An Unexpected Role for the Clock Protein Timeless in
Linda P. O’Reilly1, Simon C. Watkins2, Thomas E. Smithgall1*
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 2Department of
Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
Background: Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of
the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells.
Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse
embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian
ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the
differentiation of mES cells.
Methodology/Principal Findings: To determine whether Tim is involved in ES cell differentiation, Tim protein levels were
stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which
models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-
knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the
pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced
basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-
knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and
programmed cell death during cavitation.
Conclusions/Significance: Our data demonstrate a novel function for the clock protein Tim during a key stage of early
development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a
consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These
findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that
circadian rhythms and early development may be intimately linked.
Citation: O’Reilly LP, Watkins SC, Smithgall TE (2011) An Unexpected Role for the Clock Protein Timeless in Developmental Apoptosis. PLoS ONE 6(2): e17157.
Editor: Maurizio Pesce, Centro Cardiologico Monzino, Italy
Received August 19, 2010; Accepted January 24, 2011; Published February 17, 2011
Copyright: ? 2011 O’Reilly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by National Institutes of Health grant GM077629 (to TS). 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.
* E-mail: email@example.com
Virtually all mammalian cell types express multiple members of
the Src protein-tyrosine kinase family, which regulate diverse
pathways involved in cell growth, survival, differentiation,
adhesion, and migration . The mammalian Src family consists
of eight members, some of which exhibit fairly broad tissue
distribution patterns in adults (c-Src, c-Yes, and Fyn) with the
balance showing more lineage-restricted expression patterns
particularly in cells of hematopoietic origin (e.g., Hck and Fgr in
myeloid leukocytes, Lck in T-lymphocytes, Blk in B-cells, and Lyn
in multiple hematopoietic cell types). Strict control of Src-family
kinase (SFK) activity is essential to normal cellular function and
development , and loss of kinase regulation contributes to
several forms of cancer and other diseases .
The SFKs exhibit homologous domain organization which
includes a myristoylated (and in most cases palmitoylated) N-
terminal region, modular SH3 and SH2 domains, a helical SH2-
kinase linker, a bilobed kinase (catalytic) domain, and a C-terminal
negative regulatory tail. The crystal structures of near full-length
versions of c-Src and Hck show that the SH3 and SH2 domains
both contribute to negative regulation of kinase activity [4–7].
These non-catalytic domains pack against the back of the kinase
domain to stabilize a closed, inactive conformation. In this state,
the SH3 domain engages the SH2-kinase linker, which adopts a
polyproline type II helical conformation typical of SH3 docking
sites. The SH2 domain interacts with the C-terminal tail, which is
phosphorylated on a conserved tyrosine residue by the indepen-
dent regulatory kinases, Csk and Chk [8,9]. Relevant to the
current study is the observation that SFK activity must be carefully
regulated during mammalian development. While genetic knock-
outs of individual SFKs produce well-defined phenotypes in most
cases, these SFK-null animals remain fertile and viable . In
contrast, knockout of Csk, one of the upstream regulators of the
Src kinase family, results in embryonic lethality with concomitant
upregulation of SFK activity in the non-viable embryos .
Recent work has implicated SFKs in mouse and human ES cell
growth and differentiation [12–14]. In mES cells, seven of the
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eight mammalian SFKs are expressed and appear to serve non-
redundant functions . In this previous study, differentiation of
mES cells to EBs was shown to correlate with transcriptional
silencing of Hck and Lck, whereas c-Src and Fyn were expressed
and remained active in both pluripotent mES cells and
differentiated EBs cultured from them . Suppression of all
SFK activity with the broad-spectrum pyrrolo-pyrimidine SFK
inhibitor A-419259 substantially delayed EB formation from mES
cells, and the inhibitor-treated cultures retained the characteristics
of pluripotent ES cells . Extending these earlier findings with a
chemical genetics approach, our group recently established a non-
redundant role for c-Src activity in mES cell differentiation .
Here, mES cells were engineered to express an inhibitor-resistant
variant of c-Src; when these cells were treated with the inhibitor,
they underwent differentiation to primitive ectoderm despite the
presence of LIF. Similar experiments with inhibitor-resistant
variants of other SFKs had no observable effects. Thus, these
studies strongly suggest that c-Src activity alone is sufficient to
allow mES cells to exit from the self-renewal program and begin to
differentiate. In the present study, we applied a proteomics
approach to identify unique binding partners for c-Src in self-
renewing vs. differentiated ES cells as a way to define signaling
pathways linking Src-family kinases and ES cell fate. These studies
identified Tim, a circadian rhythm protein, as a previously
unrecognized binding partner and substrate for c-Src and a key
regulator of EB formation.
