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An unexpected role for the clock protein timeless in developmental apoptosis.

O'Reilly LP, Watkins SC, Smithgall TE - PLoS ONE (2011)

Bottom Line: Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

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.

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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 µM 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 ± 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 ± 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).
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pone-0017157-g007: 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 µM 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 ± 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 ± 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).

Mentions: 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 [30]. ES cells lacking the Casp3 gene are defective for differentiation, which can be rescued by expression of a cleavage-resistant Nanog variant.


An unexpected role for the clock protein timeless in developmental apoptosis.

O'Reilly LP, Watkins SC, Smithgall TE - PLoS ONE (2011)

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 µM 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 ± 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 ± 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).
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getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g007: 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 µM 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 ± 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 ± 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).
Mentions: 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 [30]. ES cells lacking the Casp3 gene are defective for differentiation, which can be rescued by expression of a cleavage-resistant Nanog variant.

Bottom Line: Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus