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First published online 13 August 2008
doi: 10.1242/dev.024588
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Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Author for correspondence (e-mail:
k.poss{at}cellbio.duke.edu)
Accepted 16 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Blastema, Fgf, Fin, Homeostasis, Regeneration, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Poss et al., 2003;
Stoick-Cooper et al., 2007a).
First, the amputation injury is healed through migration of epidermal cells,
covering the wound within an hour of trauma. Formation of the regeneration
blastema, a mass of undifferentiated, proliferative mesenchymal cells, is the
second and defining stage of regeneration. Here, presumptive blastemal cells
are stimulated to disorganize, migrate distally and accumulate within 1-2 days
of injury. It is currently unclear whether the blastema consists of a
homogeneous population of multipotent progenitor cells, or heterogeneous
subpopulations. During the final stage called regenerative outgrowth, a
repeated series of proliferation, patterning and differentiation events in
blastemal tissue leads to deposition of new bone-secreting scleroblasts and
distal addition of all lost bone segments within 
2 weeks.
Multiple studies in recent years have identified key molecular regulators
of blastemal formation and function. Several different approaches revealed
that signaling by fibroblast growth factors (Fgfs) is crucial for regeneration
(Lee et al., 2005;
Poss et al., 2000;
Tawk et al., 2002;
Thummel et al., 2006;
Whitehead et al., 2005). The
ligand fgf20a, which was found in a genetic screen for
temperature-sensitive regeneration mutants, is required for normal
morphogenesis of the regeneration epidermis and for mesenchymal proliferation
during blastema formation (Whitehead et
al., 2005). During regenerative outgrowth, the Fgf receptor (Fgfr)
fgfr1, as well as Fgf target genes mkp3 (dusp6 -
Zebrafish Information Network), sef (il17rd - Zebrafish
Information Network) and spry4, are expressed in blastemal mesenchyme
and in the surrounding basal epidermal layer. As regeneration proceeds, Fgf
signaling tightly controls the amount of blastemal proliferation and the rate
of growth, resulting in different regenerative rates dependent on the
proximodistal level of amputation (Lee et
al., 2005; Poss et al.,
2000). Other studies have shown that a suite of signaling
molecules, such as sonic hedgehog (Shh), bone morphogenetic proteins,
activin-βA, and canonical and noncanonical Wnts, influences blastemal
proliferation and patterning during regenerative outgrowth
(Jazwinska et al., 2007;
Laforest et al., 1998;
Quint et al., 2002;
Smith et al., 2006;
Stoick-Cooper et al.,
2007b).
Vertebrate organs generally exhibit two forms of regeneration: facultative
and homeostatic. Facultative regeneration describes mechanisms that are
activated by stimuli like amputation or chemical injury: following the initial
trauma, progenitor and/or structural cells near the injury site proliferate to
replace dead or lost tissue. By contrast, homeostatic regeneration refers to
regular replacement of cells lost through apoptosis, daily wear and aging
(Jones and Wagers, 2008).
Interestingly, surveys of regenerative capacity among mammalian organs have
found that the capacity of an organ for facultative regeneration often
correlates positively with its baseline level of cell turnover
(Rando, 2006). For instance,
blood and skin undergo frequent cell loss and replacement through the activity
of self-renewing stem cells, and use similar processes to quickly regenerate
after injury (Blanpain and Fuchs,
2006; Scadden,
2006). Conversely, the mammalian brain and heart possess low
levels of cellular turnover, and, despite evidence for resident stem cells
(Alvarez-Buylla and Lim, 2004;
Beltrami et al., 2003;
Laugwitz et al., 2005), there
is little or no regeneration after major injury. Furthermore, facultative
regenerative capacity in mammalian organs tends to decrease with age, a
phenomenon observed in concert with age-dependent reductions in the frequency
of homeostatic structural cell or progenitor cell proliferation
(Janzen et al., 2006;
Krishnamurthy et al., 2006;
Molofsky et al., 2006). These
correlations indicate that common cellular and molecular mechanisms are
responsible for recurrent cell replacement and injury-induced regeneration in
many tissues. However, neither the vigor nor the mechanisms of homeostatic
regeneration have been examined in complex tissues that regenerate through a
blastema-based mechanism, such as urodele or teleost appendages.
