|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 16 August 2006
doi: 10.1242/dev.02533
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, Saint Louis, MO 63110, USA.
* Author for correspondence (e-mail: sjohnson{at}genetics.wustl.edu)
Accepted 12 July 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Melanocyte, Regeneration, Chemical ablation, Stem cell, Cell division
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) (reviewed by
Lenhoff and Lenhoff, 1991).
More recently, attention has focused on the origins of cells contributing to
regeneration, revealing roles for both differentiated and undifferentiated
precursors in various modes of regeneration. This distinction, combined with
Morgan's original distinction based on growth and cell proliferation, suggests
four classifications for regeneration mechanisms subject to cell division and
differentiated state of regeneration precursors, as follows. (1) Regeneration
via direct cell division from differentiated cells
(Fig. 1A). This mechanism is
sometimes called compensatory regeneration, exemplified by mammalian liver
regeneration (reviewed by Michalopoulos
and DeFrances, 1997). (2) Regeneration via cell division from
undifferentiated precursors or stem cells
(Fig. 1B). This mechanism
closely matches the current usage of epimorphosis, and is best exemplified by
the stage of limb regeneration following formation of the blastema
(Chalkley, 1954;
Hay and Fischman, 1961). (3)
Regeneration without cell division by transdifferentiation from otherwise
differentiated cells, as occurs in hydra following resection
(Park et al., 1970;
Holstein et al., 1991)
(Fig. 1C). (4) A fourth, as yet
hypothetical, mode of regeneration is formally suggested by this
classification scheme and involves regeneration without cell division from
late-stage undifferentiated precursors
(Fig. 1D). We note that
additional mechanisms, such as dedifferentiation of differentiated cells to
form a blastema are also employed in some regenerating systems. Additionally,
regeneration schemes may combine some of these mechanisms, either sequentially
in the same lineage [for instance, in limb regeneration, dedifferentiation of
myofibers to form the blastema is then followed by proliferation and new
growth (Kintner and Brockes,
1984)] or use different mechanisms for the different cell types or
lineages contributing to regeneration of more complex tissues [for instance,
in zebrafish fin regeneration, keratinocytes divide directly
(Poleo et al., 2001), whereas
melanocytes are thought to be derived from undifferentiated precursors or stem
cells (Rawls and Johnson,
2000)].
Another opportunity for understanding regeneration and the underlying
mechanisms of regulation comes from the analysis of homeostasis of single cell
types in mammals, such as erythrocytes (reviewed by
Morrison et al., 1995),
epidermis (Jones and Watt,
1993) (reviewed by Fuchs and
Raghavan, 2002), and epithelium in the lining of the small
intestine (Cheng and Leblond,
1974) (reviewed Potten and
Loffler, 1990). For each of these examples, the differentiated
cells turn over rapidly and homeostatic mechanisms act on stem cells to
generate new cells. This ability to replenish certain single cell types is
crucial for maintaining the normal physiology and lifespan in humans. For
instance, the deficient replenishment of erythrocytes results in anemia,
whereas excessive turnover or misregulation of epidermal cells may cause
psoriasis or skin cancer (Weinstein and
Frost, 1968). Other human diseases result from deficits in single
cell types with less capacity for regeneration, such as pancreatic ß
cells in autoimmune type I diabetes, or oligodendrocytes in multiple sclerosis
(for reviews, see Bach, 1994;
Compston and Coles, 2002). An
understanding of the mechanisms regulating single cell type regeneration in
model systems might therefore be used to develop cures for relevant human
diseases.
An attractive cell type for this approach is the zebrafish melanocyte,
because it is easily visualized, dispensable for viability in the laboratory,
and multiple mutations have been generated that affect different aspects of
its development at different stages of the fish life cycle. As in other
vertebrates, zebrafish melanocytes are derived from the embryonic neural crest
(Raible et al., 1992).
Melanocytes first appear at 24 hours postfertilization (hpf), and, by
approximately 60 hpf, the larval pigment pattern is established with
approximately 460 post-mitotic melanocytes. This number of larval melanocytes
remains nearly constant, with only minimal birth and death of melanocytes
occurring, until the onset of adult pigment pattern metamorphosis at 14 days
postfertilization (dpf) (Milos and Dingle,
1978; Johnson et al.,
1995) (reviewed by Rawls et
al., 2001). Previously, we demonstrated the homeostatic regulation
of larval melanocytes by larval melanocyte regeneration following melanocyte
ablation with a dermatology laser. We further revealed that this melanocyte
regeneration is achieved through the recruitment of undifferentiated cells,
which tends to exclude division of differentiated melanocytes (i.e.
compensatory regeneration, Fig.
1B) as the mechanism for larval melanocyte regeneration
(Yang et al., 2004). Despite
the power of such laser ablation, our attempts to further investigate the
mechanisms of larval melanocyte regeneration were hampered by the limiting
number of regenerating melanocytes that could be generated for thorough
analyses of cell division events.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). All developmental staging is reported in hours and days
postfertilization (hpf and dpf, respectively), corresponding to staging at the
standard temperature of 28.5°C. Staging at permissive temperature
(25°C) is translated to 28.5°C stages as previously described
(Kimmel et al., 1995).
kitj1e99 and fmsj4e1 have been
previously described (Rawls and Johnson,
2003; Parichy et al.,
2000). sdyj9s1 is a spontaneous, homozygous
viable allele of the shady locus
(Kelsh et al., 1996) that has
approximately 10% of the normal number of embryonic or larval iridophores
(C.-T.Y., unpublished). All references to these mutant alleles correspond to
homozygous animals.