Tim was first described in the context of the Drosophila circadian
cycle, where clock proteins regulate their own syntheses in a
rhythmic fashion . Proteosomal degradation of circadian
proteins, including Tim, eliminates negative feedback on tran-
scription and allows RNA levels to rise. Regulation of Drosophila
Tim levels by targeted degradation is preceded by tyrosine
phosphorylation, although the precise kinase(s) involved have not
been identified [16,17]. Although not definitively placed in the
circadian pathway in mammals, Tim expression exhibits 24-hour
oscillation and it associates with the core clock components Period
and Cryptochrome [18,19]. Furthermore, conditional knockdown
of Tim in the rat suprachiasmatic nucleus disrupted the rhythms of
neuronal activity and altered levels of clock elements, suggesting a
role similar to that of its Drosophila counterpart . Mammalian
Tim also appears to link the circadian and cell cycles and may
have additional functions in DNA damage control [20,21].
Genetic studies show that Tim is essential for early embryonic
development, as homozygous knockout produces early embryonic
lethality in mice . In this study, we show that Tim is expressed
in mES cells and is essential for the differentiation of ES cells to
Results and Discussion
Previous studies summarized above point to the c-Src protein-
tyrosine kinase as an important regulator of the earliest stages of
mES cell differentiation. These findings led to the question of the
signaling pathways controlled by c-Src that account for its role in
ES cell fate. To address this question, c-Src target protein capture
experiments were performed using an immobilized, recombinant
c-Src SH3 domain fusion protein and soluble protein extracts from
both self-renewing mES cells as well as differentiated EBs. SH3
domains contribute not only to SFK regulation (see Introduction)
but also to substrate recruitment by binding to target proteins
containing polyproline type II helices . Unique SH3-
interacting proteins were captured by the c-Src SH3 domain but
not by an inactive mutant control domain, indicative of specific
binding (Fig. 1). Three prominent bands were excised from the gel,
digested with trypsin, and identified by MALDI-TOF MS and
MS/MS sequencing: 1) Dynamin II, a GTPase involved in
vesicular trafficking ; 2) hnRNPK, which regulates transcrip-
tion, pre-mRNA processing, mRNA transport and translation
; and 3) a 54 kDa N-terminal fragment of Tim. Both Dynamin
II and hnRNPK have been identified previously as c-Src SH3-
binding proteins [26,27], validating our experimental approach. In
contrast, the SH3-dependent association of Tim with c-Src or
other SFKs has not been reported, suggestive of a novel
interaction. To confirm that Tim is an SH3-binding partner for
c-Src, SH3 capture experiments were repeated using lysates from
ES cells and EBs, followed by immunoblotting with an antibody to
the Tim protein. Full-length Tim was captured in each case, as
well as a prominent cleavage product that corresponds in size to
the fragment originally identified by tryptic fingerprinting (Fig. 2A).
In contrast, no binding was detected with the inactive mutant of
the c-Src SH3 domain or with GST alone, indicative of a specific
SH3-mediated binding event.
We next investigated whether Tim is a substrate for c-Src. For
these experiments, full-length Tim was co-expressed with wild-
type c-Src or a kinase-defective mutant in the human epithelial cell
line, 293T. Tim was then immunoprecipitated from transfected
cell lysates, and analyzed for tyrosine phosphorylation by
immunoblotting with anti-phosphotyrosine antibodies. As shown
in Figure 2B, Tim was strongly phosphorylated in the presence of
active c-Src but not with a kinase-dead mutant, demonstrating that
Tim is a Src substrate. To investigate a possible relationship
between Tim tyrosine phosphorylation and ubiquitylation, as
observed previously in Drosophila , aliquots of the same Tim
immunoprecipitates were also immunoblotted with ubiquitin
antibodies (Fig. 2B). Co-expression with c-Src strongly enhanced
Tim ubiquitylation, suggesting that Src-mediated Tim phosphor-
ylation enhances this process. Interestingly, co-expression of Tim
with kinase-inactive c-Src suppressed Tim ubiquitylation below
control levels observed in the absence of c-Src expression,
suggestive of a dominant negative effect (Fig. 2B).