In this study, we tested the idea that the molecular pathways that control blastema formation and function during regeneration in amputated zebrafish fins have additional homeostatic functions in the absence of injury. We found that long-term inhibition of Fgfrs in uninjured zebrafish led to the progressive loss of distal fin structures, revealing homeostatic maintenance of fins by this pathway. Homeostatic regeneration was characterized by low-level expression of several mediators of facultative regeneration, including shh, msxb and mkp3, in areas of cell proliferation and apoptosis. Using a conditional mutant strain, we found that Fgf-dependent homeostatic regeneration is mediated at least in part by the specific ligand Fgf20a. Taken together, our findings reveal robust new requirements for Fgfs in the day-to-day homeostatic preservation of zebrafish appendages, and have implications for why elevated regenerative capacity has been selectively preserved in some vertebrate species.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Poss et al., 2002a;
Shkumatava et al., 2004;
Whitehead et al., 2005).
Heterozygous hsp70:dn-fgfr1 fish and wild-type clutchmates received
daily heat shocks using an electric heater to raise the water temperature from
26°C to 38°C, as described previously
(Lee et al., 2005). Homozygous
mps1 and fgf20a mutants, and wild-type controls, were raised
to adulthood at 25°C before transfer to aquaria with recirculating water
heated to 33°C. Homozygous mps1 mutants were identified by
phenotyping for regenerative defects at 33°C, and then allowed at least 1
month to regenerate at 25°C before use in homeostasis experiments. Some
zebrafish heterozygous for mps1 mutations also showed loss of distal
fin structures in our experiments (data not shown). For fin amputations,
50% of the caudal fin was removed using a razor blade and animals were
allowed to regenerate for 3 days at 33°C or 3-4 days at 25°C.
Fin length measurement and analysis
Fish were anesthetized in 0.1% tricaine and the fins were imaged at various
timepoints. Imaging software (Openlab) was used to measure the length of the
two rays flanking the central-most ray, from the end of the most proximal
visible ray segment to the distal tip of the ray. The two values were then
averaged to obtain a value for each fin. Each value was divided by the average
fin length at day 0 (for the same group) to give a normalized (percentage of
starting length) value of fin length. Central rays were chosen because they
are the most protected from any potential spontaneous injury, although such
injuries rarely occurred in our studies (see Fig. S1 in the supplementary
material), and because the degree of tissue loss often varied between the two
lobes of the fin, confounding statistical analysis. Tests of statistical
significance were performed using Student's t-test, with two-tailed
distribution assuming unequal variance. At least eight fins were assessed at
each timepoint for each group.
Spontaneous injury analysis
Fish were separated into groups of four or five fish and placed in a 1 l
tank so that individual fish were recognizable over the course of the
experiment. Every 2-3 days, fins were imaged on a dissecting microscope, over
a total of 14-24 days. A low-magnification image of the whole fin and a series
of high-magnification images of groups of fin rays were acquired for each
fin.
Scleroblast visualization
To visualize scleroblasts in tissue sections, fins were fixed in
paraformaldehyde (PFA) overnight at 4°C and cryosectioned. Fin sections
were then stained with the monoclonal zns-5 antibody as described
(Johnson and Weston, 1995;
Poss et al., 2002b). For
whole-mount visualization of scleroblasts, the monoclonal zn-3 antibody was
used, which marks the scleroblast cell membrane (A.A.W. and K.D.P.,
unpublished). Fins were fixed in Carnoy's solution overnight at 4°C, and
stained as described (Newmark and Sanchez
Alvarado, 2000; Poss et al.,
2002a).