Chemicals
(2-morpholinobutyl)-4-thiophenol (MoTP) was custom synthesized by Gateway
Chemical Technology (St Louis, MO). 4-hydroxyanisole (4-HA) (or
4-methoxyphenol) was purchased from Sigma-Aldrich (M1865-5, St Louis, MO). The
dose responses of both chemicals in melanocytotoxicity and fish lethality were
tested, and the most melanocytotoxic concentrations with low fish lethality
were chosen for zebrafish melanocyte ablation experiments. MoTP and 4-HA were
dissolved in dimethyl sulfoxide (DMSO) to make stock solutions, which were
then diluted in egg water at 14 µg/ml (50 µM) and 2 µg/ml (16 µM)
final concentrations, respectively. For phenylthiourea (PTU) treatment,
0.1-0.2 mM PTU was added to egg water and changed every 2 days
(Milos and Dingle, 1978).
Whole-mount in situ hybridization
In situ hybridization with antisense digoxigenin (DIG)-labeled riboprobes
was performed as described (Jowett and
Yan, 1996), using 68°C hybridization and stringency washes,
alkaline phosphatase-conjugated secondary antibodies and NBT/BCIP (Roche,
Indianapolis, IN). The riboprobes of mitf
[microphthalmia-associated transcription factor
(Lister et al., 1999)],
kit [kit receptor tyrosine kinase
(Parichy et al., 1999)] and
dct [dopachrome tautomerase
(Kelsh et al., 2000)] were
previously described. A partial tyrosinase (tyr) cDNA
(Page-McCaw et al., 2004) was
obtained by RT-PCR with primers 5'-CATCATCATGTCTCTCCATCTCC-3' and
5'-CAGCATAATGCTTGCATCCTTC-3', and cloned into a pBluescript SK
vector (Strategene) for riboprobe synthesis. For in situ hybridization
performed on larval samples older than 72 hpf, larvae were fixed in 4%
paraformaldehyde (PFA) with 1% DMSO overnight, and probes were fragmented to
300 nucleotides (Parichy et al.,
2003). For some rounds of staining (Figs
2,
3), normally reared embryos
were treated with 0.1-0.2 mM PTU to completely block melanin synthesis,
thereby allowing easier visualization of NBT/BCIP precipitates in otherwise
dark melanocytes. Note that dct+ melanoblasts are first
detected at 19 hpf and they remain dct+ and lack melanin
for approximately 5 hours, until the beginning of melanogenesis at 24 hpf
(Kelsh et al., 2000).
dct expression continues to be detected within the pigmented
melanocytes through 72 hpf. Therefore, in our experiments
(Fig. 3), melanin production
was blocked by PTU for easier visualization and photography of dct
expression; the dct+ cell counts show both
dct+ melanoblasts and differentiated (but PTU-masked)
melanocytes.
BrdU incorporation and detection
To detect and quantify cell division events in melanocyte lineages during
larval melanocyte regeneration, normally reared and MoTP-treated larvae at
various developmental stages were immersed in a solution of
5-bromo-2'-deoxyuridine (BrdU; 5 mM) for 24 hours in pulse labeling
experiments. In the continuous labeling experiment, we changed the BrdU
solution every 24 hrs. At the harvest stages, animals were fixed, imbedded in
paraffin wax, and then cut into series of 5-µm-thick sagittal sections.
Sections were deparaffinized and incubated with mouse anti-BrdU monoclonal
antibody (1:300 dilution, Santa Cruz), followed by a secondary incubation with
Alexa Fluor 594-conjugated goat anti-mouse Ig (1:300 dilution, Molecular
Probes), and bisbenzimide (100 ng/ml; Sigma) for nuclear staining
(Rawls et al., 2004). BrdU
incorporation was determined by first identifying bisbenzimide-positive nuclei
(blue fluorescence) in pigmented melanocytes, then assessing BrdU
incorporation (red fluorescence) in each identified nucleus
(Fig. 5).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). These
researchers observed that embryos developing in the presence of MoTP lacked
body melanocytes, whereas their retinal pigment epithelium (RPE) was lightly
pigmented. This differential effect on RPE and neural crest (NC)-derived
melanocytes suggests that MoTP does not merely block general melanin
production, for instance as does a tyrosinase inhibitor such as PTU.
Furthermore, Peterson et al. reported that when larvae were removed from MoTP
after a three-day incubation, melanocytes gradually repopulated the larval
body during the ensuing two days. Together, these observations led to the
suggestion that MoTP reversibly blocks or delays NC melanocyte development in
zebrafish (Peterson et al.,
2000).
To test the hypothesis that MoTP causes developmental delay in the
melanoblast lineages, we examined their development in MoTP-treated larvae by
performing a series of whole-mount RNA in situ hybridization with the early
stage melanoblast markers mitf
(Lister et al., 1999) and
kit (Parichy et al.,
1999), and the late stage melanoblast markers tyr
(Page-McCaw et al., 2004) and
dct (Kelsh et al.,
2000) (C.-T.Y., unpublished). For these experiments, we incubated
14 hpf embryos in 14 µg/ml MoTP solution and fixed them at 27 hpf for in
situ analysis. Following in situ hybridization, we were unable to distinguish
differences in the developmental patterns or numbers of labeled cells in
MoTP-treated larvae compared with untreated larvae for each of the markers
(Fig. 2A-H). These results
indicate that the differentiation of NC-derived melanoblasts remains normal
and proceeds to late developmental stages (tyr+ or
dct+) in the presence of MoTP. Interestingly, untreated
late-stage melanoblasts typically begin to melanize prior to 27 hpf, but in
MoTP treated larvae, this terminal marker (melanin) fails to form.
MoTP is cytotoxic to larval melanocytes
Because our foregoing analysis revealed that MoTP-treated melanoblasts
proceed to late stages of differentiation but fail to reach their terminal
stage of melanin production, we next explored their fate at subsequent
developmental periods in MoTP-treated larvae. We found that the number of
dct+ cells in treated larvae declined from a maximum of
100 at 31 hpf to an average of 15 dct+ cells by 60 hpf
(Fig. 3B,D,F-H). By contrast,
in the untreated larvae, the number of dct+ cells steadily
increased to more than 300 during the same time period
(Fig. 3A,C,E,H). Note that the
majority of the dct+ cells in the untreated larvae are
differentiated melanocytes, whose subsequent melanin production was blocked by
incubation with PTU, allowing for easier visualization of the in situ marker
(see Materials and methods). The disappearance of dct+
cells in treated larvae raised the possibility that, in the presence of MoTP,
melanoblasts reach the tyr and dct expression stage, at
which time they become sensitive to MoTP and die.
|
;
Sugimoto et al., 2000), we
interpret these results to indicate that MoTP induces melanocyte cell
death.
The melanocytotoxicity of MoTP is dependent on tyrosinase activity
We sought to investigate the mechanism underlying MoTP cytotoxicity in
melanocytes. One class of phenolic compounds has been previously reported to
cause cytotoxicity specific to melanocytes (melanocytotoxicity) in mammals
(reviewed by Riley, 1985).
These compounds feature a phenolic ring with a functional group at the para
position, a structure that is similar to tyrosine, the initial substrate of
tyrosinase during the biochemical synthesis of melanin. Biochemical studies
have suggested that this class of phenolic compounds, such as 4-hydroxyanisole
(4-HA; Fig. 4G), competes with
tyrosine for hydroxylation by tyrosinase and, consequently, is converted to a
cytotoxic form, mainly o-quinone, which is associated with the initiation of
cellular damage in melanocytes (Riley,
1975; Naish et al.,
1988). Thus, these studies demonstrated that the
melanocytotoxicity of such phenolic compounds is mediated by tyrosinase
activity (Fig. 4I). Like known
members of this class of phenolic compounds, MoTP also has a phenolic ring
with a functional group, in this case (morpholinobutyl)-thio, at the para
position. Therefore, we sought to test whether MoTP acts similarly to the
4-HA-related class of phenolic compounds. If so, we reasoned that the
chelation of copper, an essential cofactor for tyrosinase activity, by
addition of the copper chelator PTU, should block the melanocytotoxicity of
MoTP (Fig. 4I). Accordingly, we
co-incubated 48 hpf larvae in MoTP solution with 0.1-0.2 mM PTU. We found that
the melanocytotoxicity of MoTP was completely suppressed in the presence of
PTU, as lightly pigmented and dendritic melanocytes were observed, but we
observed no evidence of punctate melanocytes or their subsequent extrusions in
these larvae (Fig. 4F).
|
|
Melanocyte regeneration following MoTP-induced ablation
As described above, incubating zebrafish embryos in MoTP results in the
loss of melanoblasts or melanocytes. By 72 hpf, when untreated larvae have
established the larval melanocyte pattern, MoTP-treated larvae lack all
melanocytes. When MoTP-treated larvae are transferred to fresh egg water at 72
hpf, melanocytes gradually develop over the next 4-5 days (as described in the
ensuing sections). Such regeneration of larval melanocytes now provides an
opportunity to explore how regenerating cells are derived from their
precursors, including the role for cell division and differentiation state of
precursors (below).
Melanocytes regenerate from the division of undifferentiated melanocyte precursors following MoTP treatment
We first sought to test whether, following melanocyte ablation by MoTP,
melanocytes regenerate through mechanisms involving cell division.
Accordingly, we incubated larvae in MoTP from 4-5 dpf, to ablate
differentiated melanocytes. We also incubated these larvae continuously in
BrdU from 4-10 dpf, to reveal whether the differentiated melanocytes that
arise have undergone rounds of DNA synthesis, BrdU incorporation, and thus,
cell division. Counting the percentage of BrdU-positive, melanin-positive
melanocytes in 5 µm paraffin sagittal sections at 10 dpf (5 days post-MoTP
treatment) showed that 97.2±2.5% of regenerated melanocytes are BrdU
positive, compared with only 3.9±3.5% BrdU-positive melanocytes in
untreated animals (Fig. 5).
This finding indicates that virtually all regenerated melanocytes develop from
precursors that have undergone one or more rounds of cell division following
MoTP-induced melanocyte ablation.
We next explored models of regeneration that do not involve cell division (Fig. 1C,D). We reasoned that if the embryo sets aside late-stage precursors that could differentiate directly into melanocytes without cell division for regeneration and regulation (Fig. 1D), these late stage precursors might express a late-stage melanoblast marker, such as dct, in untreated (non-regenerating) larvae. In situ analysis of dct expression showed very few dorsal dct+ melanoblasts (0.2-0.3 per animal) in untreated fish between 74 and 128 hpf (Fig. 6C). By contrast, in MoTP-treated larvae (14-72 hpf), we detected 8- to 20-fold more dorsal dct+ melanoblasts (two to three per animal) at the same developmental stages (Fig. 6A-C). This result indicates that large numbers of late-stage (dct+) melanoblasts are not available for regenerating the larval melanocyte pattern, and also that ablation of the embryonic melanoblasts and melanocytes results in the recruitment of dct+ melanoblasts from less-differentiated precursors during melanocyte regeneration.
An alternative model of regeneration without cell division is regeneration
by transdifferentiation from one differentiated cell type to another. For
example, in amphibians, other NC-derived chromatophores have been suggested to
transdifferentiate into melanophores under various experimental conditions
(Thidaudeau and Holder, 1998; Ide and
Akira, 1988); thus, differentiated iridophores and xanthophores
may be candidates for transdifferentiation into melanocytes in this
regeneration system (Fig. 1C).
Evidence for or against such transdifferentiation might be gained by assessing
regeneration in the absence of iridophores or xanthophores. Accordingly, we
investigated whether melanocyte regeneration occurred in mutant larvae that
lacked most or all iridophores (sdyj9s1) or xanthophores
(fmsj4e1), but that have a normal embryonic and larval
melanocyte pattern. In each mutant, following melanocyte ablation by MoTP
treatment (14-72 hpf), we observed that melanocyte regeneration occurred
identically to that in wild-type larvae (data not shown), which suggests that
transdifferentiation from iridophores and xanthophores is not responsible for
melanocyte regeneration. We note, however, that these results do not exclude
the possibility of transdifferentiation from other cell types.
|
),
iridophores or xanthophores, or from late-stage (dct+)
precursors.
|
|
Recruitment of melanocyte precursors or stem cells in wild-type and mutant animals
We were also interested in the distribution of quiescent melanocyte
precursors or stem cells that contribute to melanocyte regeneration following
melanocyte ablation by MoTP treatment. Ideally, this knowledge could be gained
by examining markers for the melanocyte stem cells. Lacking such stem cell
markers, we have instead examined the distribution of differentiated
melanocytes in wild-type and mutant animals for less direct inferences. Thus,
in wild-type animals, we observed the first appearance of regenerated
melanocytes 24 hours post-MoTP treatment. The number of melanocytes continues
to steadily rise for the ensuing 4 days and reaches a plateau of approximately
350-400 melanocytes by 9 dpf (6 days after the removal of MoTP). This number
reveals a deficit of approximately 60-110 melanocytes in MoTP-treated versus
untreated larvae (average 460 melanocytes;
Fig. 8A). To further explore
this deficit, we examined the patterns of melanocytes in the MoTP-treated and
untreated larvae at 9 dpf. We found MoTP-treated larvae regenerate an almost
identical pigment pattern compared to that of the untreated larvae, with a
similar number and distribution of melanocytes in the dorsal, lateral and
ventral larval melanocyte stripes. One exception was that MoTP-treated larvae
failed to regenerate the majority of the ventral yolk sac melanocytes
(Fig. 8B-E). Typically,
untreated larvae have approximately 65 melanocytes in the ventral yolk sac
stripe at this stage; therefore, the absence of those melanocytes in
MoTP-treated larvae accounts for the observed melanocyte deficit
(Fig. 8A). When we allowed
these MoTP-treated larvae to develop to stages immediately prior to the onset
of adult pigment pattern metamorphosis, we observed that melanocytes slowly
begin to repopulate the ventral yolk sac area, resulting in approximately 35%
of the normal number of ventral yolk sac stripe melanocytes by 14 dpf (11 days
post-MoTP treatment; data not shown). These results suggest either that the
region of the ventral yolk sac is devoid of quiescent melanocyte precursors or
stem cells, or that they are less responsive to the presence or absence of
differentiated melanocytes in this area (see Discussion).
|
). The kit receptor tyrosine kinase is
required for ontogenetic melanocyte development in zebrafish embryos. In
kitb5 (a null mutation) embryos, NC-derived melanocytes
differentiate, but fail to migrate to proper locations and subsequently
undergo programmed cell death, resulting in larvae devoid of melanocytes until
pigment pattern metamorphosis (Johnson et
al., 1995; Parichy et al.,
1999). Previously, by using a temperature-sensitive allele,
kitj1e99, at the restrictive temperature (30-33°C), we
showed that kit is also required to fill in gaps in the larval
melanocyte pattern created by laser ablation. Interestingly,
kitj1e99 animals also failed to regenerate melanocytes
after laser ablation at temperatures described as permissive for embryonic
melanocyte development or for melanocyte regeneration in the regenerating
adult caudal fin (23 or 25°C) (Yang et
al., 2004; Rawls and Johnson,
2001; Rawls and Johnson,
2003). Because the laser ablations resulted in animals with only
small regions devoid of melanocytes, we revisited the role of
kitj1e99 in larval melanocyte regeneration using MoTP
treatment to ablate all the melanocytes in the larvae. When we reared
kitj1e99 animals in MoTP solution (14-72 hpf) at the
permissive temperature (25°C), we found that new melanocytes appeared
significantly more slowly than in wild-type larvae after MoTP treatment
(Fig. 8A,
Fig. 9A), achieving a plateau
of 15.9±5.7 melanocytes at 9 dpf. We discuss the inferences that can be
drawn from the number and distribution of these few regenerated melanocytes
more extensively below (see Discussion). |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Here, we
have developed a different method, which can ablate the entire melanocyte
population in zebrafish larvae. We demonstrated that a small molecule, MoTP,
is cytotoxic to late stage melanoblasts and melanocytes in zebrafish larvae,
and that this melanocytotoxicity is dependent on tyrosinase activity. The
melanocyte specificity of MoTP ablation is also stage specific; we found that
exposure of larvae to MoTP after 5 dpf had no effect on melanocytes (data not
shown), presumably because of the decline of tyrosinase activity in mature
melanocytes. Although tyrosinase is also expressed in the RPE
(Camp and Lardelli, 2001), we
found that the RPE is relatively insensitive to MoTP-induced cell death, as
the RPE develops normally, albeit with lighter pigment, at the MoTP
concentration that causes NC-derived melanocyte cell death
(Fig. 4C). This differential
sensitivity between types of melanocyte could result from differential
tyrosinase expression or activity, or possibly from differences in the
accessibility of these cells to MoTP during the treatment.
