1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA
2 Department of Cardiology, Children's Hospital, Boston, MA 02115, USA
* Author for correspondence (e-mail: k.poss{at}cellbio.duke.edu)
Accepted 22 September 2005
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SUMMARY |
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Key words: Zebrafish, Fin, Regeneration, Blastema, Fibroblast growth factor
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Introduction |
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One of the most striking features of appendage regeneration is the
recognition and replacement of only those structures removed by amputation.
This phenomenon, often called positional memory, has been studied most in the
regenerating newt or axolotl limb. During limb regeneration, developmental
regulation of regenerative growth rate is a prominent component of positional
memory. For example, when a salamander is given an upper arm amputation on one
limb and a digit level amputation on the other, regeneration of both limbs is
completed in approximately the same time period
(Spallanzani, 1769). Thus, the
greater amount of tissue that is amputated, the faster is the rate of
regeneration. This phenomenon has been observed in many other lower vertebrate
species, including teleosts goldfish, killifish and gourami, and in
invertebrates such as starfish (Morgan,
1906
; Tassava and Goss,
1966
). The evolutionary persistence of position-dependent growth
rate suggests a fundamental role for this regulatory mechanism in the process
of regeneration.
Studies from the past several decades have attempted to identify
morphological factors that distinguish proximal regenerates (the more proximal
amputation level) from distal regenerates (the more distal amputation level).
For example, although proximal regenerates with high growth rate usually have
greater stump dimensions after amputation, Tassava and Goss
(Tassava and Goss, 1966) found
that stump diameter showed no consistent correlation with rates of lizard tail
regeneration. Furthermore, young salamanders with smaller limbs can regenerate
considerably faster than older animals with large limbs
(Goodwin, 1946
). In other
studies, Maden (Maden, 1976
)
found no differences in volume or proliferation characteristics between the
proximal and distal axolotl limb blastema, the so-called mass of
undifferentiated mesenchymal tissue that ultimately gives rise to new
structures. Similarly, Iten and Bryant (Iten and Bryant, 1973) did not detect
growth rate differences in initial formation of the salamander limb blastema,
but instead saw the greatest difference in growth rates during later
morphogenesis and differentiation phases. Although morphological studies have
pointed out useful correlations between anatomy and growth rate, there has
been very little molecular definition of the underlying regulation responsible
for position-specific regenerative properties.
Over the past several years, the zebrafish, which regenerates fins
(Johnson and Weston, 1995),
spinal cord tissue (Becker et al.,
2004
) and heart muscle (Poss
et al., 2002a
; Raya et al.,
2003
), has gained popularity as a model for teleost appendage
regeneration. Indeed, molecular genetic analysis in zebrafish has a unique
potential to facilitate dissection of classic developmental problems such as
positional memory (Grunwald and Eisen,
2002
). Zebrafish fins are relatively simple, nearly symmetric
structures composed of several segmented fin rays of intramembranous bone.
Each fin ray comprises concave, facing hemirays that surround connective
tissue, including fibroblasts and scleroblasts (osteoblasts), and nerves and
blood vessels. The process of fin regeneration involves continual, coordinated
proliferation and differentiation events. During regenerative growth, new
segments are progressively added to the distal end of each ray until the
original length of the fin is achieved, usually in about 2 weeks
(Akimenko et al., 2003
;
Poss et al., 2003
).
During zebrafish fin regeneration, as in other examples of appendage
regeneration, the blastema is the engine for regenerative growth
(Tsonis, 1996). Both classic
and recent studies have indicated that a signal(s) released by the overlying
regeneration epidermis controls or contributes to proliferation of the
blastema. Previously, we and others found evidence that signaling by
fibroblast growth factors (Fgfs) regulates blastemal proliferation during fin
regeneration (Poss et al.,
2000
; Tawk et al.,
2002
). The Fgf receptor (Fgfr) subtype fgfr1 is expressed
in pre-blastemal mesenchymal cells during blastema formation, and maintained
in subpopulations of blastemal and epidermal cells during outgrowth.
fgf24 (originally called wfgf)
(Draper et al., 2003
), is
expressed in the wound epidermis, indicating the presence at least one Fgf
during regeneration. In addition, treatment with a pharmacological inhibitor
of Fgfrs, SU5402, blocked blastemal proliferation when applied at any stage of
regeneration (Poss et al.,
2000
). Thus, Fgf signaling is a prime candidate for influencing
regenerative growth rate in a position-dependent manner.
Here, we show that regenerative growth rate, blastemal proliferation and blastemal length are each highly dependent on the level at which the zebrafish fin is amputated, with greater proximal values than distal. Furthermore, proximal regenerates show higher expression than distal of the Fgf target genes mkp3 (dusp6 Zebrafish Information Network), sef (il17rd Zebrafish Information Network) and spry4. By way of a new transgenic strain that facilitates specific, inducible blockade of signaling through Fgfrs, we generate an artificial gradient of Fgf signaling that is capable of tightly controlling blastemal proliferation and regenerative rate. Finally, although an extended depletion of Fgf signaling potently inhibits regenerative growth, it does not erase or reprogram the positional information necessary for restoration of correct structures. Our molecular genetic experiments demonstrate that amputation level-specific amounts of Fgf signaling determine position-dependent growth rates in the regenerating vertebrate appendage.
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Materials and methods |
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For measurement of regenerative length, two rays (rays 2 and 3 with respect to the most lateral ray; see Fig. 1) from each of the ventral and dorsal portions were measured using Openlab software and the average length between the two rays was recorded. For statistical comparisons, the proximal regenerate lengths were pooled from multiple fish and averaged, to compare with averages of the distal regenerate lengths. To calculate regenerative growth rate, the changes in these averages as time progressed were divided by the time period between measurements.
Analysis of BrdU incorporation and mitosis
A 2.5 mg/ml solution of bromodeoxyuridine (BrdU) in saline was injected
intraperitoneally 30 minutes prior to collection. The brief BrdU exposure
limits labeling (Nechiporuk and Keating,
2002), an approach that facilitates distinction of highly
proliferative areas in immunostained fin regenerates. Staining was performed
as described previously (Poss et al.,
2002b
), using whole double-amputated fins that had been fixed in
Carnoy's solution. A rat-derived anti-BrdU monoclonal antibody (Accurate) and
a rabbit-derived polyclonal anti-H3P antibody (Upstate Biotechnology) were
used for primary antibodies. Laser confocal microscopy (510 LSM, Zeiss) was
used to image and analyze 1 µm slices and 10 µm projections of
whole-mount samples. The lengths of the BrdU-dense blastemal regions of
ventral and dorsal fin rays 2 and 3 were measured with Openlab software, using
the middle slice of each projection. The number of H3P-positive cells was
counted by hand within an outlined and quantified area (or volume, as it is a
projection covering a depth of 10 µm) of BrdU-dense blastemal mesenchyme.
Mitoses in proximal regenerate rays 2 and 3, and the corresponding distal rays
from each fish were counted and the averages recorded.
In situ hybridization
Whole-mount in situ hybridization was performed on double-amputated fins as
described previously (Poss et al.,
2000), using digoxigenin-labeled probes for mkp3, sef and
spry4 (Furthauer et al.,
2001
; Furthauer, 2002; Tsang
et al., 2004
). When assaying fin regenerates for graded
expression, development of the staining reaction was monitored carefully and
stopped immediately after a distinct signal developed in all of the fins (Figs
3,
6 and
8). Cryosectioning of fin
regenerates was performed as described previously
(Poss et al., 2000
).
In experiments where the length of the mkp3 expression domain was measured (Fig. 4), development of the staining reaction was allowed to progress further, until background staining was detectable. In these fins, mkp3-positive areas were measured using Openlab software.
To simultaneously assess mkp3 expression and BrdU labeling, we
cryosectioned fin regenerates (from BrdU-injected animals) that had been
stained for mkp3 expression by whole-mount in situ hybridization.
Sections were then stained for BrdU immunoreactivity as described
(Poss et al., 2002a).
Construction of hsp70:dn-fgfr1 animals
A zebrafish dn-fgfr1 cassette was designed based precisely on the
X. laevis dominant-negative Fgfr1 (Amaya and Kirschner, 1991), with
the tyrosine kinase domain replaced by egfp-coding sequence. The
construct is predicted to heterodimerize with all Fgfr subtypes, thereby
competitively blocking signaling downstream of all Fgfr subtypes. Briefly, a
3' truncated fragment of the zebrafish fgfr1 gene was amplified
by PCR using the primers 5' GTT GAA TTC ATG ATA ATG AAG ACC ACG CTG
3' and 5' GTT GGA TCC AGA GCT GTG CAT TTT GGC CAG 3'. This
1.2 kb fragment was directionally cloned into the
EcoRI/BamHI site of the pEGFP-N3 vector (Clontech). Then, a
2.2 kb NheI/AflII fragment containing the
fgfr1-egfp fusion gene was prepared from this plasmid and subcloned
behind the 1.5 kb zebrafish hsp70 promoter
(Halloran et al., 2000).
Transgenic zebrafish were made by microinjection of the
hsp70:dn-fgfr1 construct using published techniques
(Higashijima et al., 1997
).
Transgenic animals were identified by Egfp fluorescence owing to natural
hsp70 promoter activity in the lens.
Adult heat induction experiments
An electric heater placed in a stand-alone recirculating aquarium unit
(Aquatic Habitats) was used for all heat induction experiments. A digital
timer automatically activated and deactivated the heater once per day. Water
flow adjustment allowed the tuning of peak tank temperatures to 35°C,
36°C, 37°C or 38°C, from a room temperature of 26°C. Exposure
to this peak temperature was for about 1 hour, before gradual return to room
temperature. To determine effects on Egfp fluorescence, BrdU incorporation or
gene expression, a single heat shock was given to animals 5 hours before
collecting fins. To detect effects on regenerative growth, animals were
maintained in the heat induction unit after amputation and exposed daily to
heat induction.
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Results |
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Position-dependent indices of blastemal length and proliferation during fin regeneration
Intuitively, regenerative growth rate is expected to be highly dependent on
blastemal proliferation. During fin regeneration, there is a much greater
amount of mesenchymal proliferation than epidermal proliferation, with cycling
cells being preferentially localized to the proximal portion of the blastema
(Nechiporuk and Keating, 2002;
Poss et al., 2002a
). We used
whole-mount analysis of immunostained, double-amputated fins to assess
blastemal morphology and proliferation at 3 dpa, when the regenerative growth
rate difference is greatest between proximal and distal regenerates. Thirty
minutes prior to collection, animals were injected with BrdU. In confocal
slices of whole-mount fins stained for BrdU, we clearly distinguished
blastemal mesenchyme with especially high BrdU density (brackets in
Fig. 2A,B), versus more
proximal, non-blastemal regions with lower BrdU density.
While performing these experiments, we noticed that the length of the blastema appeared greater in proximal regenerates than in distal (Fig. 2A,B). We used digital imaging and computer-assisted measurements to compare this length between proximal and distal rays 2 and 3. We found that the average blastemal length of proximal regenerates from many fish was 113±5 µm (mean ± s.e.m.), compared with 97±3 µm for the distal, a proximal:distal ratio of 1.16 (Fig. 2E; n=17; t-test: P<0.05). We also compared blastemal length within the same fin to control for interfish differences and found that the average proximal:distal length ratio was 1.18±0.07. We then used an antibody against phosphorylated histone-3 (H3P) to count the number of mitoses within BrdU-dense, blastemal mesenchyme in double-amputated fins (Fig. 2C,D). Proximal regenerate blastemas had an average of 535±73 H3P-positive cells/mm2, versus 356±35 for distal regenerate blastemas, a proximal:distal ratio of 1.50 (Fig. 2E; n=10; t-test: P<0.05;). In intrafin proximal-to-distal comparisons, the average proximal:distal ratio for blastemal mitoses was 1.58±0.23. Thus, position-dependent differences in regenerative growth rates are likely to reflect differences in blastemal length and mitotic index.
|
|
To test whether amputation level determines the amount of Fgf signaling, we examined expression of mkp3, sef and spry4 in double-amputated fins (Fig. 3B). By carefully monitoring the in situ hybridization reaction, we found that the majority of fins displayed a clearly higher expression level in proximal regenerates versus distal for each gene (mkp3, 15 out of 24 fins; sef, 8 out of 11; spry4, 12 of 18). No fins displayed a higher level of gene expression in distal regenerates. These results indicate that each PD position is assigned a different amount of Fgf signaling after amputation.
In addition, we used target gene expression to assess the domain of active
Fgf signaling in proximal and distal regenerates. After fully developing in
situ hybridization reactions (see Materials and methods), we observed distinct
differences in how far proximally the mkp3 signal extended in 3 dpa
proximal and distal regenerates. In these experiments, we measured the
distance from the most distal tip of the mkp3 expression domain to
the most proximal limits. We found that the proximal regenerate expression
domain extended 28% further than the distal
(Fig. 4A-C; n=8,
P<0.005). In intrafin comparisons, the average proximal:distal
ratio of this length was 1.31±0.10. Thus, the length of the active Fgf
signaling region is determined by amputation level. Interestingly,
single-amputated fins stained for both mkp3 expression and BrdU
incorporation (30 minutes exposure) showed very little or no co-labeling in
blastemal cells. In other words, the mesenchymal mkp3 expression
domain, likely corresponding to the nonproliferative distal blastema reported
by Nechiporuk and Keating (Nechiporuk and
Keating, 2002), was located distal to the BrdU-positive proximal
blastema (Fig. 4D-F). Instead,
BrdU-positive blastemal tissue correlated better with adjacent epidermal
mkp3 expression, suggesting a potential paracrine relationship (see
Discussion). In summary, our results revealed position-dependent values of
regenerative growth rate, blastemal length and mitotic index, and properties
of Fgf signaling. In each case, proximal regenerate values were greater than
distal regenerate values.
|
|
|
To determine how different Fgf signaling amounts impacted blastemal proliferation, we gave hsp70:dn-fgfr1 fish the same heat-induction protocols and assessed blastemal integrity by BrdU incorporation. We found that blastemal proliferation was affected in a dose-dependent manner by Fgfr inhibition (Fig. 6D-F). A single 36°C or 37°C treatment markedly reduced the number of BrdU-positive cells in the blastema, while the 38°C treatment nearly abolished this structure. Interestingly, only cellular proliferation within the blastema was affected by Fgfr inhibition, while cell populations proximal to the blastema appeared to proliferate normally. Accordingly, we could easily identify an Fgf-dependent proliferative blastemal region that corresponded to an area normally flanked by epidermal Fgf target gene expression (brackets in Fig. 6F).
Next, we applied daily heat inductions to test the effects of this experimental Fgf signaling continuum on regenerative growth rate. We found that regenerative growth rate was also highly sensitive to 1°C temperature increments (Fig. 6G-K). The 37°C incubation slowed regeneration down to about half the rate of uninduced hsp70:dn-fgfr1 fish at 5 dpa (Fig. 6J,K). Interestingly, 36°C and 37°C regenerates appeared grossly normal, albeit small, with fin ray segments of approximately normal size (Fig. 6H). This observation suggested that rate, and not ray patterning, were the main targets of the inhibition. Our experiments together support the idea that PD disparities in Fgf signaling between proximal and distal regenerates directly translate into different blastemal proliferation and regenerative growth rates. In other words, the amount of Fgf signaling represents an amputation level-specific instruction for position-dependent regenerative growth rates.
|
Positional memory is maintained during Fgfr inhibition
While our experiments demonstrated an intimate relationship between
position-dependent Fgf signaling properties and growth rate, we were also
curious about whether the Fgf signaling profile represented positional memory
in total. That is, does it define both growth rate and the structures to be
regenerated?
To examine the validity of this idea, we assessed mkp3 expression at several timepoints in mid-amputated fins throughout the duration of regeneration (15 dpa). In these experiments, mkp3 expression was a robust indicator of PD position. Regenerates displayed a gradual loss of mkp3 expression intensity, with scarcely detectable marker expression by 15 dpa (Fig. 8A-C). The PD length of the mkp3 expression domain also decreased gradually during regeneration (Fig. 8D). These initial observations, without functional validation, were consistent with the notion that Fgf signaling properties might indeed encode positional memory.
|
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Discussion |
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Amputation of the zebrafish caudal fin stimulates formation of a wound epidermis, blastemal morphogenesis and rapid growth. This growth is dependent on synthesis of Fgfs and signaling through Fgfrs. The more proximal the amputation, the longer the region of active Fgf signaling in epidermal cells. Consequently, more proximally amputated fins establish a greater proximal extension of proliferative blastemal cells adjacent to these epidermal domains. Furthermore, greater amounts of Fgf signaling activity in proximal regenerates translate into higher blastemal mitotic indices. Such position-dependent differences in blastemal function explain position-dependent growth rate, defining Fgf signaling as a graded component of the positional instructions required for accurate regeneration.
