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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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EBs formed from Tim knockdown cells retain pluripotent cells.EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. A, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. B, Side profiles from merged images in part A; the location of the cavity in the control EB is indicated with a “c”. C, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. D, Secondary EB assay. Six-day EBs from part A were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).
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pone-0017157-g008: EBs formed from Tim knockdown cells retain pluripotent cells.EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. A, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. B, Side profiles from merged images in part A; the location of the cavity in the control EB is indicated with a “c”. C, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. D, Secondary EB assay. Six-day EBs from part A were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).

Mentions: We next investigated whether the cells present in the failed cavity retained characteristics of pluripotent mES cells. Wild-type and Tim knockdown EBs were fixed and immunostained for the pluripotency marker Oct4. In addition, the EBs were immunostained for the zeta isoform of protein kinase C, which is expressed in the tight junctions of the outer visceral endoderm layer and thus defines the outer edge of the EB [31]. Cells remaining in the centers of the Tim knockdown EBs exhibited strong staining for Oct4 after six days, suggesting that the cavity remains filled with undifferentiated cells (Fig. 8A, B). After 12 days, EBs derived from control ES cells showed little Oct4 staining, and 2D projections of the confocal Images revealed substantial cavitation (Fig. 8C). In contrast, 12 day EBs from both Tim knockdown ES cell lines retained very thick walls of Oct4-positive cells, with little to no cavitation evident.


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

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

EBs formed from Tim knockdown cells retain pluripotent cells.EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. A, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. B, Side profiles from merged images in part A; the location of the cavity in the control EB is indicated with a “c”. C, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. D, Secondary EB assay. Six-day EBs from part A were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).
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Related In: Results  -  Collection

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pone-0017157-g008: EBs formed from Tim knockdown cells retain pluripotent cells.EBs were cultured from control and Tim knockdown ES cell lines Lenti:87-22 and Lenti:89-18 for 6 and 12 days. A, Fixed 6 day EBs were immunostained for the pluripotency marker Oct4 (green) and the zeta isoform of PKC (red) which marks tight junctions in the outer layer of visceral endoderm surrounding the EB. Nuclei were stained with DAPI (blue), and three-color images were obtained by confocal microscopy; a merged image is also shown. Optical sections from the middle of the EB show that cells present in the failed cavity of the Tim-knockdown EBs retain Oct4 staining, indicative of undifferentiated cells. B, Side profiles from merged images in part A; the location of the cavity in the control EB is indicated with a “c”. C, Side profiles from merged images obtained from 12 day EBs and stained as for 6 day EBs. D, Secondary EB assay. Six-day EBs from part A were trypsinized to single cells, replated in methylcellulose at the cell numbers indicated, and the number of secondary EBs present were counted ten days later. The mean number of secondary EBs formed from each culture ± S.E.M. is shown in the bargraph (n = 8). Both Tim knockdown cell lines produced significantly more secondary EBs than the parental control in each case (p<0.01).
Mentions: We next investigated whether the cells present in the failed cavity retained characteristics of pluripotent mES cells. Wild-type and Tim knockdown EBs were fixed and immunostained for the pluripotency marker Oct4. In addition, the EBs were immunostained for the zeta isoform of protein kinase C, which is expressed in the tight junctions of the outer visceral endoderm layer and thus defines the outer edge of the EB [31]. Cells remaining in the centers of the Tim knockdown EBs exhibited strong staining for Oct4 after six days, suggesting that the cavity remains filled with undifferentiated cells (Fig. 8A, B). After 12 days, EBs derived from control ES cells showed little Oct4 staining, and 2D projections of the confocal Images revealed substantial cavitation (Fig. 8C). In contrast, 12 day EBs from both Tim knockdown ES cell lines retained very thick walls of Oct4-positive cells, with little to no cavitation evident.

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