A similar melanocytotoxicity has been described for a related class of
compound, including 4-HA. Like MoTP, 4-HA is a prodrug that is converted by
tyrosinase to a cytotoxin. In the case of 4-HA, the cytotoxic product is
o-quinone (Naish et al.,
1988). Thus, we predict that tyrosinase converts MoTP to a similar
molecule. The melanocytotoxicity of 4-HA was explored for use in melanoma
chemotherapy, but discontinued because of severe liver damage resulting from
liver cytochrome P450 conversion of 4-HA to toxic p-quinone
(Rustin et al., 1992;
Moridani et al., 2002). The
difference in structure of MoTP from 4-HA in the functional group at the para
position raises the possibility that MoTP or related derivatives may not show
the same liver toxicity. The recent development of a melanoma model in
zebrafish may provide a useful platform for evaluating MoTP as a
chemotherapeutic, or for the search for other tyrosinase-dependent prodrugs
for the treatment of melanoma (Patton et
al., 2005).
In addition, the finding that MoTP is a prodrug converted by tyrosinase to
a cytotoxic form suggests the potential application that specific and
non-invasive ablation of any cell type in complex tissues or organs can be
achieved by expressing tyrosinase under the control of cell- or
tissue-specific promoters, then exposing the fish to MoTP. A similar use for
transgenic expression of a prodrug converting enzyme, which we are exploring,
is expression of bacterial nitroreductase in specific cells
(Medico et al., 2001) (M. T.
Saxena and S. L. Johnson, unpublished), followed by exposure to bacterial
enzyme-specific substrates metronidazole or CB1954, that in turn are converted
to cytotoxins.
We note that Harris et al. (Harris et
al., 2003) have previously demonstrated an example of single cell
type ablation by small molecules in zebrafish; aminoglycoside antibiotics,
including neomycin, can induce hair cell death in larval zebrafish lateral
line neuromasts, which is then followed by hair cell regeneration.
Larval melanocyte regeneration is achieved via the division of otherwise quiescent melanocyte precursors
We explored the developmental mechanisms by which melanocytes regenerate
following melanocyte ablation by MoTP treatment, particularly in regards to
the role of cell division and the differentiated state of the regeneration
precursor cells (Fig. 1). The
finding that melanocyte regeneration occurs after essentially complete
ablation of the larval melanocytes by MoTP, and our previous findings
(Yang et al., 2004) that
larval melanocytes regenerate from undifferentiated precursors, rather than
differentiated melanocytes, following laser ablation, rule out that melanocyte
regeneration is achieved via the proliferation of differentiated melanocytes
(Fig. 1A). This finding,
together with our finding that all or virtually all regenerated melanocytes
arise via cell division after ontogenetic melanocyte ablation
(Fig. 5I), provides strong
support for the model outlined in Fig.
1B that larval melanocyte regeneration occurs via the recruitment
of undifferentiated precursors to divide and then differentiate to produce the
new larval melanocyte population (an example of epimorphosis).
Our findings also provide evidence against models of regeneration that do
not involve cell division (Fig.
1C,D). The notion that late stage (dct+)
precursors are available to differentiate directly into melanocytes without
cell division (Fig. 1D) is
dispelled by our finding of few dct+ melanoblasts in
untreated larvae, especially when compared with the numbers of
dct+ melanoblasts found in MoTP-treated larvae
(Fig. 6C). The notion of direct
transdifferentiation (Fig. 1C)
from other pigment cells playing a role in melanocyte regeneration is
suggested by findings of transdifferentiation between chromotaphores in
salamanders (Thidaudeau and Holder, 1998;
Ide and Akira, 1988). However,
our finding that sdy and fms mutants that lack most or all
iridophores or xanthophores, respectively, regenerate melanocytes identically
to wild-type larvae does not support the notion that melanocyte regeneration
is derived from these other pigment cell types.
|
Melanocyte stem cells in zebrafish larvae
Homeostasis or regeneration of many tissues has been shown to be achieved
via the recruitment of stem cells. Stem cells have two important
characteristics: they are undifferentiated and they self-renew
(Siminovitch et al., 1963).
These two key characteristics of stem cells were elegantly demonstrated by
multiple reconstitutions of hematopoietic cells in X-ray-irradiated mice
(Spangrude et al., 1988).
