
BACKGROUND: Thermal plasticity is pivotal for evolution in changing climates and in mediating resilience to its potentially negative effects. The efficacy to respond to environmental change depends on underlying mechanisms. DNA methylation induced by DNA methyltransferase 3 enzymes in the germline or during early embryonic development may be correlated with responses to environmental change. This developmental plasticity can interact with reversible acclimation within adult organisms, which would increase the speed of response and could alleviate potential mismatches between parental or early embryonic environments and those experienced at later life stages. Our aim was to determine whether there is a causative relationship between DNMT3 enzyme and developmental thermal plasticity and whether either or both interact with short-term acclimation to alter fitness and thermal responses in zebrafish (Danio rerio). RESULTS: We developed a novel DNMT3a knock-out model to show that sequential knock-out of DNA methyltransferase 3a isoforms (DNMT3aa-/- and DNMT3aa-/-ab-/-) additively decreased survival and increased deformities when cold developmental temperatures in zebrafish offspring mismatched warm temperatures experienced by parents. Interestingly, short-term cold acclimation of parents before breeding rescued DNMT3a knock-out offspring by restoring survival at cold temperatures. DNMT3a knock-out genotype interacted with developmental temperatures to modify thermal performance curves in offspring, where at least one DNMT3a isoform was necessary to buffer locomotion from increasing temperatures. The thermal sensitivity of citrate synthase activity, an indicator of mitochondrial density, was less severely affected by DNMT3a knock-out, but there was nonetheless a significant interaction between genotype and developmental temperatures. CONCLUSIONS: Our results show that DNMT3a regulates developmental thermal plasticity and that the phenotypic effects of different DNMT3a isoforms are additive. However, DNMT3a interacts with other mechanisms, such as histone (de)acetylation, induced during short-term acclimation to buffer phenotypes from environmental change. Interactions between these mechanisms make phenotypic compensation for climate change more efficient and make it less likely that thermal plasticity incurs a cost resulting from environmental mismatches.
Assuntos
Adaptação Fisiológica/genética , Mudança Climática , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/fisiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Feminino , Masculino , Fenótipo , Termotolerância/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Transgenic zebrafish models are efficiently used to study the effects of endocrine disrupting chemicals (EDC); thereby informing on their mechanisms of action. However, given the reported differences between zebrafish strains at the genetical, physiological and behavioral levels; care should be taken before using these transgenic models for EDC testing. In the present study, we undertook a set of experiments in different transgenic and/or mutant zebrafish strains of interest for EDC testing: casper, cyp19a1a-eGFP, cyp19a1a-eGFP-casper, cyp11c1-eGFP, cyp11c1-eGFP-casper. Some behavioral traits, and some biochemical and reproductive physiological endpoints commonly used in EDC testing were assessed and compared to those obtained in WT AB zebrafish to ensure that transgene insertion and/or mutations do not negatively modify basal reproductive physiology or behavior of the fish. Behavioral traits considered as anxiety and sociality have been monitored. Sociality was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the Novel tank diving test. No critical difference was observed between strains for either sociality or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 5 transgenic/mutants and the WT AB zebrafish studied. In summary, the transgene insertion and the mutations had no influence on the endpoints measured in basal conditions. These results were a prerequisite to the use of these transgenic/mutant models for EDC testing. Next step will be to determine the sensitivity of these biological models to chemical exposure to accurately validate their use in existing fish assays for EDC testing.
Assuntos
Animais Geneticamente Modificados/fisiologia , Animais Selvagens/fisiologia , Disruptores Endócrinos/farmacologia , Modelos Animais , Reprodução/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Feminino , Masculino , Peixe-Zebra/genéticaRESUMO
The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.