To investigate the role of Tim in mES cells, we first over-
expressed the protein and observed a reduction in cell viability and
enhanced apoptosis compared to untransfected control cells (data
not shown). Because of the negative impact of Timeless expression
on ES cell viability, we turned to the complementary approach of
gene silencing. In these experiments, Tim expression was knocked
down by lentiviral transduction of shRNAs targeting two distinct
regions of the Tim transcript. Both lentiviral vectors yielded ES
cell populations with substantial reductions in endogenous Tim
protein levels (data not shown). Six Tim knockdown cell lines were
subsequently cloned from each shRNA-transduced mES cell
population, and screened for the extent of Tim knockdown by
immunoblot analysis (Fig. 3). The two cell lines exhibiting the
greatest extent of full-length Tim knockdown without changes in
undifferentiated colony morphology were selected for further
analysis. These lines were designated as lenti:87-22 and lenti:89-
Morphologically, self-renewing cultures of both Tim-knock-
down ES cell lines exhibited less spontaneous differentiation when
compared to wild-type ES cells (Figs. 3 & 4A). To investigate
whether the loss of Tim influenced the expression of self-renewal
markers, we examined the levels of Oct4, Sox2, Nanog, and KLF4
by quantitative immunoblotting. As shown in Figure 4B, both
Tim-knockdown lines exhibited modest increases in the expression
of Oct4 and Sox2, while levels of Nanog and KLF4 were
essentially unchanged. Conversely, expression of the differentia-
tion marker AFP was decreased by more than 60% relative to
control ES cells. These changes most likely reflect loss of
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spontaneous differentiation as a consequence of Tim knockdown,
although an influence on the ES cell self-renewal program cannot
be ruled out.
To determine whether the presence of Tim is essential for early
development, we next tested the ability of the Tim knockdown ES
cell lines to form EBs. In the EB assay, ES cells are plated in
suspension in the absence of LIF, the cytokine required for the
maintenance of mES cell pluripotency. Under these conditions, the
cells differentiate into organized cysts that recapitulate the initial
stages of pre-implantation development, including formation of an
endodermal surface layer, differentiation of columnar epithelium,
and hollowing out of a central cavity via apoptosis [28,29]. While
wild-type ES cells differentiated into a typical heterogenous EB
population with respect to size, both Tim knockdown lines
produced smaller EBs of more uniform size (Fig. 5). Under normal
conditions, cystic EBs undergo expansion as they differentiate. Thus
the consistent formation of smaller EBs from the Tim knockdown
ES cells suggested a failure to expand and a possible differentiation
defect. To address this issue, we harvested control and Tim
knockdown EBs after six days of development and imaged them for
cavitation by confocal microscopy. As shown in Figure 6, the
number of EBs exhibiting cavity formation was substantially
reduced in both Tim knockdown ES cell lines. This result may
help to explain the early embryonic lethality previously observed in
Tim knockout mice. At ED 7.5, homozygous Tim knockout
embryos lack cellular organization, with necrotic cell debris filling
the amniotic cavity . As cavitation is a prerequisite for
gastrulation, the failure to cavitate resulting from loss of Tim may
prevent subsequent development as well.
To determine if loss of cavity formation was linked to an
apoptotic defect, Caspase 3/7 activity was determined in wild-type
and Tim knockdown EBs following induction with staurosporine
(Fig. 7). EBs generated from both of the Tim knockdown mES cell
lines exhibited a markedly blunted response to staurosporine in
terms of caspase activity as well as production of the cleaved,
active form of Caspase 3 (Fig. 7). Interestingly, Caspase activity has
been linked to the cleavage of Nanog, one of the core transcription
factors known to regulate self-renewal . ES cells lacking the
Casp3 gene are defective for differentiation, which can be rescued
by expression of a cleavage-resistant Nanog variant.
