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Endocrinología , Humanos , Endocrinología/tendencias , Animales , Biología Evolutiva/tendenciasRESUMEN
Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.
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Absolute measurement of light intensity is sought for in multiple areas of chemistry, biology, physics, and engineering. It can be achieved by using an actinometer from analyzing the time-course of its reaction extent on applying constant light. However, most reported actinometers exploit the absorbance observable for reporting the reaction extent, which is not very sensitive nor relevant in imaging systems. In this work, we report a series of hydrophobic and hydrophilic caged fluorophores that overcome the preceding limitations. Based on the robust pyranine backbone, they can easily be synthesized on a large scale in one to a few steps. Their brightness increases over illumination and their uncaging cross-sections have been thoroughly characterized upon one- and two-photon excitation. As a demonstration of their use, we calibrated light intensity in various chemical and biological samples, which have been observed with epifluorescence and confocal imaging systems.
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Reactive oxygen species (ROS) were originally described as toxic by-products of aerobic cellular energy metabolism associated with the development of several diseases, such as cancer, neurodegenerative diseases, and diabetes [...].
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Oxygen deprivation induces multiple changes at the cellular and organismal levels, and its re-supply also brings another special physiological status. We have investigated the effects of hypoxia/re-oxygenation on embryonic growth using the zebrafish model: hypoxia slows embryonic growth, but re-oxygenation induces growth spurt or catch-up growth. The mitogen-activated kinase (MAPK)-pathway downstream insulin-like growth factor (IGF/Igf) has been revealed to positively regulate the re-oxygenation-induced catch-up growth, and the role of reactive oxygen species generated by environmental oxygen fluctuation is potentially involved in the phenomenon. Here, we report the role of NADPH-oxidase (Nox)-dependent hydrogen peroxide (H2O2) production in the MAPK-activation and catch-up growth. The inhibition of Nox significantly blunted catch-up growth and MAPK-activity. Amongst two zebrafish insulin receptor substrate 2 genes (irs2a and irs2b), the loss of irs2b, but not its paralog irs2a, resulted in blunted MAPK-activation and catch-up growth. Furthermore, irs2b forcedly expressed in mammalian cells allowed IGF-MAPK augmentation in the presence of H2O2, and the irs2b deficiency completely abolished the somatotropic action of Nox in re-oxygenation condition. These results indicate that redox signaling alters IGF/Igf signaling to facilitate hypoxia/re-oxygenation-induced embryonic growth compensation.
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Somatomedinas , Pez Cebra , Animales , Peróxido de Hidrógeno , Hipoxia/metabolismo , Mamíferos/metabolismo , NADP/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxígeno/metabolismo , Somatomedinas/metabolismoRESUMEN
Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.
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Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Hydrogen peroxide (H2O2), the major ROS produced by cells, acts as a second messenger to modify redox-sensitive proteins or lipids. After caudal fin amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways remain poorly understood. We focused on the early dialog between H2O2 and Sonic hedgehog (Shh) during zebrafish fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, the means of this tight reciprocal control change during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of an early positive crosstalk between Shh and H2O2 that might be more generally involved in various processes paving the way to improve regenerative processes, particularly in vertebrates.
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Proteínas Hedgehog , Pez Cebra , Animales , Proteínas Hedgehog/metabolismo , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno , Cicatrización de Heridas , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Although a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H2O2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H2O2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H2O2 could participate in the patterning of the embryo. We find that perturbations of endogenous H2O2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H2O2 levels and taken up by cells with low H2O2 levels where it leads to increased H2O2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events.
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Proteínas de Homeodominio/fisiología , Peróxido de Hidrógeno/metabolismo , Proteínas del Tejido Nervioso/fisiología , Comunicación Paracrina/fisiología , Colículos Superiores/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Oxidación-Reducción , Colículos Superiores/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrolloRESUMEN
Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when ß-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra, acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/ß-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via ß-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/ß-catenin signaling, a function conserved across eumetazoan evolution.
