Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation 1, 2, via the coordinated activity of numerous growth factor signaling pathways, including the Wnt, Fgf, BMP, notch, and TGFβ pathways 3-6. Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, via pharmacological or genetic approaches, reduces cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/β-catenin signaling and the activation of one of its major downstream targets, fgf20 7, which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.

SummaryWhile it is 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 oscillate with each cell division. Furthermore, we show that the fertilization-induced Ca2+ wave is 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 a role for oscillating ROS levels in early cell cycle regulation in Xenopus embryos.

Lens development provides a good model system for studying cellular and molecular mechanisms underlying embryonic induction and morphogenesis. Members of the large Maf family of transcription factors, L-Maf and c-Maf, have been shown to play key roles in chick and mouse lens development. Here we report identification of two Xenopus maf genes, XmafB and XL-maf, which exhibit unique temporal and spatial expression patterns during lens formation. XmafB can first be detected in the presumptive lens-forming ectoderm, when the primary eye vesicle makes contact with the head ectoderm. XL-maf expression appears a little later, just before thickening of the lens placode, and both XmafB and XL-maf can be detected in the lens placode. During lens vesicle formation, the expression domains of XmafB and XL-maf segregated from each other, resulting in restricted expression in lens epithelial and fiber cells, respectively. When the optic cup anlagen was removed, only XmafB expression is detected. Both Mafs can induce the lens fiber cell-specific markers, betaA4- and gamma-crystallins. In animal cap assays, XmafB can induce Pax6, Xlens1 and Sox3 expression, but XL-maf fails to induce Pax6 and Xlens1 expression. These results suggest that these maf genes are involved in the regulation of cell-type specific gene expression and play roles in inductive events during Xenopus lens development.

SummaryIn the past decade, Xenopus tropicalis has emerged as a powerful new amphibian genetic model system, which offers all of the experimental advantages of its larger cousin, Xenopus laevis. Here we investigated the efficiency of transcription activator-like effector nucleases (TALENs) for generating targeted mutations in endogenous genes in X. tropicalis. For our analysis we targeted the tyrosinase (oculocutaneous albinism IA) (tyr) gene, which is required for the production of skin pigments, such as melanin. We injected mRNA encoding TALENs targeting the first exon of the tyr gene into two-cell-stage embryos. Surprisingly, we found that over 90% of the founder animals developed either partial or full albinism, suggesting that the TALENs induced bi-allelic mutations in the tyr gene at very high frequency in the F0 animals. Furthermore, mutations tyr gene were efficiently transmitted into the F1 progeny, as evidenced by the generation of albino offspring. These findings have far reaching implications in our quest to develop efficient reverse genetic approaches in this emerging amphibian model.