We next investigated whether the cells present in the failed
cavity retained characteristics of pluripotent mES cells. Wild-type
and Tim knockdown EBs were fixed and immunostained for the
pluripotency marker Oct4. In addition, the EBs were immuno-
stained for the zeta isoform of protein kinase C, which is expressed
in the tight junctions of the outer visceral endoderm layer and thus
defines the outer edge of the EB . Cells remaining in the
centers of the Tim knockdown EBs exhibited strong staining for
Oct4 after six days, suggesting that the cavity remains filled with
undifferentiated cells (Fig. 8A, B). After 12 days, EBs derived from
control ES cells showed little Oct4 staining, and 2D projections of
the confocal Images revealed substantial cavitation (Fig. 8C). In
contrast, 12 day EBs from both Tim knockdown ES cell lines
retained very thick walls of Oct4-positive cells, with little to no
To test whether the Oct4-positive cells present in the Tim
knockdown EBs remain pluripotent, we assayed for secondary EB
formation. This assay allows for a quantitative comparison of the
number of pluripotent ES cells remaining in each culture of
primary EBs, as only these cells can give rise to secondary EBs
[12,32]. Six-day EBs from control and Tim knockdown ES cell
lines were trypsinized to single cells, plated in methylcellulose and
Figure 1. Identification of Src SH3-binding proteins in ES cells and embryoid bodies (EBs). The mouse c-Src SH3 domain as well as a
binding-defective control were expressed in bacteria as GST fusion proteins and immobilized on glutathione-agarose beads. Lysates from self-
renewing ES cells and 6 day EBs were incubated with the immobilized wild-type and mutant Src SH3 domains, and associated proteins were eluted
and resolved by SDS-PAGE (see Materials and Methods). A, Image of a Coomassie blue-stained gel of the purified wild-type (WT) and mutant (Mut) Src
GST-SH3 proteins (No Lysate). SH3 target proteins captured from ES cell and EB lysates are indicated in the next four lanes. Unique bands were
excised and identified via tryptic fingerprinting and MALDI-TOF MS, including the known Src SH3 interacting proteins hnRNP K (RNP) and Dynamin II
(Dyn), as well as a fragment of Timeless (Tim; arrows). B, Details of the MS data obtained for Tim. The excised band from ES cells in Part A was halved
and subjected to two separate MS runs. Tim was the top hit from both runs, with two peptides of identical sequence identified in each case. The
sequence as well as the calculated and observed masses for each peptide are shown. C, The six numbered peptides from Part B map within the N-
terminal region of the Tim protein. Three major domains of Tim are illustrated, including the N-terminal Timeless region, the DDT (Domain binding
homeobox and Different Transcription factors) domain, and the C-terminal Timeless C region.
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the number of secondary EBs counted 10 days later. EBs from
Tim knockdown cells produced substantially more secondary EBs
compared to control EBs (Fig. 8D), providing strong evidence that
the cells present in the failed cavity remain undifferentiated. These
data support a model in which Tim is required for cavitation and
removal of pluripotent cells from the developing EB; without Tim,
cavitation and subsequent development are arrested.
In a final series of experiments, we investigated whether Tim
protein levels varied as a function of EB formation. Lysates were
prepared from self-renewing ES cells as well as 3, 6 and 12 day
EBs, and aliquots were analyzed for Tim protein levels by
immunoblotting. Overall Tim levels began to decrease after 6 days
of EB formation, and were dramatically reduced after 12 days
(Fig. 9A and B). The timing of Tim protein loss correlates with
cavity formation, which is clearly evident after 6 days and
complete after 12 days (Figs. 6 and 8) [28,29]. We also observed
that tyrosine-phosphorylated Tim is present in ES cells as well as 3
day EBs, and diminishes as Tim protein levels decrease (Fig. 9C).
Treatment of ES cells with two inhibitors previously shown to
block all Src-family kinase activity in ES cells (PP2 and SKI-1) 
substantially reduced endogenous Tim tyrosine phosphorylation,
strongly implicating c-Src or another member of the Src-kinase
family as the kinase responsible for Tim phosphorylation (Fig. 9D).
This result is consistent with the tyrosine phosphorylation of Tim
following co-expression with active c-Src in 293T cells as shown in
Data presented here provide the first evidence that the circadian
rhythm protein Tim also has a central role in one of the first
morphogenic changes that accompanies early development.
Knockdown of Tim results in ES cells that are capable of forming
EB-like structures, but these EBs fail to expand or cavitate and
remain filled with undifferentiated cells. While the specific
mechanism by which Tim regulates cavitation and subsequent
development will require further investigation, our results clearly
implicate Tim in the first wave of Caspase 3-dependent apoptosis
essential for EB cavitation and morphogenic progression.
In addition to early embryogenesis, other studies suggest that
Tim may regulate apoptosis required for organ formation later in
development. In the mouse embryonic kidney, for example, strong
Tim expression has been observed in regions of active ureteric bud
branching. Conditional knockdown of Tim with antisense
oligonucleotides in both kidney rudiments and isolated ureteric
bud cells profoundly inhibits embryonic kidney growth and
ureteric bud morphogenesis . Interestingly, blocking Caspase
activity also prevents ureteric bud formation, suggesting that Tim
may also regulate apoptotic signals involved in organogenesis .
Because Tim was identified as a c-Src SH3 domain-binding
protein in ES cells and EBs, Src kinase activity may regulate Tim
protein levels and activity during embryogenesis. In support of this
hypothesis, we show that full-length Tim is a substrate for c-Src
following co-expression in 293T cells, and that Src-mediated
phosphorylation correlates with enhanced Tim ubiquitylation.
This finding suggests that tyrosine phosphorylation controls Tim
protein levels in a manner analogous to circadian control of Tim
in Drosophila . Furthermore, we observed that endogenous Tim
is tyrosine-phosphorylated in self-renewing ES cells and early stage
EBs, and that tyrosine phosphorylation precedes a dramatic
decrease in Tim protein levels after EB cavitation has been
completed. These observations are consistent with the idea that
Src-family kinase-mediated phosphorylation regulates Tim ubi-
quitylation and proteasomal degradation during differentiation of
ES cells to EBs. Interestingly, our previous studies have shown that
Src-family kinase inhibitors reversibly delay EB formation [12,13].
Here we show that these same inhibitors block endogenous Tim
phosphorylation in ES cells, consistent with a role for a Src-Tim
connection in the ES cell to EB transition.
How Tim is linked to the apoptotic machinery and the
relationship of that connection to ES cell differentiation is not
Figure 2. Tim is a c-Src SH3 domain binding protein and
substrate. A, Lysates were prepared from ES cells and EBs and
incubated with immobilized GST, the Src GST-SH3 fusion protein, or the
corresponding inactive GST-SH3 mutant. Following washing, bound
proteins were separated by SDS-PAGE, transferred to PVDF membranes
and probed with an anti-peptide antibody to Tim. Full-length Tim and a
discrete cleavage product (CP) were found to associate with the GST-
SH3 fusion protein, but not with GST alone or with the mutant GST-SH3
domain. B, Tim is a substrate for c-Src. Human 293T cells were
transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD),
or with the empty expression plasmid (Con) together with V5 epitope-
tagged mouse Tim as indicated. Tim was immunoprecipitated from the
transfected cell lysates with a V5 antibody and immunoblotted for Tim
protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin
(Ub). Tranfected Src protein expression was confirmed in the cell
lysates, with actin as a loading control.
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clear. Clues to a possible mechanism may come from cellular
responses to environmental insults, such as DNA damage, which
can also trigger apoptosis. Under these conditions, activation of
checkpoint pathways initially induces cell cycle arrest, allowing
time for DNA repair to occur. However, if DNA damage is severe,
apoptosis is triggered instead (reviewed in ). In addition to its
circadian role, Tim also functions in S-phase checkpoint control
and can induce cell cycle arrest by localizing to the stalled
replication fork via interaction with Tipin . This observation
suggests the possibility that Tim may cause cell cycle arrest in ES
cells via a similar mechanism, thus indirectly inducing apoptosis as
required for cavity formation.
In summary, our data provide the first evidence that a protein
previously implicated in the control of circadian rhythms also has a
unique role to play in mES cell differentiation to EBs, two
processes that require tight temporal regulation. Remarkably little
is known about the presence or role of circadian rhythms in ES
cells or in the developing embryo. One very recent study suggests
Figure 3. Generation of Tim knockdown ES cell lines. Endogenous Tim expression was suppressed in mES cells by transduction with lentiviral
particles carrying shRNA sequences targeting independent regions of the Tim locus (Lenti:87 and Lenti:89). Following puromycin selection, 12
undifferentiated colonies were picked, expanded, and the levels of Tim protein expression determined by quantitative immunoblotting. A,
Morphology of representative Tim-knockdown lines isolated from the Lenti:87 and Lenti:89 ES cell populations. Control ES cell colony morphology is
also shown for comparison. B, The relative level of full-length Tim in lysates from each of the Tim knockdown lines shown in Part A was determined
by quantitative immunoblotting (Tim; arrow). Immunoblots were also probed with an actin antibody as a loading control, and the relative levels of
each protein were quantitated using the LI-COR Odyssey system and secondary antibodies conjugated to infrared fluorphores. C, Bargraph showing
the Tim:actin protein ratios. Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 were used in subsequent experiments based on unchanged ES
cell colony morphology (Part A; red outline) and extent of Timeless knockdown.
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that circadian clock genes are regulated differently in adult versus
embryonic cells . These authors showed that although genes
associated circadian rhythms are expressed in the developing
embryo in vivo, they were not expressed in a synchronized fashion
characteristic of circadian cycling. However, when embryonic
heart, liver and kidney tissues were placed into culture, rhythmic
expression of one of these clock proteins (PER2) was observed.
These observations suggest that ex vivo culture of embryonic
tissues, as well as in vitro differentiation of ES cells to EBs as
described in our study, may require coordination by circadian
rhythm proteins. The more general idea that circadian rhythm
proteins may coordinate cell lineage and tissue development
should be of significant interest to those exploring the directed
differentiation of ES cells in vitro. This process often requires EB
formation to allow for coordinated differentiation of the three
primitive germ layers . Support for this concept comes from
recent work by Yagita, et al., who showed that circadian feedback
loops fail to oscillate in self-renewing cultures of mES cells, but
become activated upon differentiation . Interestingly, oscilla-
tion was lost again upon genetic reprogramming of the cells back
to the pluripotent state, providing a strong connection between
circadian oscillations of clock protein promoter activity and
Materials and Methods
Culture of ES cells and EBs
Culture conditions for ES cells and EB formation have been
described previously [12,13]. Briefly, the D3 line of mouse ES cells
was obtained from the American Type Culture Collection
(Manassas, VA) and maintained in Dulbecco’s modified Eagle’s
medium supplemented with 15% fetal bovine serum, 2 mM L-
Figure 4. Knockdown of Tim suppresses spontaneous differentiation of mES cells. A, Representative images of control and Tim
knockdown ES cell lines 87-22 and 89-18 after 24 and 48 h in culture. Note that spontaneously differentiating mES cell clusters are readily apparent in
control ES cell cultures by 48 h (flat colonies with ragged edges; ‘‘d’’) but are absent from the Tim knockdown cultures. B, Analysis of self-renewal and
differentiation marker expression in Tim knockdown cell lines. Expression levels of the self-renewal markers Oct4, Sox2, Nanog, KLF4, the
differentiation marker AFP, as well as Tim were assessed by quantitative immunoblotting (LI-COR Odyssey infrared imaging system) of cell lysates
from parental ES cells as well as the Tim knockdown ES cell lines 87-22 and 89-18. Actin immunoblots served as loading control. Immunoblots were
performed in triplicate and the level of each protein was normalized to actin and is shown in the bargraph as the mean 6 S.E.M. Sox2 expression
levels were significantly increased in the Tim knockdown cells relative to parental ES cells (p,0.05), while AFP showed a statistically significant
decrease (p,0.05). While small increases in Oct4 expression were also observed in the Tim knockdown cell lines, these changes were not statistically
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glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate,
0.1 mM 2-mercaptoethanol, and 1000 U/ml LIF (Chemicon
International). For EB formation, ES cells were plated in growth
medium in the absence of LIF in bacterial-grade Petri dishes. To
assay for secondary EB formation, primary EBs were trypsinized
to single cells and cultured in the presence of 0.3% methylcellulose
and secondary EBs counted 10 days later.
Identification of Src SH3 domain binding proteins in
lysates from ES cells and EBs
The SH3 domain of Src was expressed in bacteria and purified
as a GST fusion protein as described elsewhere [38,39]. Briefly,
soluble protein extracts were prepared from pluripotent mES cells
and 6 day EBs in lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 5 mM EDTA, 1% Triton X-100] supplemented with
2 mM sodium orthovanadate, 2 mM NaF and Protease Inhibitor
Cocktail Set III (Calbiochem). Extracts were clarified by
centrifugation and incubated with GST-SH3 fusion proteins
immobilized on glutathione agarose beads. Following incubation,
the GST-SH3 resins were washed extensively with lysis buffer, and
bound proteins were eluted in sample buffer, separated on 4–20%
SDS-polyacrylamide gradient gels and visualized with Imperial
Coomassie protein stain (ThermoFisher Scientific). To define non-
specific binding, capture reactions were performed in parallel with
a mutant GST-SH3 fusion protein (W118A) that fails to bind
polyproline type II helices typically found in target proteins for
SH3 domains. Specific SH3-bound proteins were digested directly
from gel slices with trypsin and identified by MALDI-TOF mass
spectrometry (Genomics and Proteomcs Core Laboratory, Uni-
versity of Pittsburgh School of Medicine).
Analysis of Tim phosphorylation and ubiquitylation
A full-length, V5 epitope-tagged mouse Tim cDNA clone 
was expressed in 293T cells from the mammalian expression
vector pCDNA3.1 (Invitrogen) following transient transfection
either alone or in the presence of wild-type or kinase-defective c-
Src as per our published protocols [12,13]. Cell lysates were
prepared in RIPA Buffer [50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium
deoxycholate] supplemented with 2 mM sodium orthovanadate,
2 mM NaF, and Protease Inhibitor Cocktail Set III (Calbiochem).
Tim was immunoprecipitated via the C-terminal V5 epitope tag,
and immunoblotted with anti-phosphotyrosine or ubiquitin
antibodies. The V5 epitope tag (AB37292) and actin (MAB1501)
antibodies were purchased from Chemicon. Ubiquitin (sc-8017),
anti-phosphotyrosine (PY99; sc-7020), and c-Src (B12; sc-8056)
antibodies were purchased from Santa Cruz Biotechnology.
Endogenous Tim protein levels were determined in lysates of ES
cells and EBs by immunoblotting with a polyclonal rabbit anti-
peptide antiserum raised against the mouse C-terminal Tim
peptide sequence GTPRVHRKKRFQIEDEDD (Tim-P92 anti-
serum; Bio-Synthesis). To analyze tyrosine-phosphorylated Tim,
tyrosine phosphorylated proteins were immunoprecipitated from
ES cell and EB lysates using a 1:1 mixture of the anti-
phosphotyrosine antibodies PY20 (BD Biosciences) and PY99
(Santa Cruz). Tyrosine-phosphorylated Tim present in the
immunoprecipitates was visualized by immunoblotting with the
Tim-92 antiserum. For the inhibitor studies, ES cells were plated
on gelatin-coated dishes and allowed to attach for 6 h prior to
addition of the Src-family kinase inhibitors PP2 or SKI-1. Each
inhibitor was added at 10 mM, a concentration previously shown
Figure 5. Tim knockdown EBs are smaller and of more uniform size. EBs were grown from control and Tim knockdown ES cell lines Lenti:87-
22 and Lenti:89-18 for 6 and 12 days. EBs were fixed, DAPI-stained and imaged with an Olympus F500 confocal microscope. The EB sizes were then
estimated by determining the EB surface area from the images using the ImageJ 1.43U software suite. Scattergrams of the resulting data were
analyzed using the Kruskal-Wallis test (nonparametric unpaired analysis of variance by ranks; Prizm Software, GraphPad, Inc.) and a significant
difference in the median EB size was observed across both groups (P,0.0001). The median EB size is shown in each group by the red bar.
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to block SFK activity in mES cells . Cells were incubated for
an additional 16 h followed by analysis of tyrosine-phosphorylated
Tim as described above.
Caspase 3/7 activity was determined in cell lysates using the
Apo1 assay (Promega) in response to staurosporine treatment
Figure 6. Tim knockdown prevents EB cavitation. EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18
for 6 days. Fixed EBs were then stained with DAPI (nuclei; blue) plus Alexa Fluor 488-phalloidin (F-actin; green) and imaged by confocal microscopy
using an Olympus Fluoview 1000 confocal microscope. A, Optical sections (5 mm) were taken from the bottom through the middle of the EB where
cavitation is the greatest. Images of representative sections are shown of the bottom as well as one-quarter and halfway through the EB. B, Side
profiles from merged images reveal cavitation (‘‘c’’) in the control but not the Tim knockdown EBs. C, Bargraph shows the percentage of cavitated EBs
formed from the parental mES cell line, mES cells transduced with a nontargeted shRNA lentivector (Non-T) as well as the two Tim knockdown ES cell
Figure 7. Timeless-knockdown EBs are resistant to apoptosis. A, Control and Tim-knockdown EBs were grown for 6 days from the
corresponding ES cell lines and incubated in the presence or absence of 0.5 mM staurosporine for 16 h. Cell lysates were analyzed for Caspase 3/7
activity using the Apo-1 assay. The data are normalized to the activity observed with control EBs in the presence of staurosporine. The experiment
was repeated three times, and the results are presented in the bargraph as mean percent of control 6 S.E.M. B, Lysates from staurosporine-treated
control and Tim-knockdown EBs were analyzed for the presence of the active form of Capsase-3 by immunoblotting with an antibody specific for the
cleaved, active form of this protease. Signal intensities were quantified using the LI-COR Odyssey system, and normalized to control values. The
experiment was repeated in triplicate, and the bargraph shows the mean values 6 S.E.M. EBs derived from both Tim knockdown cell lines showed a
significantly reduced apoptotic response to staurosporine treatment in each of these assays (p#0.02 in each case).
Timeless and ES Cell Differentiation
PLoS ONE | www.plosone.org8 February 2011 | Volume 6 | Issue 2 | e17157
(0.5 mM for 16 h). Levels of active Caspase 3 were determined by
immunoblotting cell lysates with an antibody specific for the
cleaved, active form of Caspase 3 (Abcam). Band intensities were
quantified using the LI-COR Odyssey infrared imaging system as
described elsewhere .
Lentiviral shRNA-based knockdown and EB imaging
Lentiviral particles carrying five distinct shRNAs targeting
mouse Tim (Mission; Sigma) were used to infect mES cells.
