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1.
Nat Commun ; 14(1): 3970, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37407549

RESUMO

During early development of the sea urchin embryo, activation of ERK signalling in mesodermal precursors is not triggered by extracellular RTK ligands but by a cell-autonomous, RAS-independent mechanism that was not understood. We discovered that in these cells, ERK signalling is activated through the transcriptional activation of a gene encoding a protein related to Kinase Suppressor of Ras, that we named KSR3. KSR3 belongs to a family of catalytically inactive allosteric activators of RAF. Phylogenetic analysis revealed that genes encoding kinase defective KSR3 proteins are present in most non-chordate metazoa but have been lost in flies and nematodes. We show that the structure of KSR3 factors resembles that of several oncogenic human RAF mutants and that KSR3 from echinoderms, cnidarians and hemichordates activate ERK signalling independently of RAS when overexpressed in cultured cells. Finally, we used the sequence of KSR3 factors to identify activating mutations of human B-RAF. These findings reveal key functions for this family of factors as activators of RAF in RAS-independent ERK signalling in invertebrates. They have implications on the evolution of the ERK signalling pathway and suggest a mechanism for its co-option in the course of evolution.


Assuntos
Sistema de Sinalização das MAP Quinases , Transdução de Sinais , Animais , Humanos , Filogenia , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo
2.
Integr Environ Assess Manag ; 19(1): 63-82, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35581168

RESUMO

This review examines the presence and evolution of thyroid-like systems in selected aquatic invertebrates to determine the potential use of these organisms in screens for vertebrate thyroid hormone axis disrupting chemicals (THADCs). Such a screen might support the phasing out of some vertebrate testing. Although arthropods including crustaceans do not contain a functional thyroid signaling system, elements of such a system exist in the aquatic phyla mollusks, echinoderms, tunicates, and cephalochordates. These phyla can synthesize thyroid hormone, which has been demonstrated in some groups to induce the nuclear thyroid hormone receptor (THR). Thyroid hormone may act in these phyla through interaction with a membrane integrin receptor. Thyroid hormone regulates inter alia metamorphosis but, unlike in vertebrates, this does not occur via receptor activation by the ligands triiodothyronine (T3) and thyroxine (T4). Instead, the unliganded nuclear receptor itself controls metamorphosis in mollusks, echinoderms, and tunicates, whereas the T3 derivative tri-iodothyroacetic acid (TRIAC) acts as a THR ligand in cephalochordates. In view of this, it may be possible to develop an invertebrate-based screen that is sensitive to vertebrate THADCs that interfere with thyroid hormone synthesis or metabolism along with interaction with membrane receptors. The review makes some recommendations for the need to develop an appropriate test method. Integr Environ Assess Manag 2023;19:63-82. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).


Assuntos
Glândula Tireoide , Hormônios Tireóideos , Animais , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Invertebrados/fisiologia , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo
3.
Dev Biol ; 490: 86-99, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35841952

RESUMO

In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound.


Assuntos
Imageamento Tridimensional , Peixe-Zebra , Animais , Encéfalo/diagnóstico por imagem , Fertilização , Microscopia Confocal/métodos , Peixe-Zebra/genética
4.
Pharmaceuticals (Basel) ; 14(6)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208572

RESUMO

Zebrafish embryos (ZFE) have increasingly gained in popularity as a model to perform safety screenings of compounds. Although immersion of ZFE is the main route of exposure used, evidence shows that not all small molecules are equally absorbed, possibly resulting in false-negative readouts and incorrect conclusions. In this study, we compared the pharmacokinetics of seven fluorescent compounds with known physicochemical properties that were administered to two-cell stage embryos by immersion or by IY microinjection. Absorption and distribution of the dyes were followed at various timepoints up to 120 hpf by spatiotemporal fluorescence imaging. The concentration (10 µM) and dose (2 mg/kg) used were selected as quantities typically applied in preclinical experiments and zebrafish studies. The data show that in the case of a lipophilic compound (log D: 1.73) the immersion procedure resulted in an intrabody exposure which is similar or higher than that seen after the IY microinjection. In contrast, zero to low intrabody exposure was reached after immersion of the embryos with less lipophilic compounds. In the latter case IY microinjection, a technical procedure that can be easily automated, is highly recommended.