TUNEL staining
For comparison of cell death in the distal and proximal regions of the fin,
wild-type fins were fixed and cryosectioned, and slides were dried overnight
at room temperature. Slides were then incubated in PBS for 30 minutes at
37°C. DNaseI-treated slides were used as a positive control. Slides were
transferred to 0.3% Triton X-100 (Sigma) in PBS for 10 minutes, and then
covered with 150 µl of 1x TdT buffer (Invitrogen) for 5 minutes. The
buffer was then removed and replaced with 150 µl of 1x TdT buffer
containing 0.3 U/µl of TdT enzyme (Invitrogen) and 8 µM Biotin-14-dCTP
(Invitrogen). Slides were incubated at 37°C for 1 hour, followed by
termination in stop buffer (300 mM NaCl, 30 mM sodium citrate) for 15 minutes
at room temperature. Slides then rinsed three times in PBS and covered with 20
µg/ml Texas Red Streptavidin (Vector Laboratories) in PBS (pH 8.2), and
incubated in the dark at room temperature for 30 minutes. Slides were then
washed four times in PBS and coverslipped using Vectashield with DAPI to stain
nuclei.
Five different sections were imaged at distal and proximal regions of each
fin (n=10 fins). Distal regions represent approximately 350 µm at
the distal end of the fin, and proximal samples represent tissue 700
µm to 1050 µm from the distal end of the fin. This length was chosen
because it is the length of a frame at 20x magnification using our
imaging equipment. Epidermal and mesenchymal regions were carefully outlined
in each image, and the area quantified using Openlab software. Then,
TUNEL-positive nuclei, co-stained with DAPI, were counted by hand within these
regions. The areas and TUNEL-positive cell counts from these 5 sections were
summed, giving each animal four indices: distal epidermal, distal mesenchymal,
proximal epidermal and proximal mesenchymal cell death.
BrdU incorporation
For comparison of proliferation in distal and proximal regions of the fin,
fish were allowed to swim for 24 hours in a 50 µg/ml solution of
bromodeoxyuridine (BrdU) in fish water. After collection, fins were fixed,
cryosectioned and stained as described
(Poss et al., 2002b). For each
fin, five different sections were analyzed for both distal and proximal
samples as described above. A total of eight fins were analyzed for the number
of BrdU-positive cells per area of epidermal or mesenchymal tissue.
BrdU incorporation was also analyzed in wild-type or
hsp70:dn-fgfr1 animals given 14 days heat shock followed by 5 days
recovery at room temperature. For these experiments, a 2.5 mg/ml solution of
BrdU was injected intraperitoneally 2 hours before fin collection. Fins were
fixed in Carnoy's solution overnight at 4°C, and stained as described
(Newmark and Sanchez Alvarado,
2000; Poss et al.,
2002a). A total of 10 wild-type and eight hsp70:dn-fgfr1
fins were analyzed.
In situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Poss et al., 2000), using
digoxigenin-labeled probes for mkp3, msxb, fgf20a and mps1
(ttk - Zebrafish Information Network)
(Akimenko et al., 1995;
Lee et al., 2005;
Poss et al., 2002a;
Whitehead et al., 2005).
Wild-type and transgenic fins were hybridized and developed simultaneously.
Section in situ experiments were performed as described, using mkp3
and msxb probes (Poss et al.,
2002b). The msxb probe was developed overnight, as is
standard in our laboratory (16 hours); for mkp3, the exposure
time was shortened to 4 hours to limit nonspecific staining.
RT-PCR
RNA samples were prepared from uninjured fins and regenerating fins using
TRI Reagent (Sigma) according to the manufacturer's protocol. Total RNA (5
µg) was used for reverse transcription reactions using Superscript III and
Oligo-dT20 (Invitrogen) according to the manufacturer's protocol.
PCR reactions, consisting of 2 minutes at 94°C, 26-30 cycles of 94°C
for 20 seconds, 52°C for 20 seconds, 72°C for 45 seconds and a final
72°C extension for 5 minutes were performed in an Eppendorf Mastercycler,
and samples were run on 2% agarose gels.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yin et al., 2008). Adult
transgenic hsp70:dn-fgfr1 fish and wild-type clutchmates were given a
strong, daily heatshock regimen for 30 or 60 days, after which animals were
imaged to assess caudal fin length and morphology. Surprisingly, these
conditions caused rapid, easily visible loss of distal fin tissue in
transgenic animals (Fig. 1A).
Transgenic fins often terminated with damaged bone and an excess of epidermal
tissue, suggesting recent injury and epidermal repair
(Fig. 1B). Wild-type
clutchmates occasionally showed similar morphology (without tissue loss), but
only in isolated rays rather than the entirety of the fin (data not shown).
The degree of damage was variable in hsp70:dn-fgfr1 outer rays but
generally consistent within the inner rays; therefore, we quantified the
lengths of centrally located rays and found reductions of fin length of
10% after 30 days, and 17% after 60 days. Under identical
conditions, wild-type clutchmates maintained their fin length
(Fig. 1C). Isolated
hsp70:dn-fgfr1 animals, removed from possible aggressive interactions
with other fish, also displayed progressive loss of fin structures (data not
shown). Furthermore, similar tissue loss also occurred in dorsal, anal, pelvic
and pectoral fins, indicating that Fgf signaling is required for homeostasis
in all fin types (Fig. 1D; data
not shown). If Fgf signaling was restored to these animals for 30 days
following long-term Fgfr inhibition, the majority of fin rays were able to
recover lost structures (see Fig. S1 in the supplementary material).
|
Interestingly, upon examining fins at high magnification after 30 days of Fgf inhibition, we found that over half of the transgenic fins exhibited swelling, discoloration and/or separation or slipping of rays at the intersegmental joints (16/30 fins, Fig. 2A). Joint swelling or segment separation was only occasionally observed in wild-type clutchmates (2/30 fins), and never with the degree of severity exhibited by transgenics. This suggested that pathology or weakness at segment joints contributes to major tissue loss in hsp70:dn-fgfr1 fins. We examined the joints more closely by confocal microscopy and by histology to identify disturbances in cellular organization. The zebrafish fin lepidotrichia consist of two bony, facing hemirays, which are initially formed by mineralization of bone matrix secreted by the flattened scleroblasts that encase them. At the intersegmental joints between lepidotrichial segments, there exist clusters of scleroblasts with a different rounded morphology. In projections of confocal slices and in tissue sections, these rounded scleroblasts appear as a small bulge in the intraray mesenchyme around the joints (Fig. 2B,C). Scleroblasts in regenerating fins also have a similar rounded morphology (Fig. 2C). In confocal projections and sections through hsp70:dn-fgfr1 fin rays, the bulge at the intersegmental joints was often enlarged, with large numbers of rounded scleroblasts protruding deep into the surrounding mesenchyme (Fig. 2B,C). We observed this hypertrophy in areas of severe joint dysmorphology (Fig. 2C, bottom left), as well as in transgenic rays that lacked major anatomical changes (Fig. 2B, middle and bottom right). Our observations indicate that one way in which Fgf signaling maintains fin integrity is through control of scleroblast activity and/or patterning, particularly at intersegmental joints.
Homeostasis mechanisms include distally focused areas of cell turnover and developmental signaling
The fin atrophy exposed by Fgfr blockade indicated vigorous homeostatic
regeneration; thus, we searched for regions of cell turnover and activated
developmental programs in uninjured fins. Previous studies have described cell
proliferation in the uninjured zebrafish caudal fin, reporting qualitatively
higher levels of proliferation at the distal fin structures and lower levels
in more proximal regions. This graded proliferation is purported to reflect
growth; specifically, coordinated bursts or saltations of proliferation that
periodically add new ray segments as part of adult indeterminate growth
(Goldsmith et al., 2003;
Iovine and Johnson, 2000;
Nechiporuk and Keating, 2002).
