Mps1 defines a proximal blastemal proliferative compartment essential for zebrafish fin regeneration
Kenneth D. Poss1,*,
,
Alex Nechiporuk1,2,*,
Ann M. Hillam1,
Stephen L. Johnson3 and
Mark T. Keating1,
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
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Fig. 6. The mps1 regeneration defect is caused by severe blastemal
proliferative abnormalities. (A) Indices of proliferation at 2 days
postamputation. (Left) BrdU incorporation data were obtained from counting
500-3,000 mesenchymal nuclei from 6-10 sections of each of five whole-mount
immunostained regenerates. (Middle) A total of 14 regenerating rays from six
wild-type fish and 21 rays from eight mps1 animals were used to count
H3P-positive nuclei. (Right) A total of 1,355 H3P-positive nuclei from eight
wild-type regenerates and 704 H3P-positive nuclei from 10 mps1
regenerates were scored for mitotic phases at 500x magnification.
Results are shown as mean ± s.e.m. (t-test;
*P<0.05, **P<<0.001). (B)
(Left) Confocal projections of 2-day postamputation fin regenerates stained
with anti-H3P to indicate mesenchymal mitoses. The bright points are
individual mitotic nuclei, severely reduced in mps1 regenerates. Both
fins show non-specific epidermal fluorescence at the distal edge (see
Materials and Methods). (Middle) High magnification confocal images of
H3P-positive mesenchymal nuclei. An mps1 fin ray with an unusually
high number of mitoses is shown. Arrowheads point to late phase mitoses,
deficient in mps1 fin regenerates. (Right) Projections of single
4-day postamputation fin rays from animals that have incorporated BrdU for the
final 5 hours of regeneration. Note the reduced incorporation, and unusually
large nuclei in cycling mps1 cells that are suggestive of aneuploidy
(arrowheads in right image). Original magnification is 150x (left
panels) and 945x (middle and right panels).
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Fig. 1. Genetic screen for regeneration and identification of the ncp
mutant. (A) Illustration depicting mutagenesis and screen for
temperature-sensitive fin regeneration mutants. (B) Whole-mount wild-type and
ncp caudal fin regenerates at 2 and 7 days postamputation. The
ncp mutant showed a clear defect in fin regeneration by 7 days
postamputation. Arrows demarcate amputation plane in each photo. Original
magnification is 15x.
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Fig. 2. ncp fin regenerates display defects in proximal blastemal cells
during regenerative outgrowth. (A) Longitudinal sections of 1-day
postamputation fin regenerates during blastema formation, with the distal,
regenerating end shown at the top. At this stage, ncp regenerates
display a typical blastema with normal msxb expression (violet
stain). (B) Sections of regenerates at the onset of outgrowth. (Top)
Hematoxylin stains of ncp regenerates indicate a mesenchymal
compartment with a reduced number of blastemal cells. (Middle) msxb
expression is maintained and even expanded in ncp regenerates despite
blastemal reduction. (Bottom) The antibody Zns-5 detects scleroblasts, or
bone-depositing cells (brown stain), which align bilaterally in the patterning
zone by 2 days postamputation and begin to deposit mineral. Note that the
ncp regeneration defect was first apparent at the onset of
regenerative outgrowth and had little or no effect on the establishment of the
distal blastema or patterning zone. Arrows indicate point of amputation.
Original magnification is 250x.
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Fig. 3. Linkage of a kinase mutation in mps1 to ncp. (A) Genetic
and physical map of ncp region indicating genomic DNA, YAC clone,
cosmids, and cDNAs. Numbers in parentheses represent recombination events from
1,751 meioses in regions between ncp and linked genetic markers.
(B,C) Mutational analysis of mps1. Sequencing of cDNAs from
ncp and several wild-type strains revealed a unique thymidine to
adenosine mutation that altered isoleucine-843 to lysine (red). A portion of
the highly conserved carboxyl terminal kinase domain of Mps1 containing the
I843K mutation is shown. This isoleucine is highly conserved among
vertebrates. Thus, a mutation in mps1 is associated with the
ncp regeneration defect.
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Fig. 4. Reduced mitotic checkpoint activity in ncp cells. Histograms from
flow cytometric analysis of DAPI-stained cells from 24-hour postfertilization
wild-type and ncp embryos. (Left) FACs profile of cells collected
from embryos (raised at 25°C) after a 4-hour incubation at 33°C. The
majority of cells in wild-type and ncp samples had a 2N nuclear
content. (Right) FACs profile of cells collected from embryos treated with 1
µg/ml nocodazole during the 4-hour 33°C incubation. A large 4N peak,
representing cells that have arrested in mitosis, appeared in both wild-type
and ncp histograms. However, note the substantial >4N
subpopulation in the ncp histogram (arrows). Such cells presumably
reflect those that failed to arrest in mitosis and continued to synthesize DNA
despite the absence of mitotic spindles. High DNA content (>2% of cells
with DNA content over 4N) was not observed in cell suspensions from
nocodazole-treated wild-type embryos (n=10), but was seen in 10 of 12
suspensions from treated ncp embryos. Thus, mitotic checkpoint
activity is reduced in ncp embryonic cells, indicating that the I843K
mutation disrupts Mps1 function in zebrafish.
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Fig. 5. mps1 is induced in the proximal blastemal proliferative zone
during regenerative outgrowth. (A) Northern analysis of mps1
expression using several adult tissues as well as regenerating caudal fin
tissue. Blots were also probed for ß-actin expression as a
control to indicate the amounts of RNA loaded. (B) Whole-mount in situ
hybridization of mps1 in wild-type 1-day and 3-day postamputation fin
regenerates (violet stain indicated by arrowhead). mps1 RNA levels
were increased in the newly-formed blastema at 1 day postamputation (top) and
these levels were maintained in the blastema during regenerative outgrowht
(bottom). Whole-mount mps1 signals appeared stronger than section
mps1 signals in 1-day regenerates, an observation that is common at
that timepoint for other genes. This likely represents somewhat weak but
widespread signals in individual blastemal cells that appear stronger when
visualized en masse. (C) (Left and center) Longitudinal sections of wild-type
1- and 3-day fin regenerates co-stained for mps1 RNA and PCNA protein
(green). mps1 was upregulated in the most highly proliferative cells
during outgrowth (brackets), but was absent from the distal blastema. (Right)
msxb RNA localization (violet, arrowhead at 3 days) in the newly
formed blastema at 1 day and the distal blastema at 3 days postamputation.
Thus, in the new blastema, mps1 colocalizes with PCNA and
msxb. However, mps1 is specifically induced in the proximal
blastema during outgrowth. The morphological difference between 3-day
regenerates shown in Fig. 5C represents variation commonly seen in fin
regenerates during outgrowth. Original magnifications: 50x in B and
110x in C.
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Fig. 7. Cellular and molecular model for fin regeneration. During outgrowth, the
distal blastema (DB) is defined by msxb (orange), the proximal
blastema (PB) by mps1 (blue), and the patterning zone (PZ) by newly
patterned scleroblasts (brown) and differentiating mesenchyme (yellow).
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© The Company of Biologists Ltd 2002
