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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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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 ± 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 significant.
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pone-0017157-g004: 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 ± 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 significant.

Mentions: Morphologically, self-renewing cultures of both Tim-knockdown 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 differentiation marker AFP was decreased by more than 60% relative to control ES cells. These changes most likely reflect loss of spontaneous differentiation as a consequence of Tim knockdown, although an influence on the ES cell self-renewal program cannot be ruled out.


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

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

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 ± 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 significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g004: 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 ± 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 significant.
Mentions: Morphologically, self-renewing cultures of both Tim-knockdown 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 differentiation marker AFP was decreased by more than 60% relative to control ES cells. These changes most likely reflect loss of spontaneous differentiation as a consequence of Tim knockdown, although an influence on the ES cell self-renewal program cannot be ruled out.

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