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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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Related in: MedlinePlus

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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pone-0017157-g003: 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.

Mentions: 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:89-18.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017157-g003: 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.
Mentions: 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:89-18.

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