Zebrafish as a Model System to Study the Physiological Function of Telomeric Protein TPP1
Citation: Xie Y, Yang D, He Q, Songyang Z (
Zebrafish as a Model System to Study the Physiological Function of Telomeric Protein TPP1
Yiying Xie 0
Dong Yang 0
Quanyuan He 0
Zhou Songyang 0
Wen-Liang Zhou, Sun Yat-Sen University, China
0 1 State Key laboratory for Biocontrol, Sun Yat-Sen University , Guangzhou , People's Republic of China, 2 Verna and Marrs McLean Department of Biochemistry and Molecular biology, Baylor College of Medicine , Houston, Texas , United States of America
Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin) is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1), and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish.
Funding: This work was supported by NCI (http://www.nih.gov) CA133249, NIGMS (http://www.nih.gov) GM081627, and the Welch Foundation (http://www.
welch1.org/) Q-1673. Z.S. is a Leukemia and Lymphoma Society Scholar (http://lls.org/hm_lls). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Vertebrate telomeres are composed of duplex telomeric DNA
repeats with 39 single-stranded overhangs. Along with the
telomerase, a multitude of proteins participate in telomere
regulation. For example, six core telomeric proteins TRF1,
TRF2, RAP1, TIN2, POT1 and TPP1 can form a large molecular
weight complex telosome/shelterin that localize to the
telomere chromatin for telomere length maintenance and end
protection [1,2,3]. Using proteomic approaches that isolate
telomeric protein complexes, we identified human TPP1 as an
important regulator of telomeres, and found it to directly interact
with two other telosome components POT1 and TIN2 through
distinct domain structures on these proteins [4,5]. Furthermore,
such interactions are critical for TPP1 function and telosome
assembly [4,5]. Expression of mutants of TPP1 that failed to
interact with TIN2 or POT1 led to DNA damage responses at the
telomeres and telomere length dysregulation [4,6,7,8]. Similar
results were also obtained when TPP1 levels were inhibited by
The physiological function of telosome components has also
been examined in mice. Homozygous inactivation of TRF1,
TRF2, POT1 or TIN2 in mice led to early embryonic lethality
(,E67) [12,13,14,15,16,17], suggesting that the telosome may
play a critical role during embryonic development. TPP1 knockout
mice die perinatally . A spontaneous autosomal recessive
mutation in the mouse TPP1 gene has also been found (acd mice)
. This hypomorphic mutation occurs in the intronic region of the
TPP1 gene and results in aberrant mRNA splicing . The acd
mice typically die within 12 days after birth. The embryos have
striking defects in caudal specification, limb patterning and axial
skeleton formation. This suggests that TPP1, in addition to serving as
a telomere regulator, may also play important roles in development.
Consistent with this idea, developmental genes such as Wnt3a, Dll1
and Fgf8 displayed altered expression in acd mice .
While in vivo studies in mice and other model organisms have
offered important clues to the physiological function of telomeric
proteins, premature death and early embryonic lethality preclude
detailed analysis of the cell signaling events mediated by these
proteins in development, particularly during early embryogenesis.
Of the many model organisms, zebrafish (Danio rerio) provides
unique advantages for studying gene function and vertebrate
development, such as significant similarities with humans in
genetic information and physiology, rapid ex utero embryo
development and reproductive cycles, and the ease with which
the large and transparent embryos may be manipulated and
observed. In fact, several models of human diseases have been
developed in zebrafish [20,21].
zfTPP1-MO1-mu Translational blocker GACCTTTGATTTAAGCTGCATTCAT
Translational blocker ATCCACAGCAGCCCCTCCATCATCC
MO, morpholino. Mu, mutation.
It has been shown that zebrafish telomere sequences are very
similar to human telomeres in both length and repeat sequences
[22,23]. And the zebrafish telomerase was cloned and
demonstrated to be necessary for hematopoiesis . However, the cell
signaling pathways important for telomere regulation in zebrafish
remain to be elucidated.
We report here the identification of the zebrafish (zf) homologue
of human TPP1. We showed that zfTPP1 could localize to
telomeres, and its domain structure and interaction with other
telosome components such as POT1 and TIN2 was conserved.
Furthermore, knocking down zfTPP1 by morpholino antisense
oligos led to developmental defects including neural death, heart
malformation and caudal defect. These results indicate that TPP1
plays an important role in zebrafish embryogenesis and that
zebrafish presents a good system to study the physiological
function of telomeric proteins.
Materials and Methods
Wild-type zebrafish (strain AB) were raised and maintained in
E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2,
0.33 mM MgSO4, and 0.00001% Methylene Blue) under
standard conditions (28.5uC and 14 hr/10 hr of light/dark cycles)
. Embryo staging includes hours post fertilization (hpf) and
morphological attributes . Animal care was in accordance with
institutional guidelines, and the animal protocol was approved by
the Institutional Animal Care and Use Committee of Baylor
College of Medicine (Assurance number 3823-01) in accordance
with the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH
Publication No. 8523, revised 1996).
Morpholino antisense oligonucleotides
Morpholino antisense oligonucleotides (MO) were obtained
from Open Biosystems (Table 1), and used at a concentration of 1
2 mg/ml in injection buffer (29 mM NaCl, 0.35 mM KCl, 0.2 mM
MgSO4, 0.3 mM CaCl2, 2.5 mM HEPES pH 7.6).
Approximately 16 ng of each MO was injected into zebrafish embryos at 12
Constructs and cell lines
Full-length cDNAs encoding zebrafish (zf) TPP1, TIN2 and
POT1 (GenBank accession numbers HQ652075 and HQ652076)
were obtained from a zebrafish cDNA library (Open Biosystems)
by PCR amplification, and subsequently cloned into
pENTRTOPO vectors (Invitrogen). Primer sequences are listed in Table 2.
FLAG-, TAP- or V5-tagged full-length human TPP1 (hTPP1) or
zfTPP1 were cloned into pCL-based retroviral vectors. The
retroviral vectors were transfected into BOSC23 cells for
packaging . Viruses were collected 48 hours after transfection
and used to infect the zebrafish cell line ZF4  to generate
stable cells. zfTPP1, zfPOT1, and zfTIN2 were also cloned into
pDEST27 (Invitrogen) for the expression of GST-fusion proteins
in mammalian cells. For zfTIN2 homologues, the N-terminal
regions (zfTIN2N1, amino acid 2225; N2, amino acid 2210; N3,
amino acid 2257) were cloned into pDEST27.
Immunoprecipitation and western blotting analysis
For interaction studies, 293T cells  were co-transfected with
the appropriate constructs encoding various proteins using
Lipofectamine2000 (Invitrogen). At 48 hours post transfection, the
cells were harvested and lysed with NETN (20 mM Tris, pH 8,
1 mM EDTA, 100 mM NaCl, and 0.5% NP-40). The extracts
were then incubated for 1 hour at 4uC with anti-FLAG M2
agarose beads (Sigma) or GSH beads (GE). The bound proteins
were eluted in 2x Laemmli buffer, resolved by SDS-PAGE, and
western blotted with the indicated antibodies. The antibodies used
were anti-FLAG M2 HRP (Sigma), anti-V5 HRP (Bethyl
Laboratories), and anti-GST HRP (Roche).
Telomere chromatin immunoprecipitation
Parental ZF4 cells or those expressing zfTPP1-TAP were
crosslinked with 1% formaldehyde for 10 min at room
temperature, rinsed twice with ice-cold 1xPBS, and incubated with 2 M
Figure 2. Characterization of zfTPP1. (A) Schematic representation of domain organization of human and zebrafish TPP1. OB, oligonucleotide/
oligosaccharide binding fold. RD, POT1 recruitment domain. S/T, Serine-rich region. TID, TIN2-interacting domain. (B) Expression of TAP-tagged
zfTPP1 in zebrafish cells. Whole cell extracts from parental and TAP-tagged zfTPP1 expressing zebrafish cell line ZF4 were western blotted with
peroxisome-conjugated protein A. Actin was used as a loading control. (C) Telomere association of zfTPP1. Parental and TAP-tagged zfTPP1
expressing ZF4 cells were crosslinked and used for chromatin immunoprecipitation with protein A beads. The precipitated DNA was analyzed by
southern blotting. (D) Quantification of data in D. Error bars indicate standard error (n = 3).
Glycine to stop crosslinking. The cells were subsequently collected
using a silicon scraper, lysed in lysis buffer (50 mM Hepes, pH 7.5,
150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, and 1%
Triton X-100), and sonicated (Virsonic 600) at 4uC. Each sample
was pre-cleared with 50 ml agarose beads plus 5% BSA and
sheared salmon sperm DNA (5 mg). The supernatant was then
incubated with 20 ml of IgG beads (Amersham) plus BSA (5%) and
sheared salmon sperm DNA (25 mg) for 2 hours at 4uC. Beads
were washed sequentially with the following buffers: buffer I
(50 mM Hepes, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1%
SDS, and 1% Triton X-100), buffer II (buffer I containing
500 mM NaCl), buffer III (10 mM Tris, pH 8.0, 0.25 M LiCl,
1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, and
1 mM DTT), and buffer IV (10 mM Tris, pH 8.0, 1 mM EDTA,
and 1 mM DTT). The bound complexes were eluted and
incubated overnight at 65uC to reverse crosslinking.
Immunoprecipitated DNA and input DNA were dot-blotted onto
nitrocellulose membranes and analyzed by southern blot using the
radiolabelled (TTAGGG)3 probe as described previously .
Acridine Orange staining of zebrafish embryos
Live embryos were dechorionated with forceps and then
incubated in E3 buffer containing 5 mg/ml acridine orange
(Sigma) at room temperature for 10 minutes as described .
The stained embryos were then washed with E3 buffer, and
The TPP1 homologue in zebrafish associates with
Homologues of many human telomere proteins have been
identified in lower organisms that have proven to be important
model systems for studying telomere biology. Sequence and
structural comparisons suggest that zebrafish possess homologues
of all six components of the human telosome (data not shown).
Further analysis revealed that the gene for zebrafish TPP1
(zfTPP1) is located on chromosome 7. We subsequently cloned
the cDNA for zfTPP1, and found it to encode a polypeptide of 487
amino acids that shares extensive sequence and structural
similarities with human TPP1 (hTPP1) (Fig. 1, 2A). Importantly,
zfTPP1 appears to contain all the domains important for hTPP1
interaction with TIN2 and POT1, including the N-terminal OB
fold, the POT1-binding RD domain, and the C-terminal
TIN2interacting domain (TID).
The conservation of domain structures in zfTPP1 suggests its
possible role in regulating zebrafish telomeres. To further probe
this idea, we first sought to determine whether zfTPP1 could
localize to telomeres in zebrafish. Because antibodies against
zfTPP1 are unavailable, we examined the targeting of ectopically
expressed zfTPP1. Zebrafish ZF4 cells stably expressing
TAPtagged zfTPP1 were generated through infection with VSVG
pseudo-typed retroviruses (Fig. 2B). Chromatin
immunoprecipitation (ChIP) experiments were subsequently carried out. As shown
in Figure 2C and 2D, telomere sequences were enriched in IgG
pull-down samples from TAP-tagged zfTPP1 expressing cells,
indicating that zfTPP1 can associate with telomere DNA in
zebrafish cells. The amount of telomeric DNA that was brought
down by zfTPP1 appeared low, possibly due to lower expression of
zfTPP1-TAP in these cells.
Interactions between zfTPP1 and zfPOT1
In human cells, the interaction between TPP1 and POT1 is
mediated by the TPP1 RD domain and the POT1 PBR domain
. To determine whether such interactions also occur in
zebrafish, we first cloned the cDNA for zebrafish POT1 (zfPOT1).
As in the case of humans, the zebrafish genome contains only one
POT1-like gene, whose product exhibits high homology to human
POT1 (Fig. 3A). Moreover, zfPOT1 also harbors two N-terminal
OB folds and a C-terminal domain similar to the TPP1 binding
domain (PBR) of human POT1.
The interaction between zebrafish TPP1 and POT1 was
examined in 293T cells. GST-tagged full-length zfPOT1 was
coexpressed with FLAG-tagged full-length zfTPP1 and TPP1 RD
domain deletion mutant. Consistent with previous findings in
human cells , the interaction between zebrafish POT1 and
TPP1 also required the RD domain (Fig. 3B, C), as zfTPP1-DRD
failed to co-immunoprecipitate with zfPOT1. Similarly, the
Cterminal PBR domain of POT1 was much more efficient at
coprecipitating with zfTPP1 (Fig. 3C) . These data indicate that
zfTPP1 interacts with zfPOT1 through their respective RD and
Interactions between zfTPP1 and zfTIN2
To identify zebrafish TIN2 homologues, we utilized a zebrafish
cDNA library. Interestingly, we were able to clone three potential
TIN2 homologues that we named zfTIN2-1, zfTIN2-2, and
zfTIN2-3. The three putative TIN2 homologues share an
Nterminal domain that is homologous to hTIN2, but harbor
different C-terminal sequences. It should be noted that only
zfTIN2-1 contains a putative TRFH-binding-motif (TBM) [28,29]
The N-terminal region of TIN2 has been shown to interact with
TPP1 . We therefore co-expressed zfTPP1 with the N-terminal
regions of the three candidate zebrafish homologues in 293T cells
to examine their interactions with TPP1. As shown in Figure 4b,
only the N-terminal region of zfTIN2-1 was able to
coimmunoprecipitate with zfTPP1. Taken together with our
sequence analysis data, these findings indicate that zfTIN2-1 is
the zebrafish ortholog of hTIN2 (and hitherto referred to as
zfTIN2). Interestingly, zfTIN2 is located on chromosome 7 and in
the vicinity of the zfTPP1 locus, raising the possibility of
coevolution of these two genes. Furthermore, deletion of the
Cterminal TIN2 interaction domain (TID) on zfTPP1 (amino acids
462487) abolished its interaction with zfTIN2 (Fig. 4c), indicating
Figure 4. zfTPP1 interacts with zfTIN2-1 through its C-terminus. (A) Schematic representation of domain homology between human and
zebrafish TIN2. NTD, N-terminal domain. TBM, TRFH-binding motif. (B) zfTPP1 interacts with zfTIN2-1. Extracts from 293T cells co-expressing
V5tagged zfTPP1 or hTPP1, together with FLAG-tagged N terminal part of zfTIN2-1, zfTIN2-2, or zfTIN2-3, were immunoprecipitated with anti-FLAG
antibodies and western blotted. FLAG-tagged hTIN2 was also included. (C) The C terminal domain of zfTPP1 mediates its interaction with zfTIN2-1.
Extracts from 293T cells co-expressing GST-tagged full-length zfTIN2-1 with FLAG-tagged full-length zfTPP1 or zfTPP1-DC were analyzed by
immunoprecipitation and western blotting.
the importance of the TID domain in mediating TIN2-TPP1
interaction in zebrafish. It is also interesting to note that hTPP1
could interact weakly with zfTIN2 and vice versa (Fig. 4b), further
highlighting the evolutionary conservation of important domains/
residues for telomere protein interactions.
Collectively, our results demonstrate that zfTPP1 can localize to
telomeres and interact with zfPOT1 and zfTIN2, as is the case in
human cells. These findings suggest that the subunits and function
of the telosome may be conserved between zebrafish and human,
and provide the basis for using zebrafish as a model organism to
study the physiological function of telosome subunits.
Knockdown of zfTPP1 by morpholino oligonucleotides
resulted in developmental defects
In zebrafish, the function of genes can be assessed through
morpholino-mediated knockdown . For example, morpholino
antisense oligonucleotides are often designed to bind near the
translation initiation sites or splicing junctions on mRNA
sequences of the target gene, thereby inhibiting the expression of
morpholino-targeted genes. This block of expression does not
occur through target mRNA degradation, and therefore cannot be
assessed by examining mRNA levels. Morpholino oligos may be
injected into zebrafish embryos at different stages to determine
how disrupting expression of a particular gene may affect zebrafish
To investigate the cell signaling pathways of TPP1, we utilized
morpholino oligonucleotides against TPP1 to knockdown TPP1 in
zebrafish embryos (Fig. 5). Four different morpholino oligos
(25mer) were generated for the experiment (Table 1). MO1 targets
the translational initiation site of the zfTPP1 mRNA, while MO2
is against the 59UTR region of zfTPP1 mRNA. These two oligos
are expected to block zfTPP1translation. MO1-mu targets the
same region as MO1 but carries five missense mutations, and
Figure 5. Expression of zfTPP1 morpholinos leads to defects in embryonic development. Mock injected zebrafish embryos (a, b) and
those injected with zfTPP1 MO1 (6 ng) (c, d) and MO2 (3 ng) (e, f), zfTPP1 MO1 mutant (6 ng) (g, h), or Chordin MO (6 ng) (i, j), were observed under
the microscope at 28 and 52 hour post fertilization (hpf). Magnified images of the trunk region of mock and MO1 injected embryos were also
included. Arrows indicate blood in the embryos. Scale bar, 500 mm.
therefore serves as a control for the specificity of MO1.
ChordinMO is against the development regulator Chordin, and serves as a
positive control for morpholino knockdown. Zebrafish embryos at
one or two-cell stage were injected with the morpholino oligos and
then allowed to further develop for another 2852 hours before
Consistent with published reports, injection of Chordin-MO led
to U-shaped somites and an abnormal tail fin with multiple folds
(Fig. 5i, 5j) . In comparison, introduction of either MO1 or
MO2 morpholinos resulted in severe developmental defects in
zebrafish embryos (Fig. 5c-f). The defects caused by MO1 and
MO2 appeared similar. Notably, MO1-mu injection did not lead
to any overt phenotypical changes (Fig. 5h), suggesting that
loss of zfTPP1 activity was responsible for the defects caused
by MO1 and MO2. A lower dose of MO2 (3 ng) was needed
to produce these phenotypes compared to MO1 (6 ng), a likely
result of differences in knockdown efficiencies between these two
At 28 hours post fertilization (hpf), mock injected or MO1-mu
injected embryos developed clear heads with easily identifiable
brain structures (Fig. 6a). In contrast, most zfTPP1 morphants
(MO1 or MO2 injected embryos) (,90%) developed a dense and
opaque head (Fig. 6b), suggesting defects in neural development.
At 52 hpf, mock injected or MO1-mu injected embryos exhibited
straight torsos with somites (Fig. 5b, 6c). However, around 80% of
zfTPP1 morphants developed curly tails (Fig. 6d), suggesting a
defect in caudal development. Interestingly, a similar caudal defect
phenotype was also observed in acd mice that carry a TPP1
During normal zebrafish embryogenesis, a simple heart tube
composed of the myocardium (outer muscular layer) and the
endocardium (inner endothelial layer) emerges by 24 hpf. It then
transforms into two morphologically distinct chambers with the
linear heart tube bending gradually at the boundary between the
two chambers to create an S-shaped loop . At 52 hpf, the heart
of the mock injected embryos clearly formed a loop with two
chambers and contracted at a rate of ,180 beats/minute, driving
normal blood flow (Fig. 6e). In MO1 injected embryos, on the
other hand, looping appeared incomplete and remained central
and linear (Fig. 6f). Moreover, pericardial oedema enlargement
of the heart cavity (space surrounding the heart) was very
apparent (Fig. 6f). We observed low cardiac contractility (,50
beats/minute) and strikingly, little blood flow through the heart
(Fig. 6f). Consequently, blood became pooled elsewhere in the
morphant body (Fig. 6d). Taken together, these observations point
to profound defects in the cardiovascular development of the
zfTPP1 morphants display excessive apoptosis
In multicellular organisms, early development is a tightly
controlled balance between cell proliferation, differentiation, and
apoptosis. Given the important function of telomeric proteins in
maintaining genome stability, we reasoned that the defects in
zfTPP1 morphants could be a result of dysregulated progenitor
cells or apoptosis.
Upon close examination of the zfTPP1 morphants under
Nomarski DIC optics, we observed many button-like objects in
MO1 injected embryos (Fig. 7). We hypothesized that these objects
might in fact be apoptotic cells and tested this idea using acridine
orange, a vital dye that specifically stains apoptotic cells through its
direct binding to nucleic acid .
At 28 hpf, differences in acridine orange staining patterns
between mock injected embryos and zfTPP1 morphants became
quite pronounced (Fig. 8). The mock injected embryos generally
exhibited no staining (Fig. 8b, 8f). In the tail region where normal
developmental apoptosis occurs at this stage for shaping tails, a few
Figure 7. zfTPP1 knockdown leads to accumulation of button-like structures. Nomarski DIC images of regions of the head (a, b) and body
(c, d) of mock and zfTPP1 MO1 (6 ng) injected embryos were obtained at 28 hpf. Button-like structures are indicated by arrows. Scale bar, 5 mm.
acridine orange stained cells could be observed (Fig. 8j). These
stained cells correlated with the button-like objects observed under
Nomarski optics (Fig. 8), suggesting that the buttons were indeed
apoptotic cells. In contrast, the zfTTP1 morphants exhibited
extensive staining throughout the body (Fig. 8d, 8h, 8l). The
pattern also correlated with the button-like objects we observed
(Fig. 8o, 8p), suggesting excessive apoptosis in zebrafish embryos
knocked down for TPP1 and that extensive apoptosis may be
responsible for the phenotypes of zfTPP1 morphants.
In this study, we reported the identification of zebrafish
homologues of human TPP1, POT1, and TIN2. We presented
evidence that zfTPP1 could localize to the telomeric chromatin,
and demonstrated the interactions between zebrafish TPP1 and its
partners TIN2 and POT1. For example, we showed that the
Cterminal region of zfPOT1 was responsible for mediating its
interaction with zfTPP1. And the C-terminal tail and putative RD
domain in zfTPP1 were essential for its interaction with zfPOT1
and zfTIN2. Such mutational analysis indicates that the domains
utilized to mediate interactions between these proteins in zebrafish
are similar to those in mammalian cells. These observations
indicate that the components and organization of the telosome are
conserved from zebrafish to human, providing the molecular basis
for using zebrafish to understand the function and signal
transduction of human telomeric proteins.
Compared to mice, zebrafish represents an attractive alternative
in vivo model. The telomere length of laboratory inbred mice is
extremely long (,50150 kb) [34,35], whereas the length of
zebrafish telomeres (,420 kb) is much closer to that of human
telomeres (,810 kb) [23,24,36]. Furthermore, the availability of
large amounts of embryos within a short amount of time and the
easily observable embryogenesis and organogenesis processes
make zebrafish ideal tools for studying genes whose disruption in
mice may lead to embryonic lethality.
Mice homozygous knockout for TPP1 die perinatally . The
acd mice carrying a hypomorphic mutation in the TPP1 gene show
reduced TPP1 expression and phenotypes including urogeneital
developmental defects and caudal truncation [19,37,38]. zfTPP1
knockdown by morpholinos also led to the death of zebrafish
embryos. In this case, however, we were able to document the
morphological changes that occurred with TPP1 inactivation
during embryogenesis. Defects in multiple systems were observed
including neural development and caudal specification,
phenotypes that are strikingly similar to those observed in acd mice.
In zfTPP1 morphants, we also observed incomplete heart
development with associated progressive pericardial edema, low
Figure 8. zfTPP1 knockdown induces apoptosis. Embryos injected with zfTPP1 MO1 were stained with acridine orange and visualized (at 28hpf)
under Nomarski DIC and fluorescent microscopes (scale bar, 5 mm). Magnified Nomarski DIC and fluorescent images (28 hpf) of the tail (mock
injected) (m, n) and head region (zfTPP1 MO1 injected) (o, p) of zebrafish embryos. Arrowheads indicate button-like structures.
cardiac contractility, and blood pooling. Defects in cardiovascular
development have not been described in acd mice. Previous studies
have indicated that cardiac myocytes undergo continuous renewal,
where senescent and poorly contracting myocytes are replaced
with younger and more efficient cells . Old myocytes undergo
cellular senescence while new myocytes can derive from cardiac
stem or progenitor cells. It is interesting to note that old myocytes
appear to have much shorter telomeres compared to young
myocytes [39,40,41], suggesting that telomere maintenance
pathways may play a role in this process. In addition, the ability
of cardiac progenitor cells to divide might also be under the
regulation of the telomerase and telomeric proteins. To date,
TPP1 is the only protein in the telosome found to directly interact
with and recruit the telomerase [7,42]. One possible explanation
for the heart defects in zfTPP1 knockdown embryos is the reduced
ability of cardiac progenitor cells to produce new myocytes. As a
result, both the number and vigor of cardiac myocytes are greatly
compromised. This idea is consistent with the poor contraction we
observed in zfTPP1 knockdown embryos. Alternatively, the
cardiomyocytes in zfTPP1 knockdown embryos may undergo
accelerated senescence. Consequently, newly formed cardiac
myocytes are not sufficient to fulfill all the functional requirement
of the cardiovascular system. The role of zfTPP1 in heart
development warrants future investigation.
zfTPP1 loss of function appeared to trigger excessive apoptosis
in zebrafish embryos as well. While off-target effects cannot be
completely ruled out, the fact that two independent morpholino
oligos that target different steps during TPP1 expression argues
against this possibility. In mammalian cells, TPP1 knockdown
leads to DNA damage responses at the telomeres, where multiple
DNA damage response proteins are recruited to the telomere ends
. In fact, disruption of the telosome complex through inhibition
of its subunit in general leads to DNA damage response at the
telomeres and p53 activation [3,43]. The skin hyperpigmentation
and hair growth defects in TPP1 deletion mice can be rescued by
p53 deficiency . The caudal dysgenesis in acd mice is also
dependent on p53 pathway . These data suggesting that the
involvement of apoptosis in TPP1 deficiency is conserved across
We thank Dr. Mary Ellen Lane (Rice University) for helping us with
zebrafish studies, and Dr. Dan Liu for technical help.
Conceived and designed the experiments: YX DY. Performed the
experiments: YX DY. Analyzed the data: YX DY. Contributed
reagents/materials/analysis tools: QH. Wrote the paper: ZS.
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