JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 312B (2009)
Fin Regeneration From Tail Segment With
Musculature, Endoskeleton, and Scales
JINHUI SHAO, XIAOJING QIAN, CHENGXIA ZHANG, AND ZENGLU XU!
Department of Anatomy, Histology and Embryology, Institute of Basic Medical
Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking
Union Medical College, Beijing, P. R. China
ABSTRACT
.
It is well known that fish caudal fins can be completely regenerated after fin
amputation. Although much research on fin regeneration has been carried out, there have been very
few reports regarding fin regeneration after tail amputation. In this study, we used grass carp,
common carp, koi carp, and zebrafish as experimental organisms. Some caudal fins could be distinctly
regenerated in 2 weeks after tail amputation. After all-trans-retinoic acid treatment and tail
amputation, zebrafish were unable to regenerate caudal fins that could be seen with the naked eye.
However, after tail amputation, more than half of the zebrafish tested were able to regenerate caudal
fins. Caudal fin regeneration depended on the presence of musculature and endoskeleton at the site
of amputation. These caudal fins arose from segments of the endoskeleton, which contrast with
r 2009 Wiley-Liss, Inc.
currently accepted knowledge. J. Exp. Zool. (Mol. Dev. Evol.) 312B, 2009.
How to cite this article: Shao J, Qian X, Zhang C, Xu Z. 2009. Fin regeneration from tail
segment with musculature, endoskeleton, and scales. J. Exp. Zool. (Mol. Dev. Evol.)
312B:[page range].
All organisms mount biological responses to
damage, but they vary widely in the degree to
which they can recover from damage. These types
of homeostatic renewals are thought to be
mediated by resident stem cells of specific lineages.
In this context, teleost fish are of particular
interest, as they have a remarkable capacity to
regenerate damaged organs, including heart,
spinal cord, retina, and limbs/fins (Akimenko
et al., 2003; Poss et al., 2003; Stoick-Cooper et al.,
2007). Regeneration of organs such as amphibian
limbs and fish fins are dramatic examples, where
intricate structures consisting of multiple cell
types are patterned into complex tissues and
faithfully restored after amputation. Repair of
many organs, such as the chicken retina or mouse
liver, is thought to be mediated through activation
of resident stem cells or through proliferation of
normally quiescent differentiated cells, respectively (Fischer and Reh, 2001; Fausto et al., 2006).
Amphibian and fish appendages regenerate
through a process termed ‘‘epimorphic regeneration,’’ sometimes referred to as ‘‘true’’ regeneration (Akimenko et al., 2003; Poss et al., 2003). The
relative simplicity and the easy access of the
caudal fin and its skeletal elements make it an
excellent system for dissecting the molecular
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pathways involved in bone regeneration. While
resection of the fin at the level of the endoskeleton
is not followed by regeneration, amputation of the
dermal fin rays results in complete replacement of
the lost structures within approximately 3 weeks
(Akimenko et al., 2003; Smith et al., 2006).
Fin regeneration occurs in three steps: (1) wound
healing and formation of the wound epidermis; (2)
formation of a regeneration blastema, a population
of mesenchymal progenitor cells that are necessary
for proliferation and patterning of the regenerating limb/fin; and (3) regenerative outgrowth and
pattern reformation (Akimenko et al., 2003; Poss
et al., 2003; Stoick-Cooper et al., 2007). Many
functional genes in fin regeneration have been
researched, including a sonic hedgehog homolog
(shh), a fibroblast growth factor (fgf), and a bone
morphogenic protein (bmp) (Laforest et al., ’98;
Poss et al., 2000; Smith et al., 2006). Heat shock
!Correspondence to: Zenglu Xu, Department of Anatomy, Histology
and Embryology, Institute of Basic Medical Sciences, Chinese Academy
of Medical Sciences, School of Basic Medicine, Peking Union Medical
College, Beijing 100005, P. R. China. E-mail: [email protected]
Received 15 December 2008; Revised 25 March 2009; Accepted 1
April 2009
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/jez.b.21295
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J . SHAO ET AL.
protein 60 (hsp60) expression is increased during
formation of blastema cells, and its dysfunction
leads to mitochondrial defects and apoptosis in
these cells. Experimental data indicate that the nbl
mutation causes dilated mitochondria and apoptosis selectively in the distal blastema cells (Makino
et al., 2005).
When amputations are performed distally to the
branch points or dichotomies, where a single ray
bifurcates to extend two individual ‘‘daughter’’
rays, all-trans-retinoic acid (RA) treatment causes
a dichotomy reduction, such that the two ‘‘daughter’’ rays fuse to once again form a single ray. It is
thought that exogenous RA respecifies the pattern
in the regenerating caudal fin and the blastema
has been identified as a possible RA target tissue
(White et al., ’94). The regenerating caudal fin is
sensitive to RA treatment and shows clear
teratogenic responses on the dorso-ventral axis
under many of the experimental conditions investigated, both in wild type and long-fin mutants
(Géraudie et al., ’95). Eve1 and evx2 products are
part of the molecular signals involved in pattern
formation of the fin and fin rays in connection
with outgrowth. Therefore, RA might alter growth
and morphogenesis of the regenerating fins by a
fine regulation of these genes, among others
(Brulfert et al., ’98).
Fin regeneration from anchovy tail segments
with musculature and scales was reported in
Freshwater Fisheries (a Chinese journal) in 1980
by Chuanmi Yuan et al. They found that some
anchovies caught in the Tai lake could regenerate
irregular caudal fins after part of their tail
was bitten out by other animals in the water.
Anchovies can seldom be caught alive, because
they struggle excessively when they are in fishing
nets; consequently, it is difficult to use anchovies
as experimental animals. Four other kinds of fish,
including grass carp, common carp, koi carp, and
zebrafish (Danio rerio), were used in this study to
research further into fin regeneration from fish
tail segments with musculature and scales.
MATERIALS AND METHODS
Maintenance of animals
Grass carp, common carp, koi carp, and zebrafish were all purchased at a local pet store. Grass
carp and common carp were approximately
3.5–5 cm in length, koi carp about 5–6 cm, and
zebrafish about 2.5–4 cm. The daily photoperiod
was 14 hr of light and 10 hr of darkness, and the
J. Exp. Zool. (Mol. Dev. Evol.)
temperature was kept in the range of 24–291C.
Animals were fed once a day with a commercial
food (White et al., ’94; Santos-Ruiz et al., 2002).
Tail and fin amputation
Fish used for experiments were anesthetized by
immersion in water containing 0.2 mg/mL tricaine
(3-aminobenzoic acid ethyl ester). Tail amputation
was carried out at the proximal 1–2 mm of the far
margins of the tail that had musculature, endoskeleton, and scales. Proximal caudal fin amputations were done at the distal 1–2 mm of the far
margin of the tail that had musculature, endoskeleton, and scales. Distal caudal fin amputations
were carried out in the region just before the first
bifurcation of the fin rays. The fish were then
returned to their tanks (Akimenko et al., ’95;
Padhi et al., 2004).
Histologic processing for light microscopy
Fins were fixed for 12 hr in Bouin’s fluid,
decalcified for 3 days in 10% ethylenediamine
tetraacetic acid, embedded in paraffin, and cut into
6-mm-thick sections. These were mounted on sylanized glass slides, deparaffinized, and rehydrated
for use in immunohistochemical staining. Stained
sections were observed under a microscope (Microphot FXA, Nikon, Melville, NY), and photographed
with Nomarski optics (Santos-Ruiz et al., 2002).
Sections (thickness, 6 mm) were cut from paraffin
blocks and collected on poly-L-lysine-coated slides
(Sigma, St. Louis, MO). Sections were dewaxed in
xylene, hydrated in a descending alcohol series, and
stained by a routine hematoxylin–eosin(H–E) staining technique, as described previously (Shidham
et al., 2001; Tertemiz et al., 2005).
Alcian blue and Alizarin staining
Portions of tail and caudal fins were fixed in 4%
PFA and washed in phosphate buffer solution
(PBS), then stained for 6 hr at room temperature
with a filtered 0.1% Alcian blue (in 30% acetic acid,
70% ethanol), and dehydrated in a graded water:
ethanol series. The tissues were then stored in
100% ethanol overnight at 41C to fix the Alcian
blue in the fin rays and to de-stain the surrounding soft tissues. Fins were rehydrated in a water–
ethanol series, macerated in a fresh solution of
0.5% trypsin in 2% sodium borate for 10 min at
371C, and extensively rinsed with frequent water
changes. Bones were stained by placing the fins in
a fresh solution of 0.1% Alizarin red S in 0.5%
KOH for 4–5 hr at room temperature. Fins were
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FIN REGENERATION AFTER TAIL AMPUTATION
cleared by transfer to glycerol through a graded
series of 0.5% KOH–glycerol solutions and kept in
100% glycerol at 41C (Ferretti and Géraudie, ’95;
Laforest et al., ’98).
Bromodeoxyuridine incorporation
For studies involving bromodeoxyuridine
(BrdU), a stock concentration of 50 mg/mL BrdU
was prepared in sterile Hanks solution. The fish
were anesthetized with tricaine and injected in the
abdominal cavity with 250 mg/g weight BrdU. Six
hours postinjection, the fish were euthanized and
their caudal fins or part of the tail with musculature were amputated and fixed in Bouin’s fluid.
Slides of tissue sections were prepared and treated
with primary mouse anti-BrdU antibody (Sigma)
at a dilution of 1:500 and secondary goat antimouse antibody (ZSGB-BIO) at a dilution of 1:500.
3,30 -Diaminobenzidine (DAB) was used to visualize and photograph BrdU incorporation (Poleo
et al., 2001; Zodrow and Tanguay, 2003).
Terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate
nick end-labeling in situ apoptosis
detection assay
Apoptosis in fish caudal fins on tissue slides was
detected by enzymatic labeling of DNA strand
breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL). TUNEL was carried out using
a cell death detection kit (Roche, Mannheim,
Germany) according to the manufacturer’s instructions. Briefly, tissue sections were dewaxed in
xylene and rehydrated in a descending alcohol
series (at room temperature). Samples were
washed in PBS for 5 min, then pretreated with
3% hydrogen peroxide for inactivation of endogenous peroxidase for 10 min and 20 mg/mL proteinase
K for 30 min. The labeling reaction was performed
by adding 50 mL TUNEL reagent to each sample.
A negative control was prepared with the reagent
without enzyme. Incubation was carried out for
1 hr at 371C, and then the slides were washed with
PBS and incubated again with converter reagent
for 30 min at 371C. After washing, color development for localization of cells containing labeled
DNA strand breaks was performed by incubating
the slides with DAB substrate solution for 30 sec.
Slides were lightly counterstained with hematoxylin before permanent mounting (Selam et al.,
2002; Sasso-Cerri et al., 2006).
RA treatment
A 5 mg/mL stock solution of RA (Sigma Aldrich,
St. Louis, MO) was prepared in dimethyl sulfoxide.
An appropriate amount of RA at a final concentration of 10!6 M was added directly into the tank
water after amputation for 4 continuous days, and
the water and RA were refreshed every day. Both
control and RA-treated fish were kept in the
dark during these experiments (White et al., ’94;
Ferretti and Géraudie, ’95).
RESULTS
Interesting phenomena in caudal fin
regeneration
The caudal fins of experimental fish are shown
in Figure 1. Amputation levels are indicated by the
black horizontal lines (Fig. 1A–D). Compared with
the normal caudal fin of a grass carp, the
regenerated caudal fin rays do not branch as
normal caudal fins do, when distal caudal fin
amputations were carried out just before the first
bifurcation of the fin ray (Fig. 1E–F0 ). Regenerated
koi carp caudal fins bifurcate earlier than common
carp after caudal fin amputation at distal 1–2 mm
to the rear margin of the tail with musculature
(Fig. 1G–J). It is interesting that sometimes red common carp have a fair amount of
melanin pigment in their regenerated caudal fins
(Fig. 1K–M).
Caudal fin regeneration after tail
amputation
There are some differences in caudal fin regeneration when caudal fins or tails are anesthetized
for amputation, which was carried out at the
proximal 1–2 mm of the rear margin of the tail
with musculature, endoskeleton, and scales. The
regeneration rate of the caudal fin after tail
amputation is about 50%, while the rate after
caudal fin amputation is 100%. Only some of the
tail amputations regenerated completely, while all
of the caudal fin amputations completely regenerated. Caudal fins can be seen with the naked eye
about 2–4 weeks after tail amputation and about 3
days after caudal fin amputation. The former
regeneration comes from the segment of the tail
with musculature, endoskeleton, and scales,
whereas the latter regeneration comes from the
caudal fins with fin rays.
Some grass carp whose tails had been cut two
months before regenerated irregularly only part of
J. Exp. Zool. (Mol. Dev. Evol.)
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J . SHAO ET AL.
Fig. 1. Interesting phenomena in caudal fin regeneration. (A–D) The rear margin of normal fish tail with musculature and
distal caudal fin. Black horizontal line indicates the amputation site. Grass carp (A), common carp (B), koi carp (C), and
zebrafish (D). (E–F0 ) Caudal fin regeneration of grass carp. Normal grass carp caudal fin (E) and regenerated caudal fin of grass
carp (F) after distal fin amputation are shown. Magnified parts of E and F are shown in E0 and F0 , respectively. (G–J) Caudal fin
regeneration after proximal fin amputation. Regenerated koi carp caudal fin (G, H) bifurcate earlier than common carp (I, J).
(K–M) Three red common carp have more melanin pigment in their regenerated caudal fins after distal fin amputation.
the caudal fin. Others had not regenerated caudal
fins after five months. The stem length of the fish
is about 8 cm (Fig. 2A–D, part D). No caudal fins
completely regenerated with their original shape
after tail amputation in this experiment. Fin rays
could be seen early with the naked eye in
regenerated caudal fins of grass carp, which were
treated with tail amputation (Fig. 2A–D). Tails of
common carp had been cut about one month
before, and the regenerated caudal fins were about
1–2 mm long and could be seen easily in water but
J. Exp. Zool. (Mol. Dev. Evol.)
not out of water. After 42 days of tail amputation,
distinct fin rays still could not be seen with the
naked eye and the existing fin ray was more
similar to a blood vessel. Some fish had no
regeneration after similar amputations even after
50 days (Fig. 2E–G).
When the common carp tail was cut about three
months previously (Fig. 2H–I0 part H), it regenerated
a complete caudal fin according to its shape after one
month. However, the regenerated caudal fins had no
fin rays that could be seen with the naked eye. When
5
FIN REGENERATION AFTER TAIL AMPUTATION
Fig. 2. Caudal fin regeneration after tail amputation. (A–D) Grass carp regenerated irregular parts of caudal fins (A, B).
Others did not regenerate caudal fins (C, D). (E–G) Common carp. These had been amputated 30 days or 42 days before,
respectively (E, F). Magnified part of F is in F0 . Some show no regeneration (G) 50 days after amputation. (H–I0 ) Common carp.
Complete caudal fin of common carp with tail amputation after one month of regeneration (H). Complete caudal fin of common
carp with a proximal fin amputation (I) after two months of regeneration. H0 and I0 are higher magnifications of H and I,
respectively. (J–L0 ) Regenerated caudal fins of koi carp 28, 31, or 42 days, after tail amputation (J, K, and L, respectively).
A magnified portion of L is shown in L0 . (M–P) Zebrafish. Regenerated caudal fin 28 days and 51 days after tail amputation
(M, N, respectively). Irregularly regenerated caudal fins were also seen (O, P). (Q–T) No regeneration. A cross section of
zebrafish covered by stripe (Q); cross section of common carp with caudal endoskeleton exposed (R); Koi carp with caudal
endoskeleton exposed (S); cross section of a regenerated common carp fin covered by a scale-like substance (T).
the common carp caudal fin was cut distally 1–2 mm
behind the margin of tail with musculature about
two months before, it regenerated a complete caudal
fin according to its shape after one month. These
regenerated caudal fins had fin rays that could be
seen with the naked eye (Fig. 2H–I0 ).
Regenerated caudal fins of koi carp had abundant blood vessels but no distinct fin rays. For this
reason, these may be useful in medical experiments regarding the development of blood vessels.
The probability ratio of caudal fin regeneration
after tail amputation was 100%, and most of these
regenerated caudal fins were complete according
to their shape. The stem length of koi carp and
grass carp used in this experiment was similar,
although the stem length of koi carp was longer
than that of common carp or of zebrafish. Therefore, stem length did not appear to be a critical
factor for fin regeneration after part of the tail was
amputated (Fig. 2J–L0 ).
The probability ratio of zebrafish caudal fin
regeneration after tail amputation was about 50%.
Only part of the regenerated caudal fins was
regular and complete. When the regenerated caudal
fin after tail amputation reached 2 mm, fin rays
could be seen with the naked eye, although blood
vessels could not be seen. Most of the regenerated
zebrafish caudal fins after tail amputation were
seen between 15 and 30 days (Fig. 2M–P).
Common carp or koi carp did not regenerate
caudal fins and their caudal endoskeleton was
exposed after they were partly cut. Because of
muscular contractions around the broken ends of
the tails that had been operated on, the caudal
endoskeleton was exposed and most of these fish
died. In a few cases among common carp, the broken
ends of the tails that had been operated on were
covered with a scale-like substance (Fig. 2Q–T).
Alcian blue and Alizarin staining, H– E
staining, BrdU incorporation, TUNEL
assay, and RA treatment
After Alcian blue and Alizarin staining, tails of
koi carp and zebrafish with regenerated caudal
fins after tail amputations showed dissection
evidence that was comparable to that of normal
koi carp and zebrafish. This clearly indicates that
J. Exp. Zool. (Mol. Dev. Evol.)
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J . SHAO ET AL.
tail amputation greatly differed from caudal fin
amputation (Fig. 3A–E). At a 20 " magnification,
H–E staining of regenerated common carp caudal
fin after tail amputation indicated that the area
between the tail with musculature and the regenerated caudal fin is not smooth. In contrast, H–E
staining of untouched common carp caudal fin at
the same 20 " magnification shows a smooth area
between the tail with musculature and a caudal fin
with fin rays (Fig. 3F and G). We can see that
proliferated cells stained with DAB are located at
the margin of the regenerated caudal fin treated
with BrdU after tail amputation, while no cell
proliferation is seen in the absence of BrdU
(Fig. 3H–K0 ). Apoptotic cells occurred primarily at
the margin of the caudal fin. When magnified 400 " ,
TUNEL test results showed that there were no
DAB-stained cells in the control group (Fig. 3L–O0 ).
When RA was used, caudal fins of zebrafish that
were cut before the first bifurcation were regenerated. Caudal fins regenerated for 10 days without RA showed no deformities (Fig. 3P–Q0 , part P).
On the contrary, caudal fins regenerated for 1
month after RA treatment had distinct deformities (Fig. 3P–Q0 , part Q). This indicates that the
treatment of zebrafish with RA in the experiment
had an effect (Fig. 3P–Q0 ). Zebrafish were treated
with RA and with tail amputation. After 2 months,
only one small caudal fin was regenerated, which
could not easily be seen with the naked eye and
which showed no further growth after another
month (Fig. 3R and S). RA can cause anepithymia
in zebrafish and common carp and was seen the
day after RA treatment. Common carp are
sensitive to RA and all the treated fish died within
10 days of RA treatment, whereas common carp
without RA treatment remained alive and healthy.
No zebrafish died from RA treatment, and all
zebrafish with caudal fin amputations regenerated
deformed fins after RA treatment, although they
also showed anepithymia. The regeneration ratio
of zebrafish after tail amputation was about 50%,
whereas no zebrafish regenerated caudal fins that
could be seen easily with the naked eye after
identical tail amputation and RA treatment.
DISCUSSION
Caudal fin regeneration after fin amputation has
been researched repeatedly, in contrast to experiments on caudal fin regeneration after tail amputation (Akimenko et al., 2003; Poss et al., 2003;
Stoick-Cooper et al., 2007; Thatcher et al., 2008;
Kizil et al., 2009). Fin regeneration after tail
J. Exp. Zool. (Mol. Dev. Evol.)
amputation was demonstrated in four species of
fish, including grass carp, common carp, koi carp,
and zebrafish. Anchovies have also been observed to
have this ability. Normal fish and fish with general
caudal fin amputation have an intact rear margin of
the tail (Fig. 1A–M). It can clearly be seen that fish
with regenerated caudal fins after tail amputation
have no general rear tail margin (Fig. 2A–T). In the
experiment using Alcian blue and Alizarin staining,
tail amputation was shown to be clearly different
from caudal fin amputation (Fig. 3A–E). Fin
regeneration after tail amputation comes from tail
segment with musculature, endoskeleton, and
scales. This is different from currently held views
that resection of the fin at the level of the
endoskeleton is not followed by regeneration
(Akimenko et al., 2003; Smith et al., 2006).
