Dear Editor, Cellular senescence (or senescence) has been regarded as a stable form of cell cycle arrest by in vitro cell culture experiments. 1 Recent studies indicate that senescence is associated with aging and diseases, including cancers. 2, 3 For instances, it suppresses tumor progression by halting the growth of premalignant cells, 4 and promotes wound healing by preventing excessive tissue fibrosis or induction of cell dedifferentiation. 5, 6 Targeting senescent cells could restore tissue homeostasis in response to aging, chemotoxicity, or injury. 7 In addition to these pathological conditions in adults, cellular senescence also occurs in physiological states such as mammalian mouse 8, 9 and human 10, 11 embryonic development. Embryonic senescent cells have been reported to be non-proliferative and subjected to clearance from tissues after apoptosis at late embryonic stage. 8, 9 However, the interpretation for clearance of senescent cells at late embryonic stage is based on the disappearance of Cdkn1a (P21) expression and senescence-associated beta-galactosidase (SAβ-Gal) activity, 8, 9 two commonly used senescence markers in the field. Currently, there is no genetic fate mapping evidence for senescent cell fate in vivo. By lineage tracing of P21+ senescent cells, we found that embryonic senescent cells labeled at mid-embryonic stage gradually lost P21 expression and SAβ-Gal activity at late embryonic stage. Unexpectedly, some of the previously labeled senescent cells re-entered the cell cycle and proliferated in situ. Moreover, these previously labeled senescent cells were not cleared at late embryonic stage and remained in the tissue after birth. This study unravels in vivo senescent cell fates during embryogenesis, indicating their potential plasticity. We first performed SAβ-Gal staining on embryos and found SAβ-Gal+ signals in the apical ectodermal ridge (AER) at E10. 5-E14. 5. We hardly detected positive signals in the AER at E15. 5 and afterwards (Fig. 1 a). SAβ-Gal activity in AER was validated by staining on tissue sections (Supplementary information, Figure S1a). To confirm the specificity of SAβ-Gal staining for senescence (pH 6.0), we stained embryos at pH 6.5 and pH 7.0 for technical controls as previously described. 8 Indeed, we did not detect any positive SAβ-Gal signal at E10. 5-E14. 5 (Supplementary information, Figure S1b). These results were consistent with previous studies, 8, 9 demonstrating that senescent cells as detected by SAβ-Gal staining were present at E10. 5-E14. 5, whereas SAβ-Gal activity disappeared after E15. 5 (Fig. 1 a, b). Therefore, SAβ-Gal activity could be mainly restricted to mid-but not late embryonic stage. These experimental data have been interpreted as indicating that SAβ-Gal+ senescent cells underwent apoptosis and were cleared from tissues at late embryonic stage. 8, 9 However, an alternative explanation could be that a subset of senescent cells gradually lost SAβ-Gal activity but survived in the tissue at late embryonic stage. The in vivo senescent cell fate currently remains unknown and untested, as to date there is no fate mapping study on senescent cells.

P21 is a molecular mediator for embryonic senescence 8, 9 and is highly expressed in SAβ-Gal+ senescent cells of AER. 8 To trace the cell fate of senescent cells during embryogenesis, we generated the P21-CreER mouse line by knocking CreER cDNA into the stop codon of P21 (Fig. 1 c). 2A self-cleaving peptide sequence was used to allow simultaneous expression of CreER and P21 in P21+ cells (Fig. 1 c). Immunohistochemistry for P21 or estrogen receptor (ESR, for detection of CreER) in P21-CreER mouse forelimbs showed …