An Unexpected Role for the Clock Protein Timeless in Developmental Apoptosis

Centro Cardiologico Monzino, Italy
PLoS ONE (Impact Factor: 3.23). 02/2011; 6(2):e17157. DOI: 10.1371/journal.pone.0017157
Source: PubMed


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.
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.
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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Comment on: Smith-Roe SL, et al. Cell Cycle 2011; 10:1618-24.
    Preview · Article · Jul 2011 · Cell cycle (Georgetown, Tex.)
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: One of the core genes in the circadian regulation network is clock (clk). By forming a heterodimer with CYCLE (CYC) that binds on an E-box in the promoter region, it induces the transcription of other elements in the circadian transcriptional feedback loops and different clock output genes. In contrast to other insects, a clk double-stranded RNA (dsRNA) treatment is lethal in adults and fifth instar nymphs of the desert locust, Schistocerca gregaria, in a dose-dependent manner. Clk knock down fifth instar nymphs are able to undergo their imaginal moult but, depending on the amount of dsRNA, it takes them longer than the controls to reach adulthood. As adults, clk knock down animals do not develop their fat body and ovaries like the control animals. Therefore, we tested the expression of different genes involved in energy metabolism and reproduction to see the effect of the clk RNA interference knock down. Surprisingly, the expression of the vitellogenin gene was up-regulated in the clk knock down females who did not appear to invest their energy in egg development. Taken together, our results point out that the clk gene in the desert locust has an additional function in development besides its established role in maintaining the circadian rhythms in the brain.
    Full-text · Article · Mar 2012 · Insect Molecular Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein-tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.
    No preview · Article · Dec 2012 · Cellular Signalling
Show more