Waldner C et al. (2009), Red fluorescent Xenopus laevis: a new tool for ...

XB-ART-39960

BMC Dev Biol 2009 Jan 28;9:37. doi: 10.1186/1471-213X-9-37.

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Red fluorescent Xenopus laevis: a new tool for grafting analysis.

Waldner C , Roose M , Ryffel GU .

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Fluorescent proteins such as the green fluorescent protein (GFP) have widely been used in transgenic animals as reporter genes. Their use in transgenic Xenopus tadpoles is especially of interest, because large numbers of living animals can easily be screened. To track more than one event in the same animal, fluorescent markers that clearly differ in their emission spectrum are needed. We established the transgenic Xenopus laevis strain tom3 that expresses ubiquitously red fluorescence from the tdTomato gene through all larval stages and in the adult animal. This new tool was applied to track transplanted blastemas obtained after tail amputation. The blastema can regenerate ectopic tails marked by red fluorescence in the host animal. Surprisingly, we also found contribution of the host animal to form the regenerate. We have established a useful new tool to label grafts in Xenopus transplantation experiments.

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Species referenced: Xenopus laevis
Genes referenced: col2a1
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	Figure 1. Transgenic Xenopus laevis strain expressing ubiquitous red fluorescence. F1 animals of the tom3 strain at the neurula (A), larval (B) or froglet stage (C) seen with the red fluorescence filter set. D: Isolated tissue of a control froglet (left) and a froglet of the tom3 strain (right) seen in normal light. E: Same tissue samples seen in the red fluorescence filter set. F-H: Isolated muscle of a froglet of the blue fluorescent C5 strain [5] (left) and of the red fluorescent tom3 strain (right) seen in normal light (F), with red fluorescence filter set (G), or blue fluorescence filter set (H). Scale bars equal 1 mm.
	Figure 2. Adult animals of the tom3 strain express red fluorescence in a variety of tissues. A-D: Cryosections (10 μm) were counterstained with DAPI after methanol fixation (20 min.) to visualize cell nuclei. Overlays were done with AxioVision software using false colours. Note: Due to fixation endogenous red fluorescence as well as the tissue structure suffered. Sections were made from limb muscle (A), heart (B), kidney (C) and liver (D). E-F: Macrosection of a limb bone seen in normal light (E) or with red fluorescence filter set (F). Scale bars equal 10 μm (A-D) or 1 mm (E-F).
	Figure 3. Ectopic tail formation after blastema transplantation. 24 hours old blastemas obtained after tail amputation of stage 50 tadpoles of the tom3 strain were transplanted into wild type hosts of the same stage. After 21 days tadpoles were investigated. A-C: Tail like outgrowth in the head region: B: close-up C: red fluorescence filter set. D-F: Tail like outgrowth in the trunk: E: close-up F: red fluorescence filter set. Scale bars equal 1 mm expect in B 100 μm. The arrows mark the tail-like outgrowth.
	Figure 4. Tissue identity of blastema derived ectopic tails. 21 days old ectopic tails or control regenerates (C,F,I) were isolated and immunostained with specific antibodies directed against muscle cells (12/101)(A-C), neuronal cells (Xen-1)(D-F) or notochord (anti-Coll-2)(G-I). Scale bars equal 100 μm in A, D, G or 1 mm in C, F, I. Note, that the red fluorescence reflects the Cy3 labelled secondary antibody exclusively, as the red fluorescence of the tdTomato protein is destroyed by the fixation used for immunostaining.
	Figure 5. Contribution of the host animal to blastema derived ectopic tails. 24 hours old blastemas obtained after tail amputation of stage 50 wild type tadpoles were transplanted into hosts of the tom3 strain. After 21 days tadpoles were investigated. A-B: Tail like outgrowth in the trunk shown in the bright field (A) or with red fluorescence filter set (B). C-D: Isolated ectopic tail (upper structure) together with wild type tail shown in normal light (C) or with red fluorescence filter set (D). Scale bars equal 1 mm. The arrows mark the tail-like outgrowth.

References [+] :

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed, Xenbase