We quantified cell proliferation in distal and medial regions of uninjured
fins using assays for BrdU incorporation
(Fig. 3A). Proliferation was
low compared with that seen during regeneration of amputated structures.
Nevertheless, our data confirmed significantly higher BrdU incorporation
indices in distal structures. Epidermal BrdU incorporation was 43% higher
in the most distal 350 µm of the fin versus an identical length of medial
structures, whereas BrdU incorporation in mesenchymal tissue, which includes
connective tissue cells and scleroblasts, was 197% higher in distal
structures (Fig. 3B,D).
Although proliferation was highest at the distal ray tip, proliferating cells
within both the epidermal and mesenchymal compartments were not restricted to
a specific cell type or a concentrated area as prominent as the regeneration
blastema.
|
Next, we searched for evidence of developmental programs associated with
these turnover events, paying particular attention to programs known to
regulate regeneration of amputated fins. We first took advantage of an
transgenic EGFP reporter strain that marks expression of shh, a
reported blastemal mitogen and patterning factor synthesized in regeneration
epidermis adjacent to the blastema
(Laforest et al., 1998;
Quint et al., 2002;
Shkumatava et al., 2004). We
found that this strain visualizes shh expression during regeneration
in a manner consistent with published in situ hybridization results. Moreover,
shh was consistently detectable in similar epidermal expression
domains in the distal-most 100 µm of uninjured fins
(Fig. 3E). We similarly
examined two other markers: (1) msxb marks cells throughout the
regeneration blastema as well as regenerating scleroblasts, and was recently
shown using electroporation of antisense morpholinos to be essential for
regenerative growth (Akimenko et al.,
1995; Thummel et al.,
2006); and (2) mkp3 is an Fgf target gene that is induced
in the blastema as well as cells of the basal epidermal layer encasing the
blastema, with expression levels that correlate with the rate of regenerative
outgrowth (Lee et al., 2005).
Semi-quantitative RT-PCR demonstrated that both of these markers are expressed
in the uninjured fin, albeit at much lower levels than that seen after
amputation (see Fig. S3 in the supplementary material). By in situ
hybridization of tissue sections, we detected faint expression of both of
these molecules at the distal fin tip. Interestingly, detectable expression
was restricted to domains similar to those occupied by these molecules during
regenerative outgrowth: mkp3 was expressed in cells of the distal
epidermis and mesenchyme, while msxb expression was restricted to
mesenchymal cells, suggesting that these molecules have similar functions in
amputated and uninjured fins (Fig.
3F). Specific localization of these regeneration mediators
suggested homeostatic involvement in the cell turnover and maintenance
functions we had identified.
Do cell proliferation and low-level shh, msxb and Fgf signaling/mkp3 expression in uninjured fins reflect ongoing homeostatic events? To test whether these events rise in the face of a greater homeostatic obligation, we gave transient periods of Fgfr inhibition with the intention that it might `prime' fins for a burst of proliferation and marker expression after Fgf signaling is restored. In these experiments, a 7- or 14-day protocol of daily heatshocks was given to hsp70:dn-fgfr1 and wild-type clutchmates, followed by a 5-day recovery period at room temperature (Fig. 4A). We saw no significant difference in TUNEL-positive cells in transgenic fins versus wild-type fins during the heatshock period, suggesting little effects of Fgfr inhibition on apoptosis, as well as no significant differences in mesenchymal BrdU incorporation (data not shown). The latter observation might reflect a role for Fgfs in proliferation of just a subset of cell types within the uninjured fin. Alternatively, our assay may be limited by variability caused by very low baseline numbers of proliferating cells or the possibility of missing saltatory bursts of proliferation.