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Hydra/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Wnt3/genética , beta Catenina/genética , Animales , Evolución Biológica , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Cabeza/crecimiento & desarrollo , Cabeza/fisiología , Hydra/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Planarias/genética , Interferencia de ARN , Regeneración/fisiología , Transducción de Señal , Proteína Wnt3/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , beta Catenina/metabolismoRESUMEN
Reactive oxygen species (ROS), which were originally classified as exclusively deleterious compounds, have gained increasing interest in the recent years given their action as bona fide signalling molecules. The main target of ROS action is the reversible oxidation of cysteines, leading to the formation of disulfide bonds, which modulate protein conformation and activity. ROS, endowed with signalling properties, are mainly produced by NADPH oxidases (NOXs) at the plasma membrane, but their action also involves a complex machinery of multiple redox-sensitive protein families that differ in their subcellular localization and their activity. Given that the levels and distribution of ROS are highly dynamic, in part due to their limited stability, the development of various fluorescent ROS sensors, some of which are quantitative (ratiometric), represents a clear breakthrough in the field and have been adapted to both ex vivo and in vivo applications. The physiological implication of ROS signalling will be presented mainly in the frame of morphogenetic processes, embryogenesis, regeneration, and stem cell differentiation. Gain and loss of function, as well as pharmacological strategies, have demonstrated the wide but specific requirement of ROS signalling at multiple stages of these processes and its intricate relationship with other well-known signalling pathways.
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Inhibition of regeneration and induction of tissue fibrosis are classic outcomes of tissue repair in adult mammals. Here, using a newly developed model of regeneration in adult mammals i.e. regeneration after massive resection of an inguinal fat pad, we demonstrate that both endogenous and exogenous opioids prevent tissue regeneration in adults, by inhibiting the early production of reactive oxygen species (ROS) that generally occurs after lesion and is required for regeneration. These effects can be overcome and regeneration induced by the use of an opioid antagonist. The results obtained in both our new model and the gold standard adult zebrafish demonstrate that this mechanism can be considered as a general paradigm in vertebrates. This work clearly demonstrates that ROS is required for tissue regeneration in adult mammals and shows the deleterious effect of opioids on tissue regeneration through the control of this ROS production. It thus raises questions about opioid-based analgesia in perioperative care.
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Analgésicos Opioides/farmacología , Regeneración/efectos de los fármacos , Tejido Adiposo/patología , Analgésicos Opioides/metabolismo , Aletas de Animales , Animales , Femenino , Fibrosis/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Miocitos Cardíacos/patología , Naloxona/análogos & derivados , Naloxona/farmacología , Compuestos de Amonio Cuaternario/farmacología , Especies Reactivas de Oxígeno/metabolismo , Regeneración/fisiología , Tramadol/farmacología , Pez CebraRESUMEN
The tight control of reactive oxygen species (ROS) levels is required during regeneration. H2O2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H2O2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H2O2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H2O2 levels to their original state. A similar regulatory loop between H2O2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity.
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Proteínas Hedgehog/metabolismo , Peróxido de Hidrógeno/metabolismo , Red Nerviosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regeneración/fisiología , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.
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Orientación del Axón/efectos de los fármacos , Proteínas Hedgehog/fisiología , Peróxido de Hidrógeno/metabolismo , Neurogénesis/fisiología , Células Ganglionares de la Retina/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Orientación del Axón/fisiología , Axones/metabolismo , Catalasa/metabolismo , Cisteína/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/análisis , Transporte de Proteínas/efectos de los fármacos , Células Ganglionares de la Retina/ultraestructura , Transducción de Señal/fisiología , Superóxido Dismutasa/metabolismo , Pez Cebra/metabolismoRESUMEN
AIMS: Recent advances in redox biology have emphasized the role of hydrogen peroxide (H2O2) in the modulation of signaling pathways and revealed that H2O2 plays a role in cellular remodeling in adults. Thus, an understanding of the mechanisms that control H2O2 levels in mature tissue would be of great interest. RESULTS: We used a denervation strategy to demonstrate that sensory neurons are responsible for controlling H2O2 levels under normal conditions and after being lesioned. Moreover, we demonstrate that severed nerves respond to appendage amputation via the induction of Hedgehog signaling and that this signaling is responsible for H2O2 production in the wounded epidermis. Finally, we show that H2O2 and nerve growth are regulated via reciprocal action in adults. INNOVATION AND CONCLUSION: These data support a new paradigm for the regulation of tissue homeostasis: H2O2 attracts nerves and nerves control H2O2 levels in a positive feedback loop. This finding suggests that the peripheral nerve redox environment could be a target for manipulating cell plasticity in adults.
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Proteínas Hedgehog/metabolismo , Células de Schwann/metabolismo , Animales , Animales Modificados Genéticamente , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células de Schwann/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Pez CebraRESUMEN
Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.
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Encéfalo/embriología , Encéfalo/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.
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Adenosina/metabolismo , Aletas de Animales/fisiología , Apoptosis/fisiología , Receptor de Adenosina A2B/metabolismo , Células Madre/metabolismo , Animales , Neurogénesis/fisiología , Regeneración , Pez CebraRESUMEN
A major issue in regenerative medicine is the role of injury in promoting cell plasticity. Here we explore the function of reactive oxygen species (ROS) induced through lesions in adult zebrafish. We show that ROS production, following adult fin amputation, is tightly regulated in time and space for at least 24 hours, whereas ROS production remains transient (2 hours) in mere wound healing. In regenerative tissue, ROS signaling triggers two distinct parallel pathways: one pathway is responsible for apoptosis, and the other pathway is responsible for JNK activation. Both events are involved in the compensatory proliferation of stump epidermal cells and are necessary for the progression of regeneration. Both events impact the Wnt, SDF1 and IGF pathways, while apoptosis only impacts progenitor marker expression. These results implicate oxidative stress in regeneration and provide new insights into the differences between healing and regeneration.
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Proliferación Celular , Especies Reactivas de Oxígeno/metabolismo , Regeneración , Animales , Secuencia de Bases , Muerte Celular , Cartilla de ADN , Activación Enzimática , MAP Quinasa Quinasa 4/metabolismo , Reacción en Cadena de la Polimerasa , Pez CebraRESUMEN
Thrombin receptor, F2R or PAR1 is a G-protein coupled receptor, located in the membrane of endothelial cells. It has been initially found to transduce signals in hemostasis, but recently also known to act in cancer and in vascular development. Mouse embryos lacking PAR1 function die from hemorrhages with varying frequency at midgestation. We have performed a survey of potential PAR1 homologs in the zebrafish genome and identified a teleost ortholog of mammalian PAR1. Knockdown of par1 function in zebrafish embryos demonstrates a requirement for Par1 in cardio-vascular development. Furthermore, we show that function of Par1 requires the presence of a phylogenetically conserved proteolytic cleavage site and a second intracellular domain. Altogether our results demonstrate a high degree of conservation of PAR1 proteins in the vertebrate lineage in respect to amino acid sequence as well as protein function.
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Sistema Cardiovascular/embriología , Receptor PAR-1/fisiología , Pez Cebra/embriología , Animales , Evolución Molecular , Técnicas de Silenciamiento del Gen , Frecuencia Cardíaca , Receptor PAR-1/genética , Flujo Sanguíneo RegionalRESUMEN
DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.
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ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Desarrollo Embrionario/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Pez Cebra/embriología , Animales , Secuencia de Bases , Cristalografía por Rayos X , ADN (Citosina-5-)-Metiltransferasas/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
BACKGROUND: Cells undergoing activation or apoptosis exhibit plasma membrane changes, leading to the formation of shed vesicles (microparticles, MP). Although their effects on recipient cells in vitro, and their ability to support inflammatory or thrombotic events in the circulation have been studied, the spreading of such vesicles in tissues is still elusive. Our aim was to set up a method to examine the behavior of these vesicles in vivo. METHODS: We examined the persistence of green-fluorescent microparticles (fMP), prepared after Ca2+ ionophore activation (iono-fMP) or apoptogenic treatment (eto-fMP) of human Jurkat T lymphoblastic or non-hematopoietic embryonic kidney (HEK) cell lines, following injection in zebrafish embryos 2h after egg fertilization. RESULTS: One hour post-injection, iono-fMP issued from both cell types formed a fluorescent dispersal in the intercellular space of embryos. In contrast, eto-fMP or MP deprived of sialic acid at their membrane, gathered together at the site of injection. CONCLUSIONS: We propose a method characterizing the abilities of MP to spread in the intercellular space. We showed that MP produced by apoptosis of T cells and those deprived of sialic acid at their membrane do not diffuse within the living cells. On the contrary, MP shed upon calcium induced activation of T and HEK cells, diffuse at a distance and spread in the intercellular space. GENERAL SIGNIFICANCE: The fate of injected MP relies on the type of induction rather than the cell species and results provide a model to test the ability of vesicles to interact locally or to spread outside of the site of production.