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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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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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pone-0017157-g005: 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.

Mentions: 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 [22]. As cavitation is a prerequisite for gastrulation, the failure to cavitate resulting from loss of Tim may prevent subsequent development as well.


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

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

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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g005: 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.
Mentions: 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 [22]. As cavitation is a prerequisite for gastrulation, the failure to cavitate resulting from loss of Tim may prevent subsequent development as well.

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