By contrast, following the recovery period from heatshock, proliferation was markedly increased in distal fin regions of transgenic, but not wild-type animals, indicative of an enhanced homeostatic response (Fig. 4B). Furthermore, expression of mkp3 and msxb could be easily visualized by whole-mount in situ hybridization in transgenic fins, but not wild-type fins, during the recovery period of restored Fgf signaling (Fig. 4C). These changes would not be expected if marker expression and cell proliferation were a purely ontogenetic or saltatory growth signature of adult fins. Together, these data reveal molecular and cellular indicators of a responsive program of homeostatic regeneration in uninjured zebrafish fins.
|
). To
address this possibility and also refine our analysis of Fgf signaling during
homeostasis, we focused on fgf20a, an Fgf ligand with functions
believed to be specific for regeneration of amputated adult fins. Incubation
of developing fgf20a embryos at the restrictive temperature of
33°C has little effect on ontogenetic development, whereas incubation at
this temperature arrests adult fin regeneration during blastema formation
(Whitehead et al., 2005).
Semi-quantitative RT-PCR revealed that fgf20a is expressed at low
levels in the uninjured fin (see Fig. S2 in the supplementary material), and
in situ hybridization of fins from primed hsp70:dn-fgfr1 animals
indicated that fgf20a is a member of a homeostatic response program
that includes msxb and mkp3
(Fig. 5A). These expression
data suggested that fgf20a might mediate Fgf-dependent homeostatic
regeneration.
To test whether fgf20a is essential for appendage homeostasis, we
placed adult fgf20a mutants at 33°C for 30-60 days and assessed
fin size and integrity. Whereas wild-type controls maintained fin length at
33°C in these experiments, fgf20a zebrafish displayed a
progressive loss of distal fin structures that was quantitatively very similar
to results observed with hsp70:dn-fgfr1 animals
(Fig. 5C). Quantification of
central ray length revealed that fgf20a mutants lost 19% of
their fin tissue after 30 days at the restrictive temperature, and roughly
one-third of their fin length (35%) after 60 days
(Fig. 5D). Unlike
hsp70:dn-fgfr1 zebrafish, joint morphology appeared normal by gross
visual inspection, and tissue loss at outer fin rays was less severe. This may
be related to different strengths of genetic interventions, different
conditions for removing Fgf functions, genetic redundancy and/or functions
specific to Fgf20a apart from other Fgf ligands. In any case, our data
indicate that signaling by Fgf20a is essential to maintain zebrafish
appendages, accounting for at least part of the Fgf dependency of homeostatic
regeneration.
|
).
mps1 expression is induced in the newly formed blastema and
colocalizes with highly proliferative blastemal cells during regenerative
outgrowth. Like fgf20a, mps1 was shown to be essential for
regeneration by positional cloning of a temperature-sensitive mutation
(Poss et al., 2002a). RT-PCR
assays indicated low-level mps1 expression in the uninjured fin (see
Fig. S2 in the supplementary material), and, like fgf20a, mps1
expression was induced by a 14-day block of Fgf signaling and 5 days of
recovery in hsp70:dn-fgfr1 animals
(Fig. 5B). When mps1
mutant zebrafish were placed at the restrictive temperature of 33°C for 30
or 60 days, the animals showed severe distal tissue loss. Measurement of the
central rays revealed a 17% loss of tissue by 30 days at 33°C, and
36% after 60 days (Fig.
5C,D). Thus, Mps1, a kinase essential for proliferation of
blastemal cells after fin amputation, is also necessary to maintain tissue in
intact fins. Taken together, our data suggest that factors that enable
facultative regeneration are generally required for homeostatic regeneration
within uninjured fins. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although facultative and homeostatic regeneration appear to have many
similarities, there are also interesting differences. For example,
fgf20a mutants have a mostly penetrant defect in injury-induced fin
regeneration at 25°C (Whitehead et
al., 2005), despite developing normally to adulthood and
maintaining fins grossly normally at this temperature. This suggests that
there is a requirement for some amount or function of Fgf20a that is met
during homeostatic regeneration but not facultative regeneration. Similarly,
redundancies may be present in one type of regeneration and absent in another.
Other differences may relate to restrictive programs with greater presence in
intact fins. For example, recent work has found that signaling by epidermally
synthesized Wnt5b has an inhibitory role on blastemal proliferation
(Stoick-Cooper et al., 2007b).
