Transgenic analysis of signaling pathways required for Xenopus tadpole spinal cord and muscle regeneration

Abstract

The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue-specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet-on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt-β-catenin pathway. For the Tet-on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt-β-catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt-β-catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail.

Fig. 1

Tail regeneration in HGEM-Dkk1 spinal cord transplanted tadpoles. (A) Example of a stage 51 tadpole with a piece of spinal cord (shown in bracket) replaced. (B–G) Examples of regenerated tails in tadpoles transplanted with Dkk1 spinal cord. (B, E) Whole tail image. (C, D, F, G) H-E staining of regenerated tail, showing some tadpoles have spinal cord regenerated (G), some not (D), even though grafted spinal cord can been seen at the amputation stump (C, F). Scale bars in (A, B, E): 500 µm. Scale bars in (C, D, F, G): 100 µm.

Fig. 2

Tail regeneration in Xenopus tadpoles with HGEM-Dkk1:dsRed spinal cord transplantation. (A, B) Example of a tadpole used as donor in this study. This tadpole is transgenic for both heat shock inducible Dkk1 (HGEM-Dkk1) and dsRed (red fluorescence in (B) driven by human cytomegalovirus (CMV) promoter. Arrow in (A) indicates the green lens of the eye, which marks the integration of the HGEM transgene. (C, D) Example of a tadpole with a piece of spinal cord transplanted (shown in red) 3 days after grafting (C) and immediately after tail amputation though the graft (D). (E–H) Examples of tadpole tail regeneration in HGEM-Dkk1:dsRed spinal cord transplantation, 7 dpa. (E) An example of nonregenerating tail. (F, G) Examples of regenerating tadpole tail with partial regenerating spinal cord, as indicated by dsRed shown in red. (H) An example of fully regenerating spinal cord in a partially regenerating tail. This tadpole was subjected to a second tail amputation and fully regenerated its tail with spinal cord (I). (J) Tail regeneration in a tadpole transplanted with CFP transgenic spinal cord, 7 dpa. White arrowheads in (E–J) indicate amputation levels. dpa: day post amputation. s.c.: spinal cord. Scale bars: 500 µm.

Fig. 3

Doxycycline-inducible transgenic tadpoles. (A, B) Example of transgenic tadpoles with muscle specific expression of rtTA, indicated here by expression of tdTomato shown in red (A), and integration of transgene GfpΔTcf, by expression of GFP after doxycycline treatment (B). (C, D) Example of transgenic tadpoles with neural tissue specific expression of GFP, in the central nervous system (C) and the regenerating tadpole tail (D) after doxycycline treatment. Labeling of axons is apparent in the regenerating tail. (E–H) Cross sections of tadpole tail were collected for visualization of tdTomato (E) to indicate expression of rtTA, and immunoreacted to GFP antibody (F), followed by nuclear staining with DAPI (G). Merged image shows the colocalized expression of tdTomato and GFP (H). s.c.: spinal cord. not.: notochord. Dox.: doxycycline. Scale bars in (B, G): 500 µm. Scale bars in (C–F, H): 100 µm.

Fig. 4

Muscle regeneration in doxycycline-inducible Xfd and GfpΔTcf transgenic tadpole tails. (A, B) Example of a regenerating tails in transgenic tadpoles with muscle specific expression of Xfd, indicated here by expression of GFP shown in green. (A) Example of a tadpole tail fully regenerated but with reduced muscle. (B) Example of a tadpole tail which failed to regenerate after Xfd induction with doxycycline treatment. (C, D) Whole mount immunohistochemistry of regenerating tails with 12/101 to detect muscle tissues. (C) Example of control tadpole tail after staining. (D) Example of immunostaining of a transgenic tadpole tail with muscle specific induction of Xfd. Note the short tail regenerate. White arrows in (A–D) indicate amputation levels. White lines in (C, D) indicated the level of cross sections represented by (E, F). (E, F) Cross sections of immunostained tadpole tail counterstained with Nuclear Fast Red, showing normal and reduced muscle content. (G) Example of regenerating tail in a tadpole with muscle specific expression of ΔTcf, after doxycycline treatment. Expression of tdTomato is shown in red and overlaid to bright-field image of the regenerating tail. White arrows indicated amputation level. (H) Example of cross section through the regenerating tail of a tadpole with muscle specific expression of ΔTcf. Cross sections were obtained after whole mount immunohistochemistry with 12/101 antibody for muscle, and counterstained with Nuclear Fast Red. (I) A cross section from a wild type tadpole tail regenerate. s.c.: spinal cord. not.: notochord. Dox.: doxycycline. Scale bars in (A–D, G): 500 µm. Scale bars in (E, F, H, I): 100 µm.