5.
Sci Rep ; 11(1): 12229, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108572

RESUMO

Zebrafish (Danio rerio) is increasingly used to assess the pharmacological activity and toxicity of compounds. The spatiotemporal distribution of seven fluorescent alkyne compounds was examined during 48 h after immersion (10 µM) or microinjection (2 mg/kg) in the pericardial cavity (PC), intraperitoneally (IP) and yolk sac (IY) of 3 dpf zebrafish eleuthero-embryos. By modelling the fluorescence of whole-body contours present in fluorescence images, the main pharmacokinetic (PK) parameter values of the compounds were determined. It was demonstrated that especially in case of short incubations (1-3 h) immersion can result in limited intrabody exposure to compounds. In this case, PC and IP microinjections represent excellent alternatives. Significantly, IY microinjections did not result in a suitable intrabody distribution of the compounds. Performing a QSPkR (quantitative structure-pharmacokinetic relationship) analysis, LogD was identified as the only molecular descriptor that explains the final uptake of the selected compounds. It was also shown that combined administration of compounds (immersion and microinjection) provides a more stable intrabody exposure, at least in case of a prolonged immersion and compounds with LogD value > 1. These results will help reduce the risk of false negative results and can offer an invaluable input for future translational research and safety assessment applications.


Assuntos
Alcinos/química , Embrião não Mamífero/metabolismo , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/farmacocinética , Microinjeções/métodos , Imagem Molecular/métodos , Análise Espaço-Temporal , Animais , Embrião não Mamífero/efeitos dos fármacos , Microinjeções/classificação , Distribuição Tecidual , Saco Vitelino/metabolismo , Peixe-Zebra
6.
Environ Toxicol Chem ; 39(1): 30-41, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31598995

RESUMO

No-observed-effect concentrations (NOECs) are used in environmental hazard classification and labeling of chemicals and their environmental risk assessment. They are typically obtained using standard tests such as the fish early-life stage (FELS) toxicity test, the chronic Daphnia reproduction test, and the algae growth inhibition test. Given the demand to replace and reduce animal tests, we explored the impact of the FELS toxicity test on the determination of effect concentrations by comparing the FELS toxicity test and the Daphnia and algae acute or chronic toxicity tests. Lowest-observed-effect concentrations (LOECs) were used instead of NOECs for better comparison with median lethal or effect concentration data. A database of FELS toxicity data for 223 compounds was established. Corresponding Daphnia and algae toxicity tests were identified using established databases (US Environmental Protection Agency ECOTOX, Organisation for Economic Co-operation and Development QSAR Toolbox, eChemPortal, EnviroTox, and OpenFoodTox). Approximately 9.5% of the investigated compounds showed a 10-fold higher sensitivity with the FELS toxicity test in comparison with the lowest effect concentrations obtained with any of the other tests. Some of these compounds have been known or considered as endocrine disrupting, or are other non-narcotic chemicals, indicating that the higher sensitivity in the FELS toxicity test is related to a specific mechanism of action. Targeting these mechanisms by alternative test systems or endpoints, using fish embryos for instance, may allow reduction or replacement of the FELS toxicity test or may allow us to prioritize compounds for conduction of the FELS toxicity test. Environ Toxicol Chem 2019;39:30-41. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.


Assuntos
Alternativas aos Testes com Animais , Clorófitas/efeitos dos fármacos , Daphnia/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Peixes , Xenobióticos/toxicidade , Animais , Ecotoxicologia , Disruptores Endócrinos/toxicidade , Peixes/crescimento & desenvolvimento , Organização para a Cooperação e Desenvolvimento Econômico , Medição de Risco , Testes de Toxicidade Crônica , Estados Unidos , United States Environmental Protection Agency , Poluentes Químicos da Água/toxicidade
7.
Int J Mol Sci ; 20(7)2019 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959884

RESUMO

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at -20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau's medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.


