BMC Dev Biol
November 15, 2011;
Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration.
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
, 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
BMC Dev Biol
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Figure 1. The Xenopus tropicalis tadpole has the capacity to regenerate its tail. (A) Schematic diagram of the tissues located in the Xenopus tropicalis tadpole tail. (B) Transverse section of tadpole tail visualized with toluidine blue. (C-E) An amputated tail (C), uncut tail (D), and regenerated tail 7 days after amputation (E). (F-H) Immunostaining against skeletal muscle (12/101; F), neurons (acetylated tubulin; G), and vasculature (mTie-2::eGFP transgene; H). (I-O) A regenerated tail at one month post-amputation (I). Immunostaining showing skeletal muscle (12/101; J, K), neurons (acetylated tubulin; L, M), and vasculature (mTieGFP transgene; N, O) in non-cut and regenerated tails 28 days post amputation. Green arrowhead depicts amputation site; red arrowhead shows parallel axonal tract; orange arrowheads shows parallel blood vessels. Red scale bar is 1000 μm.
Additional file 1. Figure S1 - A transgenic Xenopus tropicalis line that expresses eGFP in its vasculature. (A) A series of merged confocal images from transgenic Xenopus tropicalis tadpole expressing eGFP under the control of the murine Tie-2 promoter; the four red boxes show the heart (B), brain and associated vasculature (C), the vasculature that surrounds the eye (D), and the vasculature in the tail (E). The single confocal slice in (E) depicts the dorsal lateral anastomosing vessel (l), spinal cord (s), dorsal aorta (d), posterior cardinal vein (p), epithelial vasculature (e), and intersomitic vessels (i).
Figure 2. Characterization of the early, intermediate, and late phases of tail regeneration. (A) Tadpole tails imaged using bright field microscopy at early (0 h, 6 h); intermediate (24 h) and late phases (48 h and 72 h) of tail regeneration. Black open arrowhead shows typical dorsal constriction at 6 h post amputation; blue arrowhead shows pre-regenerative tissue; red arrowhead shows nascent fin epidermis at the distal tip; black arrowhead = spinal cord; green arrowhead = notochord, orange arrowhead = melanophrore. (B) Tadpole tails stained with Sudan Black B (inflammatory cells). (C) Tadpole tails stained by whole-mount in situ hybridization for mmp7 (inflammatory cells). (D) Tadpole tails stained by immunohistochemistry for mitotic cells (pH3, shown in black). The tail in each panel is outlined in red and the plane of amputation is shown in green. (E) Tadpole tails stained to reveal the neuronal tissue by immunohistochemistry (acetylated tubulin, shown in black). (F) Tadpole tails stained by immunohistochemistry for vascular tissue (GFP antibody in mTie-2::eGFP transgenic line, shown in black). The open purple arrowhead shows a typical eGFP positive "clot" at the injured dorsal aorta. Green arrowheads show distally projecting blood vessels. (G) Tadpole tails stained by immunohistochemistry for muscle tissue (12/101). The open orange arrowhead shows the presence of skeletal muscle in the regenerated portion of the tail. Red scale bar for each row is 250 μm. Note that the images in panels D-F are black and white reversals (i.e. shown as negative images).
Figure 3. Array analysis of X. tropicalis tail regeneration. (A) The schematic diagrams of the early, intermediate, and late stages of tail regeneration. The circled areas depict the portion of tissue, and hence the RNA, collected and used for array analysis. (B) Shows the similarities of the eight arrays (four array time points in duplicate) using principal component analysis (PCA) mapping.
Figure 4. Validation and GO analysis of highly significant targets. (A) The validation of five highly significant array targets (blue) is plotted against normalized RT-qPCR derived expression profiles (red). Note that lines connecting each time point are meant to serve as a visual aide. (B) Shows expression patterns of RT-qPCR validated targets using whole-mounts in situ hybridization for leptin, xmenf, xcyp26a, sox9 and pdgfa at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 hours post-amputation. (C) Heat map of all highly significant and dynamic targets produced a group of 1441 targets, grouped into 12 clusters by their similar expression changes through the array time points. (D) Gene ontology (GO) analysis of the twelve clusters in (C) showed four clusters over-represented with at least three GO terms accompanied by a p-value under 1e-3 and false discovery rate (FDR) under 0.5. Error bars represent standard error of mean (SEM).
Figure 5. Systematic analysis of intracellular metabolic processes using gene ontology. (A) 66 metabolic processes are upregulated in the T24h array (gray). Five of ten highest upregulated processes in the T24h array are colored. The key to this graphic is shown below and "N" indicates the number of genes in the array corresponding to each process. "Organic acid metabolism", which was identified in an initial GO analysis (Figure 4D) is also plotted on the graph. (B) Nicotinamide, NADP, and the generation of reactive oxygen species (ROS) are connected by the metabolic pathway shown in (B) (genes regulating the pathway shown in red, proteins in green). (C) Shows the in situ hybridization patterns of g6pd, idh1, idh2, me2, me3, and nadk following tail regeneration (0 h, 1 h, 6 h, 24 h, and 60 h). (D) RT-qPCR expression profile of nadk (blue) and g6pd (red) following tail amputation. Error bars represent standard error of mean (SEM), lines connecting each error bar are meant to serve as a visual aide.
Schematic diagram of the major blood vessels and structures in the Xenopus tadpole tail (A) and in transverse section of tadpole tail visualized with toluidine blue.
Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed