Limits...
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.

Show MeSH

Related in: MedlinePlus

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 ± 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 [12] at 10 µM 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g009: 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 ± 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 [12] at 10 µM 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.

Mentions: 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) [12] 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 Figure 2B.


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

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

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 ± 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 [12] at 10 µM 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g009: 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 ± 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 [12] at 10 µM 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.
Mentions: 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) [12] 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 Figure 2B.

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