Proliferated cells were found using BrdU and DAB
in the regenerated caudal fins after tail amputation
(Fig. 3H–K0 ). This finding was similar to that
reported for regenerated caudal fins after fin amputation (Poleo et al., 2001; Zodrow and Tanguay,
2003). Cell apoptosis occurs during regeneration of
newt limbs, deer antlers, and mouse liver (Vlaskalin
et al., 2004; Mount et al., 2006; Tannuri et al., 2008).
In caudal fin regeneration after tail amputation, cell
apoptosis was also found by TUNEL (Fig. 3L–O0 ).
RA treatment increased apoptosis in the regenerated fin after fin amputation (Ferretti et al., ’95;
Géraudie and Ferretti et al., ’97).
Activation of resident muscle stem cells occurs in
regenerating salamander limbs found by processes
involving antibodies and labeling. Thus, it is likely
that de-differentiation and stem cell activation both
contribute to formation of the blastema (Morrison
et al., 2006). Although de-differentiation of cells has
not yet been demonstrated in regenerating structures other than amphibian limbs and tails, the
morphology, ontology, and gene expression profile
of the zebrafish blastema in the regenerating
caudal fin suggests that zebrafish fin regeneration
occurs by similar mechanisms (Stoick-Cooper et al.,
2007). Tail tissue that formed after tail amputation
in the region around the cross section had homologous structure to that seen in tetrapods. Therefore, fin regeneration after tail amputation has the
potential to reveal general principles that are
common to regeneration from tissue with endoskeleton in all vertebrate appendages. Other ‘‘fin
regeneration’’ studies performed on actinopterygians have been limited to the lepidotrichia, which
do not have an endoskeleton or musculature.
There are some issues with these experiments
that still require answers: First, the morphology
FIN REGENERATION AFTER TAIL AMPUTATION
7
Fig. 3. Alcian blue and Alizarin staining, H–E staining, BrdU incorporation, TUNEL assay, and RA treatment. (A–E) Alcian
blue and Alizarin staining. Tail of koi carp (A, B) and zebrafish (D) with regenerated caudal fins after tail amputation have
dissection evidence (arrow) compared with normal tails of koi carp (C) and zebrafish (E). (F, G) H–E staining. Regenerated
caudal fin (F) of common carp after tail amputation is different from normal caudal fin (G) of common carp. (H–K0 ) BrdU
incorporation. Common carp tail treated with tail amputation and BrdU (H) is positive, while normal caudal fin of koi carp
treated with BrdU (I) regenerated caudal fin of zebrafish after tail amputation (J) and normal caudal fin of koi carp (K) as
control. Magnified parts of H, I, J, and K are shown in H0 , I0 , J0 , and K0 respectively. (L–O0 ) TUNEL assay. Normal koi carp
caudal fin (L) is the control. Regenerated caudal fin of common carp (M) after tail amputation is positive. Regenerated caudal
fins of common carp after tail amputation (N, O) are also as control. Magnified part of L, M, N, and O are shown in L0 , M0 , N0 , and
O0 respectively. (P–Q0 ) Regenerated caudal fin of zebrafish after distal fin amputation. Some are treated without RA (P), while
others are treated with RA (Q). Magnified parts of P and Q are shown in P0 and Q0 , respectively. (R, S) Zebrafish treated with RA
and tail amputation.
J. Exp. Zool. (Mol. Dev. Evol.)
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J . SHAO ET AL.
and molecular changes of fin regeneration between
the time of tail amputation to the time when the
regenerated fin has reached a length of 2 mm
should be further researched. Second, for reasons
that are not clear, the regenerated caudal fin of
common carp and koi carp, after tail amputation,
regenerate fin rays quite late, and some have no fin
rays even after a complete caudal fin, according to
shape, has been regenerated. Third, some caudal
fins regenerate only very slowly after tail amputation and only parts of them regenerate, while
amputation of the dermal fin rays results in
complete replacement of the lost structures within
approximately 3 weeks (Akimenko et al., 2003;
Smith et al., 2006). This also requires further
investigation. Finally, RA was shown to have a very
powerful inhibitory effect on caudal fin regeneration after tail amputation, again for reasons that
are unclear. Zebrafish with caudal fin amputation
after RA treatment always regenerated deformed
fins. Further experiments are needed to understand these unusual regeneration phenomena.
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