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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) tadpolelimb 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.
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 tadpolelimb showing GFP expression
from pmaxGFP plasmid in green
(C) and Dicer-Mo in red (D). (C,D)
Views of the same tadpolelimb.
White line indicates the level of the
cross sections shown in (E, F). (E, F)
Distribution of Dicer-Mo and GFP in
tadpolelimb tissues. Nuclei counterstained
with DAPI are shown in blue.
(G) Detection of Dicer by Western
blotting in tadpolelimb 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 tadpolelimb 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 leftlimb 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]
