title: An unexpected role for the clock protein timeless in developmental apoptosis.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Linda P
authors.initials: LP
authors.lastname: O'Reilly
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D019521:Body Patterning / Q000187:drug effects / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D055786:Gene Knockout Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology
chemical_list: D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D056950:Period Circadian Proteins; D034741:RNA, Small Interfering; C509770:Timeless protein, mouse