Following puromycin selection, the transduced populations were
evaluated for Timeless knockdown by immunoblotting. Two of the
shRNA lentiviruses (TRCN0000097987 and TRCN0000097989;
abbreviated as Lenti:87 and Lenti:89 elsewhere in the text)
induced significant reductions in Tim expression in the overall ES
cell population. Six undifferentiated ES cell colonies were then
cloned from each culture and tested for the extent of Tim
knockdown by immunoblotting using the Tim-P92 antiserum. The
impact of Tim knockdown on ES cell pluripotency was assessed by
immunoblotting of cell lysates with antibodies against the self-
renewal markers Oct4 (sc-5279; Santa Cruz), Sox2 (ab59776;
Abcam), Nanog (ab70482; Abcam), KLF4 (sc-20691; Santa Cruz)
as well as the differentiation marker AFP (sc-8108; Santa Cruz).
Immunoblots for actin (mab1501; Millipore) served as loading
controls. Additional details of the immunoblotting proctocols have
been published elsewhere .
ES cells (56105) were plated on non-adherent plastic petri dishes
and grown in suspension in the absence of LIF to promote EB
formation as described elsewhere [12,13]. EBs were fixed 3, 6 and
Figure 8. EBs formed from Tim knockdown cells retain pluripotent cells. EBs were cultured from control and Tim knockdown ES cell lines
Lenti:87-22 and Lenti:89-18 for 6 and 12 days. A, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform
of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-
color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells
present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. B, Side profiles from merged images in
part A; the location of the cavity in the control EB is indicated with a ‘‘c’’. C, Side profiles from merged images obtained from 12 day EBs and stained
as for 6 day EBs. D, Secondary EB assay. Six-day EBs from part A were trypsinized to single cells, replated in methylcellulose at the cell numbers
indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture 6
S.E.M. is shown in the bargraph (n=8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each
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12 d later in 4% paraformaldehyde, permeabilized in RIP buffer
(50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 1 mM EDTA), and stained with
DAPI (Sigma), Alexa Fluor 488-phalloidin (Molecular Probes) as
well as antibodies for Oct4 (sc-5279; Santa Cruz) and the zeta
isoform of PKC (sc-216; Santa Cruz) based on the methods of
Denham et al. . Optical sections were captured using an
Olympus Fluoview 1000 confocal microscope; additional details of
the imaging techniques are provided in the Figure legends.
Statistical significance was determined using Student’s t-tests
where appropriate. Differences in EB size were assessed using the
Kruskal-Wallis nonparametric unpaired analysis of variance by
ranks. Statistical determinations were performed using the Prism
Software package (GraphPad, Inc.).
The authors thank Dr. Steven M. Reppert, University of Massachusetts
Medical School,forkindlyprovidingcDNAclonesformouseTimeless,as well
as Jason Devlin and Greg Gibson of the University of Pittsburgh Center for
Biologic Imaging for their help with confocal microscopy of EBs.
Conceived and designed the experiments: LOR TS. Performed the
experiments: LOR. Analyzed the data: LOR SW TS. Contributed
reagents/materials/analysis tools: LOR SW TS. Wrote the paper: LOR
Figure 9. Changes in Tim protein levels and tyrosine phosphorylation during EB formation. A and B, Tim protein levels diminish during
EB formation. Lysates were prepared from self-renewing ES cells (ESC) and 3, 6, and 12 day EBs, and immunoblotted for Tim and actin protein levels.
Full length Tim as well as two possible cleavage products (CP) are indicated by the arrows. Relative band intensities for full-length Tim and actin were
determined using ImageJ from four independent experiments and Tim:actin ratios were calculated. The results were normalized to ratios obtained
from control ES cells, and are presented in the bargraph as the mean 6 S.E.M. The level of Tim was significantly reduced after 6 and 12 days of EB
formation (p#0.01). C, Tyrosine phosphorylation of endogenous Tim in ES cells and EBs. Lysates were prepared from self-renewing ES cells and 3, 6,
and 12 day EBs, and tyrosine-phosphorylated proteins were immunoprecipitated from protein aliquots and analyzed for Tim by immunoblotting (top
panel). Actin blots were performed to verify equivalent levels of input protein for the immunoprecipitation (lower panel). This experiments was
repeated three times with comparable results; a representative example is shown. D, Inhibition of Tim tyrosine phosphorylation in ES cells by Src-
family kinase inhibitors. ES cells were incubated with the Src-family kinase inhibitors PP2 and SKI-1  at 10 mM for 16 h. Tyrosine-phosphorylated
proteins were immunoprecipitated and analyzed for the presence of Tim by immunoblotting (top panel). Tim blots (lower panel) verified equivalent
levels of Tim in each lysate prior to immunoprecipitation.
Timeless and ES Cell Differentiation
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