Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags.

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.

A series of caged fluorophores for calibrating light intensity.

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.

Hydrogen Peroxide Signaling in Physiology and Pathology.

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 [...].

NADPH-Oxidase Derived Hydrogen Peroxide and Irs2b Facilitate Re-oxygenation-Induced Catch-Up Growth in Zebrafish Embryo.

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.

Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway.

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.

An early Shh-H2O2 reciprocal regulatory interaction controls the regenerative program during zebrafish fin regeneration.

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.

H2O2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum.

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.

An evolutionarily-conserved Wnt3/ß-catenin/Sp5 feedback loop restricts head organizer activity in Hydra.

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.

Hydrogen Peroxide and Redox Regulation of Developments.

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.