Because such techniques of transplantation and reconstitution are not
generally possible for other lineages, other lines of evidence may help
evaluate the notion of stem cells or quiescent precursors. The presence of
melanocyte stem cells in zebrafish has been suggested by observations of the
unlimited capacity of melanocyte pattern re-establishment in the regenerating
caudal fin (Rawls and Johnson,
2000). Melanocyte or pigment cell stem cells have also been
suggested to contribute to the adult melanocyte population during
metamorphosis, indicating that stem cells are established during embryonic or
larval stages (Johnson et al.,
1995; Parichy,
2003). Assuming that each of these events draws upon the same
precursor population, our finding of a high proliferative capacity of the
melanocyte precursors during larval melanocyte regeneration suggests that
these melanocyte precursors have many of the characteristics expected of stem
cells. Finally, the demonstration of melanocyte stem cells in mammalian hair
follicles (Nishimura et al.,
2002) supports the idea of an analogous stem cell in the
zebrafish.
Zebrafish melanocytes begin to appear at 24 hpf, and the larval pigment
pattern is established at approximately 60 hpf. Our results suggest that in
addition to establishing the larval melanocyte pattern, embryos set aside a
population of quiescent reserve cells, henceforth melanocyte stem cells, that
can be drawn upon to generate new melanocytes for larval melanocyte
homeostasis; for instance, for filling gaps in the melanocyte pattern
(Yang et al., 2004), or for
later stages of melanocyte development, such as for adult pigment pattern
formation (Johnson et al.,
1995; Parichy,
2003). Our BrdU incorporation experiments suggest that melanocyte
stem cells are maintained in a minimal state of activity during the larval
stage. Upon melanocyte ablation by MoTP treatment, melanocyte stem cells are
released from this quiescent state, divide and produce new melanocytes to
replenish the ablated population. The activity of these melanocyte stem cells
and their descendant amplifying melanoblasts appears to be highly regulated
during melanocyte regeneration. Our BrdU incorporation experiments show that
the recruitment of melanocyte stem cells into the cell cycle is relatively
rapid, beginning less than 24 hours after melanocyte cell death. Furthermore,
we observe cell divisions throughout the 5-day regeneration period with fewer
cell divisions at the end of this period
(Fig. 7). The fact that the
pattern is largely regenerated with no local excesses or deficits of
melanocytes (with the exception of the ventral yolk sac stripe melanocytes,
see discussion below) also suggests that feedback regulation from the final
pattern regulates the later cell divisions.
One intriguing finding in our regeneration studies is that, following
melanocyte ablation by MoTP treatment, ventral yolk sac stripe melanocytes
fail to regenerate (Fig. 8).
The melanocyte stripe on the ventral yolk sac is the last larval melanocyte
stripe established, and, furthermore, these melanocytes migrate the largest
distance from the dorsum to their final ventral locations. Uneven distribution
of melanocyte progenitors has been described to account for the varying
densities of melanoblasts in different parts of the mouse embryo (reviewed by
Besmer, 1993;
Wilkie et al., 2002).
Following this logic, the failure of larval melanocyte regeneration in the
zebrafish ventral yolk sac could be due to the lack of melanocyte stem cells
in the region. Alternatively, melanocyte stem cells may in fact be established
in the ventral yolk sac but fail to survey the environment for melanocyte
homeostasis, or to re-enter developmental pathways upon the melanocyte
ablation by MoTP treatment. Interestingly, one third of the normal number of
ventral yolk sac stripe melanocytes eventually reappears by late larval stage,
during the onset of adult pigment pattern metamorphosis (14 dpf, data not
shown). An attractive possibility is that this late stage regeneration now
takes advantage of melanocyte stem cells primed by the onset of pigment
pattern metamorphosis to reconstitute the previously ignored yolk sac
melanocyte deficit. Such models can be further tested once markers that label
melanocytes precursors or stem cells are developed.
An interesting question of melanocyte regeneration is how many melanocyte stem cells in the zebrafish larvae contribute to the reconstitution of the entire larval melanocyte population following melanocyte ablation. One approach to answer this question is to estimate the number of cell divisions that each melanocyte has progressed through during regeneration. Based on our BrdU incorporation experiments, we estimate that melanocyte stem cells or the descendant amplifying melanoblasts may have gone through as many as two to four cell divisions. This is suggested in part by the accumulated percentage of BrdU-incorporated melanocytes observed over the six intervals of 24-hour BrdU labeling during and after MoTP incubation, indicating an average of 1.8 cell divisions for each melanocyte lineage (Fig. 7). This number of cell divisions per lineage is an underestimate, as some lineages are likely to have divided twice or more during any one period of 24-hour BrdU labeling. In addition, our observation that some melanocytes differentiate and leave the cell cycle during the early stage of regeneration suggests that those that differentiate at late stage of regeneration have undergone even more cell divisions. From these calculations, we estimate that there are approximately 35-145 melanocyte stem cells in larval zebrafish that give rise to the approximately 350-400 melanocytes present at the completion of regeneration.
Another estimate for the number of stem cells comes from our observation of dct-expressing cells during prolonged MoTP exposure. We typically observe 15-20 dct+ cells in the larvae during prolonged MoTP incubation (50-68 hpf; Fig. 3H). In addition, the dct+ cells on the dorsum of these larvae are distributed with a stereotyped spacing pattern (Fig. 3G). We suggest that these cells are newly progressed to dct+ stage, and that they then die once sufficient MoTP-induced cytotoxins accumulate. An attractive possibility is that these dct+ cells each mark the position or the domain of a single melanocyte precursor or melanocyte stem cell, and the that number of these dct+ cells reflects the number of melanocyte stem cells. Because, at any given time, some stem cell lineages may not have dct+ daughters, the number of melanocyte stem cells may be greater than the number of dct+ cells present in a single fish at a particular moment.