Assuntos
Engenharia Genética/métodos , Controle de Insetos/métodos , Controle Biológico de Vetores/métodos , Cromossomos Sexuais/genética , Animais , Animais Geneticamente Modificados/fisiologia , Drosophila melanogaster/genética , Feminino , Masculino , Modelos Animais , Análise para Determinação do Sexo/métodos , Processos de Determinação Sexual/genética , Transgenes/genéticaRESUMO
BACKGROUND: Release of gene-drive mutants to suppress Anopheles mosquito reproduction is a promising method of malaria control. However, many scientific, regulatory and ethical questions remain before transgenic mosquitoes can be utilised in the field. At a behavioural level, gene-drive carrying mutants should be at least as sexually attractive as the wildtype populations they compete against, with a key element of Anopheles copulation being acoustic courtship. We analysed sound emissions and acoustic preference in a doublesex mutant previously used to collapse Anopheles gambiae (s.l.) cages. METHODS: Anopheles rely on flight tones produced by the beating of their wings for acoustic mating communication. We assessed the impact of disrupting a female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; measured as flight tone) of males (XY) and females (XX) in homozygous dsxF- mutants (dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 dsxF+ controls (dsxF+/+). To exclude non-genetic influences, we controlled for temperature and wing length. We used a phonotaxis assay to test the acoustic preferences of mutant and control mosquitoes. RESULTS: A previous study showed an altered phenotype only for dsxF-/- females, who appear intersex, suggesting that the female-specific dsxF allele is haplosufficient. We identified significant, dose-dependent increases in the WBF of both dsxF-/- and dsxF+/- females compared to dsxF+/+ females. All female WBFs remained significantly lower than male equivalents, though. Males showed stronger phonotactic responses to the WBFs of control dsxF+/+ females than to those of dsxF+/- and dsxF-/- females. We found no evidence of phonotaxis in any female genotype. No male genotypes displayed any deviations from controls. CONCLUSIONS: A prerequisite for anopheline copulation is the phonotactic attraction of males towards female flight tones within mating swarms. Reductions in mutant acoustic attractiveness diminish their mating efficiency and thus the efficacy of population control efforts. Caged population assessments may not successfully reproduce natural mating scenarios. We propose to amend existing testing protocols to better reflect competition between mutants and target populations. Our findings confirm that dsxF disruption has no effect on males; for some phenotypic traits, such as female WBFs, the effects of dsxF appear dose-dependent rather than haplosufficient.
Assuntos
Anopheles , Controle de Mosquitos/métodos , Comportamento Sexual Animal/fisiologia , Acústica , Comunicação Animal , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Anopheles/genética , Anopheles/fisiologia , Voo Animal , Tecnologia de Impulso Genético/métodos , Audição , Mosquitos Vetores/genética , Mosquitos Vetores/fisiologia , Mutagênese , MutaçãoRESUMO
P130CAS/BCAR1 belongs to the CAS family of adaptor proteins, with important regulatory roles in cell migration, cell cycle control, and apoptosis. Previously, we and others showed that P130CAS mediates VEGF-A and PDGF signalling in vitro, but its cardiovascular function in vivo remains relatively unexplored. We characterise here a novel deletion model of P130CAS in zebrafish. Using in vivo microscopy and transgenic vascular reporters, we observed that while bcar1-/- zebrafish showed no arterial angiogenic or heart defects during development, they strikingly failed to form the caudal vein plexus (CVP). Endothelial cells (ECs) within the CVP of bcar1-/- embryos produced fewer filopodial structures and did not detach efficiently from neighbouring cells, resulting in a significant reduction in ventral extension and overall CVP area. Mechanistically, we show that P130Cas mediates Bmp2b-induced ectopic angiogenic sprouting of ECs in the developing embryo and provide pharmacological evidence for a role of Src family kinases in CVP development.
Assuntos
Animais Geneticamente Modificados/fisiologia , Embrião não Mamífero/fisiologia , Neovascularização Fisiológica , Veias/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/embriologia , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Embrião não Mamífero/citologia , Veias/embriologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood. RESULTS: We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN, and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model. CONCLUSIONS: We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Atrofia Muscular Espinal/genética , Proteínas de Ligação a RNA/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Animais Geneticamente Modificados/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Endocitose/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due, in part, to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be crucial in rescue are correspondingly crucial in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all crucial for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI.
