Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Xenopus laevis transgenesis by sperm nuclear injection.

The stable integration of transgenes into embryos of the frog Xenopus laevis is achieved using the procedure described here. Linear DNA containing the transgene is incorporated randomly into sperm nuclei that have had their membranes disrupted with detergent treatment. Microinjection of these nuclei into unfertilized eggs produces viable embryos that can be screened for activity of the transgene. The proportion of embryos that harbor the transgene varies from 10 to 40% of the total number of surviving embryos. Multiple copies of the transgene can integrate as a concatemer into the sperm genome, and more than one site of DNA integration might occur within resulting animals. Germ cell transmission of the transgene is routine and the procedure is well suited to the production of transgenic reporter frog lines. One day should be allocated for the preparation of the sperm nuclei, which are stored as aliquots for future use. The transgenesis reaction and egg injection take one morning.

Regulation of apoptosis in theXenopus embryo by Bix3.

Members of the Bix family of homeobox-containing genes are expressed in the vegetal hemisphere of the Xenopus embryo at the early gastrula stage. Misexpression of at least some of the family members causes activation of mesoderm- and endoderm-specific genes and it is known that some of the proteins, including Bix2 and Bix3, interact with Smad proteins via a motif that is also present in the related protein Mixer. In this paper we study the function of Bix3. Misexpression of Bix3, similar to misexpression of other members of the Bix family, causes the activation of a range of mesendodermal genes, but the spectrum of genes induced by Bix3 differs from that induced by Bix1. More significantly, we find that overexpression of Bix3 also causes apoptosis, as does depletion of Bix3 by use of antisense morpholino oligonucleotides. The ability of Bix3 to causes apoptosis is not associated with its ability to activate transcription and nor with its possession of a Smad interaction motif. Rather, Bix3 lacks a C-terminal motif, which, in Bix1, acts in cis to inhibit apoptosis. Mutation of this sequence in Bix1 causes the protein to acquire apoptosis-inducing activity.