Our study of larval melanocyte regeneration in
kitj1e99animals at permissive temperature may provide
additional evidence for the above estimate of the number and position of
melanocyte stem cells. Among its roles in melanocyte development, kit
has been suggested to be required for melanoblast proliferation in mammals
(Mackenzie et al., 1997). In
zebrafish larvae, in addition to defects in melanocyte migration and
subsequent survival, kitb5 null mutants also develop a
reduced number of embryonic melanocytes (50-60% of the wild-type melanocyte
number), consistent with a role in proliferation as well
(Parichy et al., 1999). Here,
we find that following melanocyte ablation and recovery at the otherwise
permissive temperature for the allele, kitj1e99 animals
regenerate approximately 5% (15.9±5.7) of the melanocyte number
regenerated in wild-type animals (Fig.
9). The mutant lesion for this allele is in the second kinase
domain (Rawls and Johnson,
2003), and seems to confer temperature-sensitivity on the gene
product. The different effects of the kitj1e99 allele
between ontogeny and regeneration may reveal a regeneration-specific function
of the kit receptor tyrosine kinase mapping to this site, or,
alternatively, that the mutation partially reduces kit function at
the permissive temperature, and the regeneration role has a greater demand on
kit activity. Whichever is the case, the distribution of melanocytes
that regenerate in kitj1e99 animals may be informative of
the distribution of melanocyte precursors or stem cells. We note that
regenerated melanocytes in the dorsum of kitj1e99animals
are distributed in a similar pattern to the dct+ cells
observed in the prolonged MoTP-treated animals discussed above
(Fig. 3G,
Fig. 9C). One possibility is
that each melanocyte stem cell gives rise to a single melanoblast that
differentiates without further cell division. If this were so, then the number
of regenerated melanocytes observed in the kitj1e99animals
may directly reflect the number, and possibly the position, of melanocyte stem
cells.
Taken together, our observations on BrdU incorporation during regeneration, on the position and number of dct+ cells during prolonged MoTP incubation, and on the position and number of regenerated melanocytes in kitj1e99 animals lead us to postulate that there are as few as 15, or as many as 145, quiescent melanocyte precursors or stem cells in the zebrafish larvae that contribute to the larval melanocyte regeneration described here.
Our finding that the roles of the kit receptor tyrosine kinase in the regeneration process are different from those previously described for the ontogenetic development of larval melanocytes now raises the possibility that the MoTP ablation and regeneration assay could be applied to forward genetic analysis. This may allow us to specifically isolate mechanisms involved in the regeneration process, including how the stem cell is kept in check by the presence of its target tissue, or how the stem cell is activated to re-enter the developmental pathway.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bach, J. F. (1994). Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 15,516 -542.[CrossRef][Medline]
Besmer, P., Manova, K., Duttlinger, R., Huang, E. J., Packer, A., Gyssler, C. and Bachvarova, R. F. (1993). The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev. Suppl.125 -137.
Camp, E. and Lardelli, M. (2001). Tyrosinase gene expression in zebrafish embryos. Dev. Genes Evol. 211,150 -153.[CrossRef][Medline]
Chalkley, D. T. (1954). A quantitative histological analysis of forelimb regeneration in Triturus viridescens, J. Morphol. 94,21 -70.[CrossRef]
Cheng, H. and Leblond, C. P. (1974). Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141,537 -561.[CrossRef][Medline]
Compston, A. and Coles, A. (2002). Multiple sclerosis. Lancet 359,1221 -1231.[CrossRef][Medline]
Fuchs, E. and Raghavan, S. (2002). Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3,199 -209.[CrossRef][Medline]
Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W. and Rubel, E. W. (2003). Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J. Assoc. Res. Otolaryngol. 4, 219-234.[CrossRef][Medline]
Hay, E. D. and Fischman, D. A. (1961). Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 3, 26-59.[CrossRef][Medline]
Holstein, T. W., Hobmayer, E. and David, C. N. (1991). Pattern of epithelial cell cycling in hydra. Dev. Biol. 148,602 -611.[CrossRef][Medline]
Ide, H. and Akira, E. (1988). Differentiation and transdifferentiation of amphibian chromatophores. Prog. Clin. Biol. Res. 256,35 -48.[Medline]
Johnson, S. L., Africa, D., Walker, C. and Weston, J. A. (1995). Genetic control of adult pigment stripe development in zebrafish. Dev. Biol. 167, 27-33.[CrossRef][Medline]
Jones, P. H. and Watt, F. M. (1993). Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73,713 -724.[CrossRef][Medline]
Jowett, T. and Yan, Y. L. (1996). Two-color whole-mount in situ hybridization. In A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis (ed. P. A. Krieg), pp.381 -409. New York: John Wiley.