Assuntos
Proteínas de Bactérias/metabolismo , Evolução Biológica , Citoplasma/metabolismo , Drosophila melanogaster/microbiologia , Infecções por Bactérias Gram-Negativas/metabolismo , Mutação , Wolbachia/fisiologia , Animais , Animais Geneticamente Modificados/microbiologia , Animais Geneticamente Modificados/fisiologia , Proteínas de Bactérias/genética , Citoplasma/microbiologia , Drosophila melanogaster/fisiologia , Feminino , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/patologia , MasculinoRESUMO
The roles of bioelectric signaling in developmental patterning remain largely unknown, although recent work has implicated bioelectric signals in cellular processes such as proliferation and migration. Here, we report a mutation in the inwardly rectifying potassium channel (kir) gene, kcnj13/kir7.1, that causes elongation of the fins in the zebrafish insertional mutant Dhi2059. A viral DNA insertion into the noncoding region of kcnj13 results in transient activation and ectopic expression of kcnj13 in the somite and dermomyotome, from which the fin ray progenitors originate. We made an allele-specific loss-of-function kcnj13 mutant by CRISPR (clustered regularly interspaced short palindromic repeats) and showed that it could reverse the long-finned phenotype, but only when located on the same chromosome as the Dhi2059 viral insertion. Also, we showed that ectopic expression of kcnj13 in the dermomyotome of transgenic zebrafish produces phenocopies of the Dhi2059 mutant in a gene dosage-sensitive manner. Finally, to determine whether this developmental function is specific to kcnj13, we ectopically expressed three additional potassium channel genes: kcnj1b, kcnj10a, and kcnk9 We found that all induce the long-finned phenotype, indicating that this function is conserved among potassium channel genes. Taken together, our results suggest that dermomyotome bioelectricity is a new fin-patterning mechanism, and we propose a two-stage bioelectricity model for zebrafish fin patterning. This ion channel-regulated bioelectric developmental patterning mechanism may provide with us new insight into vertebrate morphological evolution and human congenital malformations.
Assuntos
Nadadeiras de Animais/fisiologia , Animais Geneticamente Modificados/fisiologia , Padronização Corporal , Eletricidade , Regulação da Expressão Gênica , Canais de Potássio/metabolismo , Peixe-Zebra/fisiologia , Animais , Fontes de Energia Bioelétrica , Células Epiteliais/metabolismo , Músculos/metabolismo , Canais de Potássio/genética , Somitos/metabolismoRESUMO
From humans to vinegar flies, exposure to diets rich in sugar and fat lowers taste sensation, changes food choices, and promotes feeding. However, how these peripheral alterations influence eating is unknown. Here we used the genetically tractable organism D. melanogaster to define the neural mechanisms through which this occurs. We characterized a population of protocerebral anterior medial dopaminergic neurons (PAM DANs) that innervates the ß'2 compartment of the mushroom body and responds to sweet taste. In animals fed a high sugar diet, the response of PAM-ß'2 to sweet stimuli was reduced and delayed, and sensitive to the strength of the signal transmission out of the sensory neurons. We found that PAM-ß'2 DANs activity controls feeding rate and satiation: closed-loop optogenetic activation of ß'2 DANs restored normal eating in animals fed high sucrose. These data argue that diet-dependent alterations in taste weaken satiation by impairing the central processing of sensory signals.
Obesity is a major health problem affecting over 650 million adults worldwide. It is typically caused by overeating high-energy foods, which often contain a lot of sugar. Consuming sugary foods triggers the production of a reward signal called dopamine in the brains of insects and mammals, which reinforces sugar-consuming behavior. The brain balances this with a process called 'sensory-enhanced satiety', which makes foods that provide a stronger sensation of sweetness better at reducing hunger and further eating. High-energy food was scarce for most of human evolution, but over the past century sugar has become readily available in our diet leading to an increase in obesity. Last year, a study in fruit flies reported that a sugary diet reduces the sensitivity to sweet flavors, which leads to overeating and weight gain. It appears that this sensitivity is linked to the effectiveness of sensory-enhanced satiety. However, the mechanism linking diets high in sugar and overeating is still poorly understood. One hypothesis is that fruit flies estimate the energy content of food based on the degree of dopamine released in response to the sugar. May et al. compared the responses of neurons in fruit flies fed a normal diet to those in flies fed a diet high in sugar. As expected, both groups activated the neurons involved in the dopamine reward response when they tasted sugar. However, when the flies were on a sugar-heavy diet, these neurons were less active. This was because the neurons responsible for tasting sweetness were activated less in flies fed a high-sugar diet, leading to a lowered response by the neurons that produce dopamine. The flies in these experiments were genetically engineered so that the dopamine-producing neurons could be artificially activated in response to light, a technique called optogenetics. When May et al. applied this technique to the flies on a sugar-heavy diet, they were able to stop these flies from overeating. These findings provide further evidence to support the idea that a sugary diet reduces the brain's sensitivity to overeating. Given the significant healthcare cost of obesity to society, this improved understanding could help public health initiatives focusing on manufacturing food that is lower in sugar.