Candidates for analogous factors in intact fins include several miRNAs that
were recently shown to have higher levels in uninjured fins than in fins
regenerating after amputation. At least one of these miRNAs, miR-133, also
displayed functional properties of a regenerative brake
(Yin et al., 2008).
Understanding the balance between permissive and restrictive factors in
injured and uninjured appendages is likely to be important in unraveling
seminal issues in regeneration.
Fgfs, homeostatic regeneration and positional memory
One of the most fascinating aspects of appendage regeneration is positional
memory, the ability of the limb or fin stump to recognize and restore only
those structures lost by injury. Positional memory is thought to be based on a
gradient of some determinant(s) existing in uninjured tissue or quickly
established after amputation. Recently, we found that the amount of Fgf
signaling established after amputation is graded along the proximodistal axis,
with higher amounts in more proximal tissue and lower amounts distally
(Lee et al., 2005). Greater
Fgf signaling positively impacts blastemal proliferation and regenerative
rate, leading to more rapid outgrowth in proximal regenerates. Similarly, as
regeneration proceeds gradually to completion, amounts of Fgf signaling
gradually wane.
|
Evolutionary significance of homeostatic regeneration and regenerative capacity
Why is the capacity for appendage regeneration, or organ regeneration in
general, distributed unequally among vertebrate species? The selective
advantages for non-mammalian regenerative events are not uniformly obvious.
For example, the capacity for tail regeneration in lizards facilitates
repeated use of the anti-predatory tactic of autotomy, and also is thought in
some species to reduce potential reproductive, social and locomotor costs
(Clause and Capaldi, 2006). By
contrast, there is no immediate explanation for the capacity of adult
non-mammalian vertebrates to regenerate resected cardiac muscle
(Poss et al., 2002b).
Our current study, supported by previous experiments
(Nechiporuk and Keating,
2002), suggests that one contributing factor behind the high
regenerative capacity of adult zebrafish fins is the particularly dynamic
nature by which they are actively maintained. That is, the preservation of
capacity to regenerate patterned fin structures after major injury might be
the evolutionary consequence of a more crucial role for regenerative
mechanisms to balance regularly day-to-day cell loss and maintain existing
tissue. Accordingly, it is interesting to speculate that teleosts and urodeles
possess a wider array of regenerative tissues than mammals, in part, because
of greater cell turnover among those organs. This might also be related to the
capacity for indeterminate growth in many of these species, although the newt
does not grow throughout its adult life. Supporting this idea, we have found
that the adult zebrafish heart, unlike the more static mammalian heart,
actively adds cardiac cells during adult animal growth and size maintenance
(Wills et al., 2008).
Similarly, adult teleost CNS structures like the highly regenerative retina
and the brain show unusually high basal rates of neurogenesis
(Grandel et al., 2006;
Otteson and Hitchcock,
2003).
Notably, the severe fin regression phenotypes we observed after genetic
manipulations in zebrafish are highly reminiscent of those seen recently after
various gene knockdown experiments in the classic invertebrate model system
for blastema-based regeneration: freshwater planarians. Rapid facultative and
homeostatic regeneration in planarians are based on stem cell-like neoblasts,
the sole proliferative cell type responsible for renewal of all structural
cells (Birnbaum and Sanchez Alvarado,
2008). In those studies, long-term RNA interference of individual
genes frequently had similar effects on facultative and homeostatic
regeneration; that is, regeneration could be blocked after animal bisection,
while fatal `curling' or progressive changes in adult pattern occurred within
weeks of gene perturbation in intact animals
(Cebria et al., 2007;
Cebria et al., 2002;
Cebria and Newmark, 2005;
Reddien et al., 2007;
Reddien et al., 2005). It will
be interesting to compare among different vertebrate species and organs the
extent to which the capacity for injury-induced regeneration correlates with
the activity of ongoing homeostatic regeneration maintained by Fgf ligands and
other signaling pathways.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3063/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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