Kelsh, R. N., Brand, M., Jiang, Y. J., Heisenberg, C. P., Lin, S., Haffter, P., Odenthal, J., Mullins, M. C., van Eeden, F. J., Furutani-Seiki, M. et al. (1996). Zebrafish pigmentation mutations and the processes of neural crest development. Development 123,369 -389.[Abstract]
Kelsh, R. N., Schmid, B. and Eisen, J. S. (2000). Genetic analysis of melanophore development in zebrafish embryos. Dev. Biol. 225,277 -293.[CrossRef][Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Kintner, C. R. and Brockes, J. P. (1984). Monoclonal antibodies identify blastemal cells derived from dedifferentiating limb regeneration. Nature 308, 67-69.[CrossRef][Medline]
Lenhoff, H. M. and Lenhoff, S. G. (1991). Abraham Trembley and the origins of research on regeneration in animals. In A History of Regeneration Research: Milestones in the Evolution of a Science (ed. C. E. Dinsmore), pp.47 -66. Cambridge: Cambridge University Press.
Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. and Raible, D. W. (1999). nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126,3757 -3767.[Abstract]
Mackenzie, M. A., Jordan, S. A., Budd, P. S. and Jackson, I. J. (1997). Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo. Dev. Biol. 192,99 -107.[CrossRef][Medline]
Medico, E., Gambarotta, G., Gentile, A., Comoglio, P. M. and Soriano, P. (2001). A gene trap vector system for identifying transcriptionally responsive genes. Nat. Biotechnol. 19,579 -582.[CrossRef][Medline]
Michalopoulos, G. K. and DeFrances, M. C.
(1997). Liver regeneration. Science
276, 60-66.
Milos, N. and Dingle, A. D. (1978). Dynamics of pigment pattern formation in the zebrafish, Brachydanio rerio. I. Establishment and regulation of the lateral line melanophore stripe during the first eight days of development. J. Exp. Zool. 205,205 -216.[CrossRef]
Morgan, T. H. (1901). Regeneration. New York: The Macmillan Co.
Moridani, M. Y., Cheon, S. S., Khan, S. and O'Brien, P. J.
(2002). Metabolic activation of 4-hydroxyanisole by isolated rat
hepatocytes. Drug Metab. Dispos.
30,1063
-1069.
Morrison, S. J., Uchida, N. and Weissman, I. L. (1995). The biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol. 11,35 -71.[CrossRef][Medline]
Naish, S., Cooksey, C. J. and Riley, P. A. (1988). Initial mushroom tyrosinase-catalysed oxidation product of 4-hydroxyanisole is 4-methoxy-ortho-benzoquinone. Pigment Cell Res. 1,379 -381.[CrossRef][Medline]
Nishimura, E. K., Jordan, S. A., Oshima, H., Yoshida, H., Osawa, M., Moriyama, M., Jackson, I. J., Barrandon,Y., Miyachi, Y. and Nishikawa, S. (2002). Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416,854 -860.[CrossRef][Medline]
Page-McCaw, P. S., Chung, S. C., Muto, A., Roeser, T., Staub, W., Finger-Baier, K. C., Korenbrot, J. I. and Baier, H. (2004). Retinal network adaptation to bright light requires tyrosinase. Nat. Neurosci. 7,1329 -1336.[CrossRef][Medline]
Parichy, D. M. (2003). Pigment patterns: fish in stripes and spots. Curr. Biol. 13,R947 -R950.[CrossRef][Medline]
Parichy, D. M., Rawls, J. F., Pratt, S. J., Whitfield, T. T. and Johnson, S. L. (1999). Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development. Development 126,3425 -3436.[Abstract]
Parichy, D. M., Ransom, D. G., Paw, B., Zon, L. I. and Johnson, S. L. (2000). An orthologue of kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio. Development 127,3031 -3044.[Abstract]
Parichy, D. M., Turner, J. M. and Parker, N. B. (2003). Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish. Dev. Biol. 256,221 -241.[CrossRef][Medline]
Park, H. D., Ortmeyer, A. B. and Blankenbaker, D. P. (1970). Cell division during regeneration in Hydra. Nature 227,617 -619.[CrossRef][Medline]
Patton, E. E., Widlund, H. R., Kutok, J. L., Kopani, K. R., Amatruda, J. F., Murphey, R. D., Berghmans, S., Mayhall, E. A., Traver, D., Fletcher, C. D. et al. (2005). BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15,249 -254.[CrossRef][Medline]
Peterson, R. T., Link, B. A., Dowling, J. E. and Schreiber, S.
L. (2000). Small molecule developmental screens reveal the
logic and timing of vertebrate development. Proc. Natl. Acad. Sci.
USA 97,12965
-12969.
Poleo, G., Brown, C. W., Laforest, L. and Akimenko, M. A. (2001). Cell proliferation and movement during early fin regeneration in zebrafish. Dev. Dyn. 221,380 -390.[CrossRef][Medline]
Potten, C. S. and Loeffler, M. (1990). Stem
cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for
and from the crypt. Development
110,1001
-1020.
Raible, D. W., Wood, A., Hodsdon, W., Henion, P. D., Weston, J. A. and Eisen, J. S. (1992). Segregation and early dispersal of neural crest cells in the embryonic zebrafish. Dev. Dyn. 195,29 -42.[Medline]
Rawls, J. F. and Johnson, S. L. (2000). Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration. Development 127,3715 -3724.[Abstract]
Rawls, J. F. and Johnson, S. L. (2001).
Requirements for the kit receptor tyrosine kinase during regeneration of
zebrafish fin melanocytes. Development
128,1943
-1949.
Rawls, J. F. and Johnson, S. L. (2003). Temporal and molecular separation of the kit receptor tyrosine kinase's roles in zebrafish melanocyte migration and survival. Dev. Biol. 262,152 -161.[CrossRef]
