XB-ART-54607Wound Repair Regen January 1, 2018; 26 (1): 46-53.
The ontogenetic decline of regeneration capacity in the anuran amphibian Xenopus makes it an excellent model for regeneration studies. However, the cause of the regeneration ability decline is not fully understood. MicroRNAs regulate animal development and have been indicated in various regeneration situations. However, little is known about the role of microRNAs during limb regeneration in Xenopus. This study investigates the effect of Dicer, an enzyme responsible for microRNA maturation, on limb development and regeneration in Xenopus. Dicer is expressed in the developing Xenopus limbs and is up-regulated after limb amputation during both regeneration-competent and regeneration-deficient stages of tadpole development. Inactivation of Dicer in early (NF stage 53) tadpole limb buds leads to shorter tibulare/fibulare formation but does not affect limb regeneration. However, in late-stage, regeneration-deficient tadpole limbs (NF stage 57), Dicer inactivation restores the regeneration blastema and stimulates limb regeneration. Thus, our results demonstrated that Xenopus limb regeneration can be stimulated by the inactivation of Dicer in nonregenerating tadpoles, indicating that microRNAs present in late-stage tadpole limbs may be involved in the ontogenetic decline of limb regeneration in Xenopus.
PubMed ID: 29453851
Article link: Wound Repair Regen
Species referenced: Xenopus laevis
Genes referenced: actb dicer1 eef1a1 eef1a2 pcna
GO keywords: tissue regeneration
Antibodies: Dicer1 Ab1 actb Ab5
Morpholinos: dicer1 MO1
Article Images: [+] show captions
|Figure 1. Expression of Dicer in Xenopus tadpole limbs. (A, B) Dicer transcripts detected by RT-PCR and real-time PCR. Relative expression of Dicer was normalized to Ef1a and compared to stage 53 unamputated limb (St53C). RT–: no reverse transcriptase control; St33: stage 33 tailbud stage embryo; St53R: stage 53 limb regenerate, 4 days postamputation (dpa); St57C: stage 57 unamputated hindlimb, distal part; St57R, stage 57 limb stump, 8 dpa. Result in (B) was from two independent experiments. (C, D) Dicer transcript detection by in situ hybridization in the limb stumps of stage 53, 4 dpa (C, lateral view), and stage 57, 8 dpa (D, ventral view) tadpoles. In situ signals are in purple blue. Black dots in (D) are melanocytes. (E–G) Immunofluorescence analysis of Dicer expression in parasagittal sections of unoperated stage 53 limb (E) and regenerating limb, 4 dpa (F). (G) Enlarged view of Dicer expression in the regenerating blastemal. Scale bars: 0.5 mm in (C, D); 0.1 mm in (E–G). dpa: days postamputation. White arrowheads in (C, D, F) indicate amputation planes.|
|Figure 2. Dicer-Mo delivery method and validation of its effect in Xenopus tadpole limbs. (A, B) Dicer-Mo (10 nL of 125 lM) injection in dorsal animal pole of 4–8 cell stage embryos results in defect in anterior embryonic development, such as eye deformation (white arrow) in the injected side (Dicer-Mo). (A) The side view of the injected side of stage 33 tailbud embryo. (B) The side view of the uninjected side of the same tailbud embryo. Inset indicates the localization of Dicer-Mo. (C–F) Electroporation is efficient in delivering both DNA plasmid and morpholinos into tadpole limbs. Lateral view of a stage 53 tadpole limb showing GFP expression from pmaxGFP plasmid in green (C) and Dicer-Mo in red (D). (C,D) Views of the same tadpole limb. White line indicates the level of the cross sections shown in (E, F). (E, F) Distribution of Dicer-Mo and GFP in tadpole limb tissues. Nuclei counterstained with DAPI are shown in blue. (G) Detection of Dicer by Western blotting in tadpole limb regenerates (3 dpa), after injection/electroporation with control or Dicer-Mo and Dicer- Mo1Dicer DNA. (H) Real-time PCR analysis of mature miR-196a and miR-23a in tadpole limb regenerates (3 dpa) after Dicer-Mo injection/ electroporation. Data in (H) were from two independent experiments; error bars are standard deviations. Scale bars: 1 mm in (A–D) and 0.1 mm in (E, F).|
|Figure 3. Dicer inactivation in stage 53 tadpole limbs leads to limb development defects. (A) A tadpole with Dicer-Mo (50 nL of 125 lM) injection/electroporation in the left limb at stage 53 developed shortened limb. (B) Enlarged view of the Dicer-Mo injected limb, 20 days after electroporation. Red fluorescence shows the distribution of Dicer-Mo. Skeletal staining of Dicer-Mo injected (C) and control (D) limbs, 45 days after Dicer-Mo injection/electroporation. I–V indicate digit identities. Scale bars: 1 mm. [Color figure can be viewed at wileyonlinelibrary.com]|
|Figure 4. Dicer inactivation does not affect limb regeneration in earlystage tadpoles. A tadpole with Dicer- Mo injection in one side of its hindlimb, 1 day postamputation (A), and 16 days postamputation (B, C), with hindlimb regenerated similar to uninjected side (D). Dicer-Mo was present in the regenerated limb (C). Scale bars: 0.5 mm. dpa: day postamputation. White arrowheads indicate amputation planes. [Color figure can be viewed at wileyonlinelibrary. com]|
|Figure 5. Xenopus tadpole hindlimb regeneration is stimulated by Dicer inactivation. Hind limbs of a tadpole receiving control-Mo (A) or Dicer-Mo (B) injection/electroporation at stage 57, 17 days postamputation. Inset in (B) indicates distribution of Dicer-Mo. Detection of proliferating cells (by PCNA, shown in green) limbs with control (C) and Dicer-Mo (D) injection/ electroporation at stage 57, 6 dpa. A tadpole receiving control (right) and Dicer-Mo (left) injection/ electroporation at stage 57, 25 dpa, before (E) and after (F) skeletal preparation. Scale bars in (A, B, E, F): 0.5 mm; scale bar in (C, D): 0.2 mm. White arrowheads indicate amputation planes. dpa, days postamputation. [Color figure can be viewed at wileyonlinelibrary.com]|