Project description:KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.
Project description:Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.
Project description:The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.
Project description:Emerin is an inner nuclear membrane protein often mutated in Emery-Dreifuss muscular dystrophy. Because emerin has diverse roles in nuclear mechanics, cytoskeletal organization, and gene expression, it has been difficult to elucidate its contribution to nuclear structure and disease pathology. In this study, we investigated emerin's impact on nuclei assembled in Xenopus laevis egg extract, a simplified biochemical system that lacks potentially confounding cellular factors and activities. Notably, these extracts are transcriptionally inert and lack endogenous emerin and filamentous actin. Strikingly, emerin caused rupture of egg extract nuclei, dependent on the application of shear force. In egg extract, emerin localized to nonnuclear cytoplasmic membranes, and nuclear rupture was rescued by targeting emerin to the nucleus, disrupting its membrane association, or assembling nuclei with lamin A. Furthermore, emerin induced breakage of nuclei in early-stage X. laevis embryo extracts, and embryos microinjected with emerin were inviable, with ruptured nuclei. We propose that cytoplasmic membrane localization of emerin leads to rupture of nuclei that are more sensitive to mechanical perturbation, findings that may be relevant to early development and certain laminopathies.
Project description:Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype.
Project description:PurposeThe purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation.MethodsThe apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR.ResultsComet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2- and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P<0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4- and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05).ConclusionsThe results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.
Project description:We inegrated quantitative phosphoproteome, proteome, a tergeted MS analysis, in conjunction with pathway perturbation to obtain a global view of the mechano-responses of embryonic tissues. This study provides mechanistic insights into mechanical force sensing pathways.
Project description:Robust and efficient protocols for fertilization and early embryo care of Xenopus laevis and Xenopus tropicalis are essential for experimental success, as well as maintenance and propagation of precious animal stocks. The rapid growth of the National Xenopus Resource has required effective implementation and optimization of these protocols. Here, we discuss the procedures used at the National Xenopus Resource, which we found helpful for generation and early upkeep of Xenopus embryos and tadpoles.
Project description:We inegrated quantitative phosphoproteome, proteome, a tergeted MS analysis, in conjunction with pathway perturbation to obtain a global view of the mechano-responses of embryonic tissues. This study provides mechanistic insights into mechanical force sensing pathways.