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:Anuran genomes have a large number and diversity of transposable elements, but are little explored, mainly in relation to their molecular structure and evolutionary dynamics. Here, we investigated the retrotransposons containing tyrosine recombinase (YR) (order DIRS) in the genome of Xenopus tropicalis and Xenopus laevis. These anurans show 2n = 20 and the 2n = 36 karyotypes, respectively. They diverged about 48 million years ago (mya) and X. laevis had an allotetraploid origin (around 17-18 mya). Our investigation is based on the analysis of the molecular structure and the phylogenetic relationships of 95 DIRS families of Xenopus belonging to DIRS-like and Ngaro-like superfamilies. We were able to identify molecular signatures in the 5' and 3' noncoding terminal regions, preserved open reading frames, and conserved domains that are specific to distinguish each superfamily. We recognize two ancient amplification waves of DIRS-like elements that occurred in the ancestor of both species and a higher density of the old/degenerate copies detected in both subgenomes of X. laevis. More recent amplification waves are seen in X. tropicalis (less than 3.2 mya) and X. laevis (around 10 mya) corroborating with transcriptional activity evidence. All DIRS-like families were found in both X. laevis subgenomes, while a few were most represented in the L subgenome. Ngaro-like elements presented less diversity and quantity in X. tropicalis and X. laevis genomes, although potentially active copies were found in both species and this is consistent with a recent amplification wave seen in the evolutionary landscape. Our findings highlight a differential diversity-level and evolutionary dynamics of the YR retrotransposons in X. tropicalis and X. laevis species expanding our comprehension of the behavior of these elements in both genomes during the diversification process.
Project description:The molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome.We found that, like the traditionally used Xenopus laevis, the Xenopus tropicalis tadpole has the capacity to regenerate its tail following amputation, including its spinal cord, muscle, and major blood vessels. We examined gene expression using the Xenopus tropicalis Affymetrix genome array during three phases of regeneration, uncovering more than 1,000 genes that are significantly modulated during tail regeneration. Target validation, using RT-qPCR followed by gene ontology (GO) analysis, revealed a dynamic regulation of genes involved in the inflammatory response, intracellular metabolism, and energy regulation. Meta-analyses of the array data and validation by RT-qPCR and in situ hybridization uncovered a subset of genes upregulated during the early and intermediate phases of regeneration that are involved in the generation of NADP/H, suggesting that these pathways may be important for proper tail regeneration.The Xenopus tropicalis tadpole is a powerful model to elucidate the genetic mechanisms of vertebrate appendage regeneration. We have produced a novel and substantial microarray data set examining gene expression during vertebrate appendage regeneration.
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: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:In this experiment, we revealed the critical steps for regeneration initiation. We discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomics of the regenerating Xenopus laevis tail. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types that promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects.
Project description:In this experiment, we revealed the critical steps for regeneration initiation. We discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomics of the regenerating Xenopus laevis tail. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types that promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects.
Project description:In this experiment, we show transcription profiling of the Xenopus tropicalis tadpole tail tissue regeneration following removal. The tail tissues include its spinal cord, notochord, muscle, and dorsal aorta. We characterized the early, intermediate, and late stages of Xenopus tropicalis tail regeneration using the Xenopus tropicalis Affymetrix genome array in biological replicate.
Project description:Xenopus laevis tadpoles can regenerate whole tails after amputation. We have previously reported that interleukin 11 (il11) is required for tail regeneration. In this study, we have screened for genes that support tail regeneration under Il11 signaling in a certain cell type and have identified the previously uncharacterized genes Xetrov90002578m.L and Xetrov90002579m.S [referred to hereafter as regeneration factors expressed on myeloid.L (rfem.L) and rfem.S]. Knockdown (KD) of rfem.L and rfem.S causes defects of tail regeneration, indicating that rfem.L and/or rfem.S are required for tail regeneration. Single-cell RNA sequencing (scRNA-seq) revealed that rfem.L and rfem.S are expressed in a subset of leukocytes with a macrophage-like gene expression profile. KD of colony-stimulating factor 1 (csf1), which is essential for macrophage differentiation and survival, reduced rfem.L and rfem.S expression levels and the number of rfem.L- and rfem.S-expressing cells in the regeneration bud. Furthermore, forced expression of rfem.L under control of the mpeg1 promoter, which drives rfem.L in macrophage-like cells, rescues rfem.L and rfem.S KD-induced tail regeneration defects. Our findings suggest that rfem.L or rfem.S expression in macrophage-like cells is required for tail regeneration.