RESUMO
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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The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. Here, we employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, we utilised the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, our work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Feminino , Animais , Proteínas Hedgehog/genética , Membrana Celular , Drosophila , Via Secretória , MamíferosRESUMO
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.
Assuntos
Somatomedinas , Peixe-Zebra , Animais , Peróxido de Hidrogênio , Hipóxia/metabolismo , Mamíferos/metabolismo , NADP/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxigênio/metabolismo , Somatomedinas/metabolismoRESUMO
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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Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant. How this temperature independence is achieved is a mystery that we address in this study. Using RT-qPCR we show that the endogenous fgf8 mRNA concentration decreases during somitogenesis and correlates with the exponent of the shrinking pre-somitic mesoderm (PSM) size. As the temperature decreases, the dynamics of fgf8 and many other gene transcripts, as well as the segmentation frequency and the PSM shortening and tail growth rates slows down as T-Tc (with Tc = 14.4 °C). This behavior characteristic of a system near a critical point may account for the temperature independence of somitogenesis in zebrafish.
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Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra , p-Aminoazobenzeno/análogos & derivados , p-Aminoazobenzeno/farmacologiaRESUMO
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 , Peixe-Zebra , Animais , Proteínas Hedgehog/metabolismo , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio , Cicatrização , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.
Assuntos
Nadadeiras de Animais/diagnóstico por imagem , Proteínas de Bactérias/genética , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Proteínas Luminescentes/genética , Nadadeiras de Animais/lesões , Nadadeiras de Animais/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/instrumentação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Peróxido de Hidrogênio/química , Ácido Hipocloroso/síntese química , Ácido Hipocloroso/metabolismo , Proteínas Luminescentes/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Oxirredução , Fagocitose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-ZebraRESUMO
Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.
Assuntos
Corantes Fluorescentes , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Peixe-ZebraRESUMO
Catalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H2O2 concentration by accelerating its dismutation. Many physiopathological situations are associated with oxidative stress resulting from H2O2 overproduction, during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di-copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solutions and in cells using different analytical methods. The complex exhibited CAT activity in solutions and, more interestingly, on HyPer HeLa cells that possess a genetically encoded ratiometric fluorescent sensors of H2O2. These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity.
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Antioxidantes/metabolismo , Catalase/metabolismo , Cobre/metabolismo , Metaloproteínas/metabolismo , Peptídeos/metabolismo , Antioxidantes/química , Catalase/química , Cobre/química , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Metaloproteínas/química , Peptídeos/química , Células Tumorais CultivadasRESUMO
Oxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) meso-tetrakis(N-(2'-n-butoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP5+) and Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [1,4, 7,10] tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22] (Imisopasem manganese, M40403, CG4419) were found efficacious at 10 µM, while Mn(II) chloro N-(phenolato)-N,N'-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine (Mn1) requires an incubation at 100 µM. This study thus demonstrates that MnTnBuOE-2-PyP5+, M40403 and Mn1 were efficacious in suppressing inflammatory response in HT29-MD2 cells and such action appears to be related to their ability to enter the cells and modulate reactive oxygen species (ROS) levels.
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Catalase/metabolismo , Manganês/metabolismo , Compostos Organometálicos/metabolismo , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/metabolismo , Linhagem Celular , Glutationa Peroxidase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Metaloporfirinas/metabolismo , Mimetismo Molecular , Oxirredução , Estresse Oxidativo , Porfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein-protein interactions by live-cell fluorescence microscopy.
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Técnicas Biossensoriais , Corantes Fluorescentes/química , Biologia Molecular/métodos , Imagem Óptica/métodos , Plasmídeos/química , Coloração e Rotulagem/métodos , Animais , Compostos de Benzilideno/química , Células COS , Chlorocebus aethiops , Clonagem Molecular , Cor , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Expressão Gênica , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Plasmídeos/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peixe-ZebraRESUMO
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.
Assuntos
Proteínas de Homeodomínio/fisiologia , Peróxido de Hidrogênio/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Comunicação Parácrina/fisiologia , Colículos Superiores/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Oxirredução , Colículos Superiores/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.
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Corantes Fluorescentes/química , Imagem Molecular/métodos , Animais , Embrião de Galinha , Células HeLa , Humanos , Ligação Proteica , Peixe-Zebra/embriologiaRESUMO
Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.
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Movimento Celular , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Oxidantes/metabolismo , Animais , Transporte Biológico , Extensões da Superfície Celular/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional , Larva/metabolismo , Membranas Mitocondriais/metabolismo , Peixe-ZebraRESUMO
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.
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Optogenética/métodos , Compostos Policíclicos/química , Receptores de Estrogênio/genética , Ativação Transcricional , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Expressão Gênica , Luz , Processos Fotoquímicos , Receptores de Estrogênio/química , Peixe-Zebra/embriologiaRESUMO
Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied in vivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H2O2) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation.
Assuntos
Diferenciação Celular , Peroxidação de Lipídeos , Neurogênese , Espécies Reativas de Oxigênio/metabolismo , Retina/citologia , Células-Tronco/citologia , Animais , Proliferação de Células , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Oxirredução , Retina/fisiologia , Células-Tronco/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Hydra/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Wnt3/genética , beta Catenina/genética , Animais , Evolução Biológica , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/crescimento & desenvolvimento , Cabeça/fisiologia , Hydra/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Planárias/genética , Interferência de RNA , Regeneração/fisiologia , Transdução de Sinais , Proteína Wnt3/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismoRESUMO
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.