1 Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical
School, Department of Cardiology, Children's Hospital, Boston, MA 02115,
USA
2 University of Utah, Department of Human Genetics, Salt Lake City, UT 84112,
USA
3 Department of Genetics, Washington University School of Medicine, St. Louis,
MO 63110, USA
Authors for correspondence (e-mail:
mkeating{at}enders.tch.harvard.edu
or
kposs{at}enders.tch.harvard.edu).
Accepted 14 August 2002
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SUMMARY |
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Key words: Zebrafish, Fin, Regeneration, Blastema, Mps1
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INTRODUCTION |
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In contrast, because of their amenability to genetic manipulation,
zebrafish have proved to be a valuable laboratory model for understanding many
aspects of vertebrate embryogenesis. Small- and large-scale mutagenesis
screens have yielded hundreds of interesting mutants, from which dozens of
genes essential for ontogeny have been identified (see
Driever et al., 1996;
Gaiano et al., 1996
;
Haffter et al., 1996
;
Zhang et al., 1998
). Somewhat
overlooked is the fact that zebrafish can regenerate an impressive number of
structures as adults, such as spinal cord, optic nerve, scales, and each of
five types of fins (Johnson and Weston,
1995
; Bernhardt et al.,
1996
; Becker et al.,
1997
).
For several reasons, the fin is an excellent model organ for studying
regeneration. First, fins have a simple architecture, consisting of several
segmented, bony fin rays composed of concave, facing hemirays that surround
connective tissue, nerves and blood vessels
(Ferretti and Géraudie,
1998). Second, surgery is nearly effortless, so that hundreds of
amputations per hour may be performed. Third, regeneration is rapid and
reliable, with most structures replaced within 1-2 weeks. Finally, zebrafish
unable to regenerate fins survive normally despite their wounds, allowing the
recovery of mutant founders from genetic screens
(Johnson and Weston,
1995
).
Fin regeneration can be broken down into four stages
(Poss et al., 2000a). First,
epidermal cells migrate to cover the wound and form a multilayered cap.
Second, mesenchymal tissue down to two segments beneath the new epidermis
disorganizes, or dedifferentiates, and mesenchymal cells migrate distally
toward the amputation plane (Poleo et al.,
2001
). Third, these cells proliferate and accumulate to form the
regeneration blastema, a tissue mass from which the new fin structures are
ultimately derived. Regeneration is completed by a phase of outgrowth,
composed of exquisitely integrated proliferation, patterning, and
differentiation events. Recent work has demonstrated that the distal
regenerate is divided into three compartments during regenerative outgrowth
(Nechiporuk and Keating,
2002
). Distal blastemal cells are essentially nonproliferative and
express msxb, a transcriptional repressor gene that is induced during
blastema formation and might function to activate cellular dedifferentiation
in mesenchyme underlying the wound epidermis
(Akimenko et al., 1995
;
Poss et al., 2000a
;
Odelberg et al., 2000
). More
proximal blastemal cells are highly proliferative, and show high
bromodeoxyuridine (BrdU) incorporation and mitotic indices with a very brief
G2 cell cycle phase. Proximal to this region is a patterning zone,
where proliferation is less intense and bone-depositing scleroblasts align. We
have postulated that the distal blastema might function to specify the
direction of regenerative outgrowth, while the proximal blastema drives
regeneneration (Nechiporuk and Keating,
2002
). However, these regeneration zones have not been
functionally dissected, as no agents or mutations that affect a specific
region of the blastema have been discovered.
To find genes that mediate fin regeneration we treated zebrafish with N-ethyl-N-nitrosourea (ENU) and screened mutagenized families, as adults, for individuals unable to regenerate amputated caudal fins. We identified the nightcap (ncp) mutant, a strain that fails during regenerative outgrowth owing to severe blastemal proliferative defects. We then used positional cloning to demonstrate that this regenerative block is caused by a temperature-sensitive mutation in the zebrafish orthologue of mps1, a cell cycle regulator that is specifically upregulated in the proximal fin blastema. Our findings indicate that proximal blastemal cells are required to support proliferation through mps1 expression and function, and that compartmentalization of the blastema is crucial for regeneration. Thus, through a molecular genetic approach, we propose a molecular and cellular model for blastemal function during regeneration.
