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:Tissue regeneration is of fast growing importance in the development of biomedicine, particularly organ replacement therapies. Unfortunately, many human organs cannot regenerate. Anuran Xenopus laevis has been used as a model to study regeneration as many tadpole organs can regenerate. In particular, the tail, which consists of many axial and paraxial tissues, such as spinal cord, dorsal aorta and muscle, commonly present in vertebrates, can fully regenerate when amputated at late embryonic stages and most of the tadpole stages. Interestingly, between stage 45 when feeding begins to stage 47, the Xenopus laevis tail cannot regenerate after amputation. This period, termed "refractory period", has been known for about 20 years. The underlying molecular and genetic basis is unclear in part due to the difficult to carry out genetic studies in this pseudo-tetraploid species. Here we compared tail regeneration between Xenopus laevis and the highly related diploid anuran Xenopus tropicalis and found surprisingly that Xenopus tropicalis lacks the refractory period. Further molecular and genetic studies, more feasible in this diploid species, should reveal the basis for this evolutionary divergence in tail regeneration between two related species and facilitate the understanding how tissue regenerative capacity is controlled, thus with important implications for human regenerative medicine.
Project description:Maintenance of optimal conditions such as water parameters, diet, and feeding is essential to a healthy Xenopus laevis and Xenopus tropicalis colony and thus to the productivity of the lab. Our prior husbandry experience as well as the rapid growth of the National Xenopus Resource has given us a unique insight into identifying and implementing these optimal parameters into our husbandry operations. Here, we discuss our standard operating procedures that will be of use to both new and established Xenopus facilities.
Project description:Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5x10(4) cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous beta-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations. Developmental Dynamics 238:1422-1432, 2009. (c) 2009 Wiley-Liss, Inc.