Assuntos
Embrião não Mamífero/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Cloranfenicol/metabolismo , Cromatografia Líquida , Descoberta de Drogas , Malation/metabolismo , Paration/metabolismo , Peixe-Zebra
8.
Comput Biol Med ; 105: 157-168, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30654166

RESUMO

Fish embryo models are widely used as screening tools to assess the efficacy and/or toxicity of chemicals. This assessment involves the analysis of embryo morphological abnormalities. In this article, we propose a multi-scale pipeline to allow automated classification of fish embryos (Medaka: Oryzias latipes) based on the presence or absence of spine malformations. The proposed pipeline relies on the acquisition of fish embryo 2D images, on feature extraction based on mathematical morphology operators and on machine learning classification. After image acquisition, segmentation tools are used to detect the embryo before analysing several morphological features. An approach based on machine learning is then applied to these features to automatically classify embryos according to the presence of axial malformations. We built and validated our learning model on 1459 images with a 10-fold cross-validation by comparison with the gold standard of 3D observations performed under a microscope by a trained operator. Our pipeline results in correct classification in 85% of the cases included in the database. This percentage is similar to the percentage of success of a trained human operator working on 2D images. The key benefit of our approach is the low computational cost of our image analysis pipeline, which guarantees optimal throughput analysis.


Assuntos
Embrião não Mamífero , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Aprendizado de Máquina , Oryzias/embriologia , Coluna Vertebral , Animais , Embrião não Mamífero/anormalidades , Embrião não Mamífero/diagnóstico por imagem , Embrião não Mamífero/embriologia , Coluna Vertebral/anormalidades , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/embriologia
9.
PLoS Genet ; 14(9): e1007621, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30222786

RESUMO

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-ß ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a ß-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a ß-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/metabolismo , Paracentrotus/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/fisiologia , Motivo ETS , Embrião não Mamífero , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Proteína Nodal/genética , Proteólise , Proteínas Contendo Repetições de beta-Transducina/metabolismo
10.
Cell Rep ; 14(9): 2263-2272, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26923600

RESUMO

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Marcação de Genes , Humanos , Mutação INDEL , Camundongos , Oligonucleotídeos/genética , Ratos , Peixe-Zebra
11.
Curr Opin Genet Dev ; 23(4): 445-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23769944

RESUMO

Recent studies suggest that specification of the dorsal-ventral and left-right axes of the sea urchin embryo relies on Nodal-expressing signalling centres located in the ventral ectoderm and in the archenteron that share striking similarities with vertebrate organising centres. Nodal and its downstream target BMP2/4 pattern all three germ layers along the dorsal-ventral axis, repress neural fates and control morphogenesis of the larva. Moreover, Nodal establishes left-right asymmetry by repressing formation of the adult rudiment and inhibiting germline cells differentiation on the right side, while BMP2/4 promotes expression of mesodermal genes on the left side. These findings provide a framework for future studies and raise new questions regarding the events upstream and downstream of Nodal and BMP signalling during axis formation.


Assuntos
Padronização Corporal/genética , Diferenciação Celular/genética , Proteína Nodal/genética , Ouriços-do-Mar/embriologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Ouriços-do-Mar/genética , Transdução de Sinais/genética
12.
Proc Natl Acad Sci U S A ; 108(1): 155-60, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21169220

RESUMO

The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/genética , Modelos Biológicos , Crista Neural/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Humanos , Crista Neural/embriologia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Dev Biol ; 322(2): 355-67, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18721802

RESUMO

Loss of function studies have shown that the Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest formation and maintains cells in a mitotic undifferentiated state. However, its position in the genetic cascade regulating neural crest formation and its relationship with other neural crest regulators remain largely unknown. Here we find that Hairy2 is regulated by BMP, FGF and Wnt and that it is only required downstream of BMP and FGF for neural crest formation. We show that Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos. We show that Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling. Knockdown and rescue experiments indicate that Id3 protein, which physically interacts with Hairy2, negatively regulates Hairy2 activity. However, Id3 is required to allow Hairy2 to promote neural crest formation. Together, our results provide evidence that Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state, and that Hairy2-Id3 interactions play an essential role in neural crest progenitor specification.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Crista Neural/embriologia , Células-Tronco/citologia , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Ligação Proteica , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Xenopus/metabolismo
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