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MATERIALS AND METHODS |
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Immunohistochemistry
Hematoxylin staining and whole-mount in situ hybridization were performed
as described previously (Poss et al.,
2000a). To generate digoxigenin-labeled probes for this study, we
used a full-length 3.2 kb mps1 cDNA (EST fi61a12) and a full-length
1.2 kb msxb probe (Akimenko et
al., 1995
). Immunohistochemistry on sectioned tissue was performed
as described previously (Poss et al.,
2000b
), using the monoclonal antibody Zns-5
(Johnson and Weston, 1995
).
For simultaneous detection of mps1 mRNA and PCNA, a monoclonal
anti-PCNA antibody (Oncogene, 1:100 dilution) was added following in situ
hybridization during fin incubation with anti-digoxigenin antibody coupled to
alkaline phosphatase. Fins were then washed for at least 2 hours in multiple
changes of phosphate-buffered saline (PBS)-0.1% Tween 20 (PBT), followed by a
final wash in PBT with 2 mg/ml bovine serum albumin (PBTs). Fins were treated
with anti-mouse secondary antibodies coupled to Alexa-488 (Molecular Probes)
in PBTs overnight at 4°C. On the following day, fins were washed in
multiple changes of PBT for at least 2 hours and processed for an alkaline
phosphatase reaction using the NBT/BCIP substrate. Following the detection
reaction, fins were rinsed several times with PBT and processed for
cryosectioning as described previously
(Poss et al., 2000a
). Frozen
blocks were sectioned at 14 µm, mounted using Vectashield with DAPI
(Vector), and digital images were captured using an Axiocam CCD camera
equipped with Axiovision software (Zeiss).
For BrdU incorporation experiments, animals were injected intraperitoneally
with a 2.5 mg/ml solution of BrdU. To detect BrdU and H3P by whole-mount
immunostaining, fin regenerates were incubated in Carnoy's fixative (60%
ethanol, 30% chloroform, and 10% acetic acid) overnight at 4°C and stained
as described (Newmark and Sanchez
Alvarado, 2000). Fins were washed twice in methanol and rehydrated
through a methanol/PBS+0.3% Triton X-100 (PBTx) series. They were then washed
twice in 2 N HCl in PBTx and incubated in 2 N HCl in PBTx for 30 minutes,
followed by two rinses in PBTx and blocking for 4 hours in (PBTx + 0.25% BSA).
Then, fins were incubated with anti-BrdU monoclonal antibodies (Chemicon
International Inc., 1:100) and rabbit polyclonal anti-H3P antibody (Upstate
Biotechnology, 1:200) overnight at 4°C. The next day, fins were washed in
several changes of PBTx (last wash PBTx + 0.25% BSA) and incubated overnight
at 4°C in Alexa 594-coupled goat antimouse antibodies and Alexa
488-coupled goat anti-rabbit antibodies (Molecular Probes), both diluted 1:200
in PBTx. Whole-mount regenerates were washed several times in PBS and examined
by laser confocal microscopy (410 LSM, Zeiss).
The number of mitoses per regenerating ray was counted using
three-dimensional projections of confocal images through the entire depth of
the fin (100 µm). Epidermal cells developed a nonspecific cytoplasmic
fluorescence after H3P staining and secondary antibody detection. This
fluoresence was observed at the distal epidermal edges of the regenerate in
sections of both wild-type and mps1 fins (see
Fig. 6C). Therefore, it was
relatively easy to limit scans to mesenchymal tissue by setting the confocal
depth range to just within the highly stained epidermis. In wild-type
regenerates during outgrowth, this mesenchymal region was 45-55 µm.
Longitudinal sections and work from a previous study confirmed that
mesenchymal mitoses far outnumber epidermal mitoses in the distal regenerate
(see Nechiporuk and Keating,
2002).
|
Genetic mapping and positional cloning
We assigned the ncp gene to LG16 by centromere-linkage analysis,
using CA-repeat markers (Shimoda et al.,
1999) to genotype 51 progeny generated by early pressure of
ncp/+ oocytes (Johnson et al.,
1996
). CA-repeat markers and ESTs
(Hukriede et al., 2001
)
previously localized to the vicinity of the ncp were then utilized to
finely map the ncp mutation in ncp x ncp/+
crosses. All progeny were raised to 2-3 months at 25°C, before phenotyping
for regeneration defects at 33°C. Genotyping information from both
ncp and ncp/+ progeny were used in mapping experiments.
Zebrafish YAC clone pools (Research Genetics) were screened by PCR for
fc38g10 (Clark et al., 2001)
and z6506 sequences (Zhong et al.,
1998
). DNA from YAC43G3 was subcloned into the SuperCos1 cosmid
vector (Stratagene) according to manufacturer's protocol. Cosmid clones were
screened with labeled zebrafish genomic DNA to select those inserts containing
repetitive zebrafish DNA. A cosmid contig was assembled using recombination
data generated from use of cosmid end sequences. After obtaining a critical
region of 80 kb, cosmid inserts were labeled and hybridized to cDNA library
filters generated from 28-hour embryos or adult fin regenerates. mps1
and bckdhb cDNA clones were obtained through hybridization and, after
observing synteny with human 6p 14, from commercial sources (Incyte). To find
the ncp mutation, we amplified 400 bp mps1 fragments from
wild-type and ncp mutant cDNAs, and sequenced these using a 3100
Genetic Analyzer (Applied Biosystems).
Mitotic checkpoint analysis
24-hour postfertilization embryos were dechorionated, disaggregated with a
kontes pestle in 250 µl trypsin solution, and triturated for 10 minutes.
The trypsin was inactivated with 250 µl of 20% FBS/DMEM, and the suspension
passed through 100 µm mesh and then 40 µm mesh. Cells were centrifuged
for 10 minutes, and the pellet was resuspended in 250 µl PBS/embryo. 10
µl/ml of Triton X-100 was added to the suspension, followed by 20 µl/ml
of 0.1 mg/ml DAPI. Cells were kept on ice until flow cytometic analysis. Each
sample comprised cells from 1-4 embryos; 10 wild-type samples and 12
ncp samples were examined.
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RESULTS |
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The ncp mutation was inherited in a recessive manner and identified in a family from which two of seven members displayed regenerative blocks. ncp fin regenerates appeared grossly normal through 2 days postamputation at the restrictive temperature of 33°C, but typically had stalled or regressed back to the amputation plane without forming new bone by 7 days (Fig. 1B). Incubation of ncp adults at 33°C for long periods (over 3 months) had little or no effect on survival, suggesting that the ncp mutation does not hinder general cell survival or adult physiology. All ncp mutants regenerated normally at 25°C. Thus, ncp is a temperature-sensitive mutation that disrupts fin regeneration.
To test if the ncp gene is required for embryonic development, we
raised embryos from heterozygous crosses at 33°C. In our studies,
wild-type zebrafish embryos raised in a 33°C incubator following
fertilization showed considerably reduced viability compared to those raised
at 25-28°C. Although 50% of wild-type or heterozygous zebrafish reached
swimming stage normally, no homozygous ncp mutants attained this
stage (data not shown). Defects displayed by ncp mutants appeared
nonspecific and grossly indistinguishable from those in inviable wild-type or
heterozygous siblings, such as cardiac edema, hooked tail and failure to form
a swim bladder. ncp animals raised at 25°C also appeared to have
compromised viability, as only half of the expected numbers of ncp
from parental backcrosses were recovered during adulthood. This suggested that
the ncp mutation was hypomorphic at 25°C, but not strong enough
to affect regeneration at that temperature. The only consistent observation in
ncp animals raised at 25°C was that smaller adult fish from
parental backcrosses tended to be ncp homozygotes. These observations
support our initial assumption that genes required for fin regeneration are
also required for embryonic or larval development (see also,
Johnson and Weston, 1995).
ncp regenerates display proximal blastemal defects at the
onset of regenerative outgrowth
To determine the cellular mechanism of the ncp regenerative
failure, we examined the histology of ncp regenerates through
different stages of regeneration at 33°C. We found that the early stages
of wound healing and blastema formation appeared normal
(Fig. 2A). However, by 2 days
postamputation, ncp regenerates demonstrated obvious histological
abnormalities, particularly in mesenchymal cells distal to the amputation
plane. To refine our analysis of the ncp defect, we analyzed
regenerates for expression of msxb. We also examined expression of
Zns-5, which recognizes an unknown antigen on scleroblasts, or bone-depositing
cells, and thus can be used to visualize patterning events such as scleroblast
alignment (Johnson and Weston,
1995). At 1 day postamputation, msxb expression was
normal in ncp regenerates (Fig.
2A). Interestingly, msxb expression was maintained in
ncp regenerates at 2-3 days postamputation, often in an expanded
domain, indicating that the mutation spares the distal blastema during
outgrowth. The larger msxb-expressing domain might be due to (1)
failure of blastemal cells to properly condense into the distal blastema, (2)
epidermal pinching and separation of the distal blastema, or (3) compensatory
expansion of the distal blastema as a result of deficiencies in other portions
of the regenerate. Furthermore, we found that scleroblasts were present and
correctly patterned in the most proximal regenerate, suggesting that the
ncp mutation did not directly affect patterning or differentiation
functions (Fig. 2B). These data
indicate that ncp affects a specific subpopulation of cells in the
proximal blastema at the onset of regenerative outgrowth.
|
A mutation in the zebrafish orthologue of mps1 is fully
linked to ncp
To define the ncp gene, we raised 1,751 progeny from ncp
x ncp/+ mapping crosses to adulthood at 25°C, scored for
regenerative defects at 33°C, and genotyped these animals using CA-repeat
and SSCP markers. We found two closely linked markers that flanked a 0.7
centiMorgan (cM) region containing ncp on linkage group 16, and
utilized these markers to retrieve a 950 kb yeast artificial chromosome (YAC)
clone that spanned the critical region. After making a cosmid library from
this YAC clone, we generated additional SSCP markers from cosmid end
sequences. We then used these new markers to refine the genetic map to 0.11
cM. These new flanking markers were contained within two cosmids, representing
approximately 80 kb (Fig.
3A).
|
To identify the ncp gene, we screened a zebrafish cDNA library with radiolabeled cosmid inserts. We identified cDNA clones representing two genes, the zebrafish orthologues of bckdhb (corresponding to zebrafish ESTs fb34c01 and fb54e12) and mps1 (corresponding to zebrafish ESTs fi32g09, fi61a12, and fl31h05). No other genes were identified using this technique. We then searched the human genome database to identify the human syntenic region. Human mps1 (known as TTK) and BCKDHB are located on chromosome 6p14, approximately 50 kb apart. From a search of GenBank no human mRNAs were located between TTK and BCKDHB. These experiments indicate that the ncp mutation is located in either the bckdhb or mps1 genes.
BCKDHB encodes a subunit of a metabolic enzyme mutated in human
maple syrup urine disease (Indo et al.,
1987). To test the candidacy of bckdhb for the
ncp mutation, we sequenced the entire bckdhb coding sequence
from ncp mutants. No nucleotide differences were detected from
wild-type AB strain sequences (data not shown). These data suggest that
bckdhb is not the ncp gene.
Mps1 is an intracellular kinase important for cell proliferation. cDNA sequence analysis revealed that zebrafish mps1 encodes a protein of 983 amino acids. Zebrafish Mps1 is 34% identical at the amino acid level with murine Mps1 (Mpeg1), and 39% identical with X. laevis Mps1. The carboxyl terminus encoding the kinase domain (326 amino acids) is 60% and 71% identical, respectively (GenBank accession number AF488735). To test the candidacy of mps1, we sequenced cDNAs from wild-type and ncp fish. We found one nucleotide difference from wild-type AB strain mps1 cDNA sequence, a thymidine to adenosine (T to A) transversion in the mps1 gene of ncp fish that converts isoleucine 843 to lysine (Fig. 3B). Isoleucine 843 is identical in zebrafish, amphibians and mammals, and resides within kinase subdomain VII. The comparable amino acid in budding and fission yeasts is leucine, a conservative amino acid substitution (Fig. 3C). The sequence of the corresponding region in the C32 inbred strain was identical to that of the AB-derived cDNA. Since ncp was isolated in the C32 inbred background, this indicates that the T to A transversion was caused by the ENU mutagenesis that produced the ncp mutation. Furthermore, the point mutation was unique to the ncp mutant strain among all additional genetic backgrounds examined, including Ekkwill, WIK and SJD, indicating that the I843K mutation is not a variant or polymorphism among laboratory stocks of zebrafish. These data indicate that mps1 is the ncp gene.
ncp embryonic cells display reduced mitotic checkpoint
activity
Mps1 is important for the mitotic checkpoint and centrosome duplication
(Abrieu et al., 2001;
Fisk and Winey, 2001
).
mps1 was initially isolated as a budding yeast mutant that disrupts
spindle pole body duplication, leading to its name
(monopolar spindle)
(Winey et al., 1991
).
Subsequent experiments in yeast revealed a second, distinct role in the
mitotic, or spindle, checkpoint (Weiss and
Winey, 1996
). The mitotic checkpoint is a sensing mechanism that
monitors interactions between kinetochores and microtubules and prevents
sister chromatid segregation until all chromosomes are properly aligned. In
fact, six temperature-sensitive alleles of mps1 that disrupted
mitotic checkpoint activity in S. cerevisae changed residues of the
kinase domain (Schutz and Winey,
1998
). The role of Mps1 in mitotic checkpoint signaling has been
confirmed in S. pombe, X. laevis cell extracts, and human cells in
vitro (He et al., 1998
;
Abrieu et al., 2001
;
Fisk and Winey, 2001
;
Stucke et al., 2002
).
Interestingly, Mps1 was required only for mitotic checkpoint activity and not
centrosome duplication in human cells in vitro and S. pombe
(He et al., 1998
;
Stucke et al., 2002
).
To determine if Mps1 function was affected by the I843K mutation, we examined mitotic checkpoint signaling in ncp embryonic cells. We treated 24-hour postfertilization wild-type and ncp embryos (raised at 25°C) for 4 hours at 33°C with the microtubule-disrupting agent, nocodazole. Nocodazole destroys mitotic spindles and activates the mitotic checkpoint. Cycling cells with normal mitotic checkpoint activity respond to nocodazole treatment by arresting in mitotic metaphase. In our experiments, wild-type embryonic cells clearly arrested in mitosis (4N nuclear content) in the absence of mitotic spindles. While most cycling ncp cells also arrested in mitosis during this period, a subpopulation of cells continued to increase DNA content in the presence of nocodazole, suggesting that these cells entered new cell cycles despite gross spindle defects (Fig. 4). These data indicate that the I843K mutation is associated with abnormal mitotic checkpoint activity in ncp cells. Together with genetic data, these functional data indicate that mps1 is the ncp gene. We will refer to ncp animals hereafter as mps1 mutants.
|
mps1 expression is induced in proximal blastemal cells
To define the timing and pattern of mps1 expression during fin
regeneration, and to determine whether expression is consistent with
mps1 mutant pathology, we performed northern analysis and in situ
hybridization experiments. We found that zebrafish mps1 RNA was
undetectable or present at very low levels in all adult somatic tissues
examined, including unamputated caudal fins. In contrast, northern analysis of
1, 2, and 4 days postamputation fin regenerates indicated induction of
mps1 (Fig. 5A). In
situ hybridization experiments demonstrated mps1 expression beginning
at 18-24 hours postamputation (at 33°C) in the newly formed blastema
(Fig. 5B). At this point,
mps1 was coexpressed with both msxb and proliferating cell
nuclear antigen (PCNA), a marker for actively cycling cells
(Takasaki et al., 1981)
(Fig. 5C). However, during
regenerative outgrowth, mps1 RNA was limited to PCNA-positive
proximal blastemal cells, and was not detectable in the PCNA-negative,
msxb-positive distal blastema or in the patterning zone
(Fig. 5C). These data indicate
that mps1 is specifically expressed in a subpopulation of cells in
the proximal blastema during regenerative outgrowth, findings that are
entirely consistent with mps1 mutant pathology.
|
Mps1 is required for blastemal proliferation during outgrowth
To define the mechanism of the mps1 regenerative defect, we
examined cell cycle entry by BrdU incorporation. We also assayed mitosis by
the presence of phosphorylated histone-3 (H3P)
(Hendzel et al., 1997).
mps1 fin regenerates showed normal BrdU incorporation and H3P
staining at 1 day postamputation (data not shown). However, at the onset of
regenerative outgrowth (2 days postamputation), mps1 blastemal tissue
displayed a slight (30%) reduction in mesenchymal BrdU incorporation
(Fig. 6A). The decrease in
mesenchymal mitoses was more severe, as mps1 regenerates had
approximately one-fifth of the number of mitoses as wild-type at 2 days
postamputation (Fig. 6A,B).
Microscopic examination of H3P-positive nuclei indicated an unusually low
percentage of mps1 mesenchymal cells progressing into later mitotic
phases, suggesting an additional defect in transition to anaphase
(Fig. 6A,B). Finally, by 4 days
postamputation, at which point gross differences between wild-type and
mps1 outgrowth were apparent, we observed unusually large mesenchymal
nuclei in mps1 regenerates (Fig.
6B). Many of these nuclei continued to incorporate BrdU. Increased
nuclear size suggested aneuploidy, and the fact that these enlarged nuclei
continued to synthesize DNA indicated a defect in cell cycle regulation. We
saw normal TUNEL-staining in ncp fin regenerates at 3 days and 7 days
postamputation, indicating that programmed cell death did not play a major
role in the regenerative block (data not shown).
As Mps1 is required for mitotic checkpoint signaling, and mitotic
checkpoint deficiencies have been linked to similar proliferative defects in
both invertebrate and vertebrate embryos
(Kitagawa and Rose, 1999;
Dobles et al., 2000
;
Kalitsis et al., 2000
), the
mps1 proliferative failures likely reflected a reduction in
checkpoint activity. Consistent with this notion and the fact that
mps1 embryonic cells showed checkpoint abnormalities, regenerating
fin cells from mps1 animals also had a diminished response when
challenged at 33°C to arrest in nocodazole (as assayed by increases in
H3P-positive cells; data not shown). Thus, the mps1 regenerative
defect is caused by failed proliferation in proximal blastemal cells during
regenerative outgrowth, indicating that mps1-expressing proximal
blastemal cells have a critical role in regeneration.
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DISCUSSION |
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A model for blastemal function during fin regeneration
Together with our previous findings
(Poss et al., 2000a;
Nechiporuk and Keating, 2002
)
as well as those of others (Laforest et
al., 1998
; Poleo et al.,
2001
), a molecular and cellular model of zebrafish fin
regeneration is beginning to emerge (Fig.
7). After injury, the first obvious step is formation of the wound
epidermis, a non-proliferative event that does not require Mps1. Next,
mesenchymal tissue beneath the epidermis disorganizes; this event may involve
cellular dedifferentiation. The first signs of proliferation are apparent at
12-18 hours postamputation, as mesenchymal cells orient longitudinally, begin
to migrate distally toward the wound epidermis, and form a rudimentary
blastema. Blastema formation involves induction of msxb and
msxc genes, as well as mps1, and depends on intact
fibroblast growth factor signaling. At 48-72 hours postamputation, the
blastema matures to form the distal and proximal blastemal compartments. The
msxb-positive, distal blastema is approximately 5 cell diameters and
is non-proliferative. This region does not induce mps1 expression and
does not require Mps1 function. We believe that the distal blastema may
provide a source of undifferentiated cells for proliferation and
differentiation. The msxb-negative proximal blastema extends 10-20
cell diameters and is highly proliferative. Here, cells are cycling, with a
rapid median G2 cell cycle phase of approximately 60 minutes.
mps1 is induced and required to establish or maintain intense
proliferation in this region, ostensibly the engine of regenerative outgrowth.
