Biology subjects

Amaya, E.

Publications and source records attributed to Amaya, E..

6 recordsLinked to original sources

bioRxiv2021

The roles of NADPH oxidases during adult zebrafish fin regeneration

Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for whole body, appendage and organ regeneration in various organisms, including planarians, Hydra, zebrafish, axolotl, Xenopus, geckos and mice. In the majority of cases these roles have been shown via the use of NADPH oxidase pharmacological inhibitors, which generally target all NAPDH oxidases (NOXes). To identify the specific NOX or NOXes essential for ROS production during adult fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4). We also crossed these mutant lines to a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels in adult zebrafish fins. Using this approach, we found that homozygous duox mutants have significantly attenuated ROS levels following fin amputation, and this correlated with a significantly diminished rate of fin regeneration. While the other nox homozygous mutants (nox5 and cyba) showed less of an effect on ROS levels or adult fin regeneration, duox/cyba double mutants showed a more diminished rate of fin regeneration than duox mutants alone, suggesting that Nox1-4 do play a role during regeneration, but one that is secondary to that of Duox. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate during the day with a circadian rhythm.

developmental biology↗

bioRxiv2021

Aerobic glycolysis is important for zebrafish larval wound closure and tail regeneration

The underlying mechanisms of appendage regeneration remain largely unknown and uncovering these mechanisms in capable organisms has far-reaching implications for potential treatments in humans. Recent studies implicate a requirement for metabolic reprogramming reminiscent of the Warburg effect during successful appendage and organ regeneration. As changes are thus predicted to be highly dynamic, methods permitting direct, real-time visualization of metabolites at the tissue and organismal level, would offer a significant advance in defining the influence of metabolism on regeneration and healing. We sought to examine whether glycolytic activity was altered during larval fin regeneration, utilising the genetically encoded biosensor, Laconic, enabling the spatiotemporal assessment of lactate levels in living zebrafish. We present evidence for a rapid increase in lactate levels within minutes following injury, with a role of aerobic glycolysis in actomyosin contraction and wound closure. We also find a second wave of lactate production, associated with overall larval tail regeneration. Chemical inhibition of glycolysis attenuates both contraction of the wound and regrowth of tissue following tail amputation, suggesting aerobic glycolysis is necessary at two distinct stages of regeneration.

developmental biology↗

bioRxiv2021

Matriptase generates a tissue damage response via promoting Gq signalling, leading to RSK and DUOX activation

Tissues respond to damage by increasing inflammation and epithelial cell motility. How damage detection and responses are orchestrated is unclear. Overexpression of the membrane bound protease, Matriptase, or mutation of its inhibitor, Hai1, results in inflamed epithelia, in which cells have increased motility and are prone to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H2O2, Nf{kappa}B signalling, and IP3R-mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit rescues both the hai1a inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated pERK, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as pERK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

cell biology↗

bioRxiv2018

Zebrafish duox mutations provide a model for human congenital hypothyroidism

Thyroid dyshormonogenesis is a leading cause of congenital hypothyroidism, a highly prevalent but treatable condition. Thyroid hormone synthesis is dependent on the formation of reactive oxygen species (ROS). In humans, the primary sources for ROS production during thyroid hormone synthesis are the NADPH oxidase, DUOX1 and DUOX2. Indeed mutations in DUOX1 and DUOX2 have been linked with congenital hypothyroidism. Unlike humans, zebrafish has a single orthologue for DUOX1 and DUOX2. In this study, we investigated the phenotypes associated with two nonsense mutant alleles of the single duox gene in zebrafish, sa9892 and sa13017. Both alleles gave rise to readily observable phenotypes reminiscent of congenital hypothyroidism, from the larval stages through to adulthood. By using various methods to examine the external and internal phenotypes, we discovered a strong correlation between TH synthesis and duox function, beginning from the early larval stage, when T4 levels are already noticeably absent in the mutants. Loss of T4 production resulted in growth retardation, pigmentation defects, ragged fins, thyroid hyperplasia / external goiter, and infertility. Remarkably all of these defects associated with chronic congenital hypothyroidism could be rescued with T4 treatment, even when initiated when the fish had already reached adulthood. Our work suggests that these zebrafish duox mutants may provide a powerful model to understand the aetiology of untreated and treated congenital hypothyroidism even in advance stages of development.

developmental biology↗

bioRxiv2017

Ca2+-induced mitochondrial ROS regulate the early embryonic cell cycle

While it has long been appreciated that reactive oxygen species (ROS) can act as second messengers in both homeostastic and stress response signaling pathways, potential roles for ROS during early vertebrate development have remained largely unexplored. Here we show that fertilization in Xenopus embryos triggers a rapid increase in ROS levels, which oscillates with each cell division. Furthermore, we show that the fertilization induced Ca2+ wave is both necessary and sufficient to induce ROS production in activated or fertilized eggs. Using chemical inhibitors, we identified mitochondria as the major source of fertilization induced ROS production. Inhibition of mitochondrial ROS production in early embryos results in cell cycle arrest, in part, via ROS dependent regulation of Cdc25C activity. This study reveals for the first time, a role for oscillating ROS levels in the regulation of the early cell cycle in Xenopus embryos.\n\nHighlightsO_LIROS, including hydrogen peroxide, are produced after fertilization in Xenopus\nC_LIO_LICa2+ signaling after fertilization induces ROS production in mitochondria\nC_LIO_LIMitochondria are the major source of oscillating ROS levels\nC_LIO_LIROS regulate Cdc25C activity and the early cell cycle\nC_LI

developmental biology↗

bioRxiv2017

Elevated and sustained reactive oxygen species levels facilitate mesoderm formation during early Xenopus development

Fertilisation triggers embryonic development culminating with the activation of a number of highly co-ordinated and evolutionarily conserved signalling pathways, which induce and pattern the mesoderm of the developing embryo. Previous studies in invertebrates have shown that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), can act as a signalling molecule for axis specification during early development. Using a HyPer transgenic Xenopus laevis line that expresses a H2O2-sensitive fluorescent protein sensor maternally, we recently found that fertilisation triggers a rapid increase in ROS production. Here we show that this increase in ROS levels is sustained throughout early embryogenesis, lasting until the tailbud stages. In addition we show that lowering ROS levels from the blastula stage through the gastrula stages via antioxidant treatments disrupts mesoderm formation. Furthermore, we show that attenuating ROS levels during the blastula / gastrula stages affects some, but not all, growth factor signalling pathways involved in mesoderm induction and patterning, including the PI3K/Akt, TGF-{beta}/Nodal, and Wnt/{beta}-catenin signalling pathways. These data suggest that sustained elevated ROS levels during the blastula and gastrula stages are essential for early vertebrate embryonic development, at least partly, through their roles in promoting growth factor signalling.

developmental biology↗