RESUMO
Cell movements are essential for morphogenesis during animal development. Epiboly is the first morphogenetic process in zebrafish in which cells move en masse to thin and spread the deep and enveloping cell layers of the blastoderm over the yolk cell. While epiboly has been shown to be controlled by complex molecular networks, the contribution of reactive oxygen species (ROS) to this process has not previously been studied. Here, we show that ROS are required for epiboly in zebrafish. Visualization of ROS in whole embryos revealed dynamic patterns during epiboly progression. Significantly, inhibition of NADPH oxidase activity leads to a decrease in ROS formation, delays epiboly, alters E-cadherin and cytoskeleton patterns and, by 24â¯h post-fertilization, decreases embryo survival, effects that are rescued by hydrogen peroxide treatment. Our findings suggest that a delicate ROS balance is required during early development and that disruption of that balance interferes with cell adhesion, leading to defective cell motility and epiboly progression.
Assuntos
Blastoderma/metabolismo , Citoesqueleto/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra/fisiologia , Animais , Caderinas/metabolismo , Adesão Celular , Movimento Celular , Embrião não Mamífero , Morfogênese , Proteínas de Peixe-Zebra/metabolismoRESUMO
By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), ß-peltatin-A-methylether (2), 5'-desmethoxy-ß-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1â»7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.
Assuntos
Bursera/química , Citoesqueleto/química , Lignanas/química , Tubulina (Proteína)/química , Animais , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Lignanas/farmacologia , Microtúbulos , Estrutura Molecular , Peixe-ZebraRESUMO
Antioxidant cellular mechanisms are essential for cell redox homeostasis during animal development and in adult life. Previous in situ hybridization analyses of antioxidant enzymes in zebrafish have indicated that they are ubiquitously expressed. However, spatial information about the protein distribution of these enzymes is not available. Zebrafish embryos are particularly suitable for this type of analysis due to their small size, transparency and fast development. The main objective of the present work was to analyze the spatial and temporal gene expression pattern of the two reported zebrafish glutathione peroxidase 4 (GPx4) genes during the first day of zebrafish embryo development. We found that the gpx4b gene shows maternal and zygotic gene expression in the embryo proper compared to gpx4a that showed zygotic gene expression in the periderm covering the yolk cell only. Following, we performed a GPx4 protein immunolocalization analysis during the first 24-h of development. The detection of this protein suggests that the antibody recognizes GPx4b in the embryo proper during the first 24 h of development and GPx4a at the periderm covering the yolk cell after 14-somite stage. Throughout early cleavages, GPx4 was located in blastomeres and was less abundant at the cleavage furrow. Later, from the 128-cell to 512-cell stages, GPx4 remained in the cytoplasm but gradually increased in the nuclei, beginning in marginal blastomeres and extending the nuclear localization to all blastomeres. During epiboly progression, GPx4b was found in blastoderm cells and was excluded from the yolk cell. After 24 h of development, GPx4b was present in the myotomes particularly in the slow muscle fibers, and was excluded from the myosepta. These results highlight the dynamics of the GPx4 localization pattern and suggest its potential participation in fundamental developmental processes.