The patterning zone comprises the remaining portion of mesenchymal tissue, and
does not express msxb or mps1. Proliferation is less
intense, Mps1 function is not required, and patterning and differentiation
events predominate. These three compartments continue to function until the
regenerative process is completed.
|
Our findings indicate that the distribution of blastemal function into
distinct domains is essential for regeneration. We suspect that extracellular
signaling molecules released from the wound epidermis establish and maintain
these domains during outgrowth. Signaling by Fgfs appears to contribute to
distal blastemal identity, as pharmacological inhibition of Fgf receptors
diminished established blastemal msxb expression
(Poss et al., 2000a). In
addition, recent studies have suggested that Sonic hedgehog might pattern
proximal blastemal cells (Laforest et al.,
1998
; Nechiporuk and Keating,
2002
; Quint et al.,
2002
). It is likely that these molecules or others such as Wnt
factors (Poss et al., 2000b
)
participate in maintaining the proliferative properties of the proximal
blastema. The mps1 strain might be useful for evaluating these
candidate signals. For instance, the augmented msxb-expressing region
in the mps1 mutant regenerate might represent an expansion of the
distal blastema to compensate for compromised proximal function. Potentially,
the expression patterns of epidermal signals that maintain this msxb
expression are also expanded.
A conditional mutation in mps1
We predict that genetic dissection of regeneration requires the production
of conditional mutations that allow determination of in vivo gene function
beyond that gene's earliest requirement. In zebrafish, conditional mutations
can be identified by screening for temperature-sensitive phenotypes; thus,
experimental regulation of gene function is easily accomplished by changing
water temperature. Recently, temperature-sensitive mutations have been
identified in developmentally important genes such as bmp7 and
kit (Dick et al.,
2000; Rawls and Johnson,
2001
). One would also predict that the availability of conditional
mutations is essential for detailed study of Mps1 function in vertebrates, as
Mps1 is required for normal cell division in yeast, and zebrafish
mps1 mutants raised at 33°C fail to complete embryogenesis. This
prediction likely applies to all mitotic checkpoint signaling members; for
instance, mice containing null mutations in the checkpoint genes Mad2
or Bub3 fail to survive past early embryogenesis
(Dobles et al., 2000
;
Kalitsis et al., 2000
).
Accordingly, we expect that the mps1 zebrafish strain, containing a
temperature-sensitive allele of mps1, will be a useful reagent for
studying the mitotic checkpoint in vertebrate processes in addition to
regeneration, such as gametogenesis, organogenesis, and tumorigenesis.
Regeneration genetics
The study of regeneration holds great promise for the emerging field of
regenerative medicine, but to realize this promise, regenerative phenomena
must be understood in molecular terms. Genetic analysis of regeneration in
zebrafish provides a unique instrument for achieving this goal. Zebrafish are
the only genetic model system that reliably regenerates complex tissue.
Accumulating technological advances in zebrafish genetics, including the
genome sequencing initiative, stand to substantially advance discoveries
through regeneration genetics. From our genetic screen for fin regeneration
mutants, we expected to find disruptions in wound healing, tissue
dedifferentiation, blastema formation and proliferation, and organ patterning.
While the mps1 mutant represents a defect in blastemal proliferation
during outgrowth, mutants in other stages of regeneration have also been found
and should shed light on these events
(Johnson and Weston, 1995) (A.
N., K. D. P., S. L. J. and M. T. K., unpublished observations). In future
experiments, we will focus on the early events that initiate fin regeneration,
and also extend our studies to additional organ systems that have not yet been
examined for regenerative potential, such as the heart.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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