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The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable 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. We present that approach and its uses in a variety of physiological contexts.

The zebrafish has become an increasingly popular and valuable cancer model over the past few decades. While most zebrafish cancer models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression in live zebrafish. We utilize this technique to transiently or constitutively activate a typical human oncogene, kRASG12V, in zebrafish embryos and investigate the developmental and tumorigenic phenotypes. We demonstrate the spatiotemporal control of oncogene expression in live zebrafish, and characterize the different tumorigenic probabilities when kRASG12V is expressed transiently or constitutively at different developmental stages. Moreover, we show that light can be used to activate oncogene expression in selected tissues and single cells without tissue-specific promoters. Our work presents a novel approach to initiate and study cancer in zebrafish, and the high spatiotemporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells.

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. [ABSTRACT FROM AUTHOR]

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 catchup growth. The mitogen-activated kinase (MAPK)-pathway downstream insulin-like growth factor (IGF/Igf) has been revealed to positively regulate the re-oxygenationinduced 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]