Histone H3 Lysine 27 Trimethylation Leads to Loss of Mesendodermal Competence During Gastrulation in Zebrafish Ectodermal Cells.

Previous studies in Xenopus have shown that forced expression of Nodal signaling can change ectodermal cells to a mesodermal fate by the early gastrula stage, suggesting mesodermal competence in early ectoderm cells. This mesodermal competence in ectodermal cells has been shown to be regulated at the level of nucleocytoplasmic localization of Smad2 in Xenopus. However, the regulation of mesodermal competence through epigenetic mechanisms has not been fully elucidated. Here, we used a constitutively active form of zebrafish Smad2 (Smad2ca) to overcome the inhibition of Nodal signaling via the nuclear exclusion of Smad2. While heat-shock-dependent expression of Smad2ca at 5 h post fertilization (hpf) induced ectopic expression of mesendodermal genes in zebrafish ectodermal cells, responsiveness to Smad2ca was lost by 7 hpf. Chromatin immunoprecipitation-quantitative PCR analyses revealed that in ectodermal cells, levels of H3K27me3, but not H3K9me3, at both transcriptional start site (TSS) and 3'-flanking regions of mesendodermal genes at 9 hpf were markedly higher than those at 5 hpf. In contrast to mesendodermal genes, the levels of H3K27me3 at the TSS, but not 3'-flanking regions, of ectodermal genes remained low in ectodermal cells even at 9 hpf. We also found that chemical inhibition of H3K27me3 modification was able to recover the mesendodermal competence in ectodermal cells at 7 hpf, but not at 10 hpf. Taken together, our results suggest that the mesendodermal competence in zebrafish ectodermal cells is restricted by multiple mechanisms, including upregulation of H3K27me3 levels at the TSS of mesendodermal genes during early gastrulation.

Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins.

BACKGROUND: The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown. RESULTS: To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration. CONCLUSION: Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.