While most of our experiments focused on comparison of two amputation levels within a single fin, our data from single-amputated fins support this model. Both amounts and proximal extension of Fgf signaling gradually diminish during regeneration, aligning these parameters with PD position until the process is completed. Such observations explain the gradual decrease in regenerative growth rate from 3 to 15 dpa (Fig. 1). They also indicate that there is not only a mechanism to establish a position-dependent amount of Fgf signaling after amputation, but an additional related mechanism for gradual position-dependent reduction in these amounts to slow and then stop regeneration as it concludes.
Our data suggest that the effect of Fgfr activation on blastemal cells is
cell non-autonomous; i.e. Fgf signal transduction in basal epidermal cells
somehow influences nearby blastemal cells. Because the Erk signaling
inhibitors mkp3, sef and spry4 are induced in epidermal
cells and are Fgf dependent during fin regeneration, we suspect that Fgfrs
regulate blastemal function via a mechanism that involves epidermal Erk
activation. According to this model, epidermal cells then release a mitogen
that diffuses to adjacent blastemal cells
(Fig. 9B). Sonic hedgehog
(shh) is a candidate for this mitogen, as it is expressed in basal
epidermal cells and its transcription is dependent on Fgfr activation
(Laforrest et al., 1998; Poss et al.,
2000). Furthermore, cyclopamine, the pharmacological inhibitor of
Hedgehog signal transduction, was recently shown to block blastemal
proliferation in the regenerating zebrafish fin, as well as the amputated
axolotl tail (Quint et al.,
2002
; Schnapp et al.,
2005
). Continued candidate approaches should help resolve the
downstream mechanisms by which Fgfs modulate blastemal function.
|
It is reasonable to suspect that an Fgf ligand gradient is responsible for the Fgf signaling gradient described in our experiments. We have not been able to conclusively detect position-dependent regulation of fgf24 mRNA levels (Y.L. and K.D.P., unpublished), but there are many Fgf genes to be examined for position-dependent regulation at the RNA or protein level during fin regeneration. Whatever the primary rate-determinant in the Fgf signaling pathway may be, our data indicate that Fgf signaling translates position into rate, but lies downstream of the master regulator(s) that furnishes position-dependent instructions (Fig. 9A). Removal of Fgf signaling for an extended period does not irreversibly change positional values, while disruption of more upstream factors that regulate the positional memory program is predicted to do so. However, it is tempting to speculate that experimental increases in Fgf signaling levels during later stages of fin regeneration might have the effect of extending the length of the final regenerate.
|
The second molecule implicated in positional memory is CD59, a
membrane-localized protein whose expression is graded along the PD axis and
regulated by RA in the amphibian limb. When CD59 function is blocked in
blastemal explant cultures, proximal blastemal behavior engulfment of
distal blastemal explants is inhibited
(da Silva et al., 2002).
Furthermore, CD59 overexpression in electroporated axolotl limb regenerates
appears to proximalize blastemal cells
(Echeverri and Tanaka, 2005
).
The mechanism by which CD59 might control positional memory remains unclear.
Therefore, it would be interesting to functionally examine CD59 during
zebrafish fin regeneration as carried out here for Fgf signaling. Moreover,
unbiased genetic screens for defects during fin regeneration are possible in
zebrafish (Johnson and Weston,
1995
; Poss et al.,
2002b
; Nechiporuk et al.,
2003
), and can be modified to identify mutants that regenerate too
few or too many structures. Such approaches represent an attractive method for
increasing the molecular resolution of mechanisms by which positional memory
directs appendage regeneration.
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ACKNOWLEDGMENTS |
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