• 2018-07
  • 2018-10
  • 2018-11
  • These findings predict that Lgr CBCs must have a significant


    These findings predict that Lgr5-CBCs must have a significantly shorter lifespan than the reserve ISCs; otherwise, an age-related accumulation of Lgr5-CBCs would result from the constant generation of CBCs from the reserve ISCs. Indeed, this finite lifespan of Lgr5-CBCs can be observed in prior studies where the random activation of any one of four fluorophores in the Lgr5-CreER::R26R-Confetti mouse model results in multiple unique labeling events in any given crypt, and over time, clonal extinction is observed as a drift to monoclonality (Snippert et al., 2010). In the current study, we directly compare the extinction of Lgr5-CreER- and Hopx-CreER-marked clones and demonstrate that, whereas approximately two thirds of Lgr5 clones are lost over a 6-month period, there is no loss of Hopx-CreER-derived clones. This result provides additional evidence that the reserve ISC is capable of longer proliferative output than the Lgr5 CBC. Because CBCs have a shorter lifespan than the reserve Hopx-CreER ISCs, it is tempting to speculate that lost CBCs are replaced by new CBCs generated de novo from reserve ISCs. Although there is clear evidence that Lgr5 elastase inhibitor compete with one another for crypt dominance through neutral drift (existing Lgr5 cells replacing lost Lgr5 cells through symmetric division), there is also clear evidence that reserve ISCs generate Lgr5 cells, and thus, further studies are necessary to determine which mechanism of Lgr5 cell replacement occurs during homeostasis and with what frequency.
    Experimental Procedures
    Introduction In the mouse, germline specification begins around E6.25, when the expression of PR domain proteins (PRDM1 and PRDM14) mediates the transcriptional suppression of the somatic differentiation program in the nascent primordial germ cells (PGCs) (Kurimoto et al., 2008; Yamaji et al., 2008). Shortly after the specification period, the importance of posttranscriptional regulation in germ cell maintenance becomes evident. For example, in mice lacking DND1 or NANOS3, both of which are germ cell-specific RNA binding proteins, PGCs are gradually lost by apoptosis starting at E8.5, when PGCs start to migrate toward the future gonads (Youngren et al., 2005; Suzuki et al., 2008). Upon arrival in the gonad, DAZL (Deleted in azoospermia-like), a germ cell-specific RNA-binding protein, is essential for developing PGCs (Lin and Page 2005), and loss of Dazl expression again results in apoptosis of the postmigratory germ cells. The DAZ (Deleted in Azoospermia) gene family is composed of the germ cell-specific genes Boule, Dazl, and DAZ. The encoded proteins all contain RNA recognition motifs (RRM) in the N-terminal domains, which presumably interact with target RNAs, and one or multiple DAZ repeats in the C-terminal domain, of which the function is still unknown. Dazl is conserved in all vertebrates, and the transcripts of Dazl are germ plasm components in both Xenopus (Houston et al., 1998) and zebrafish (Hashimoto et al., 2004). Although a germ plasm analog was never identified in mammalian PGCs, loss of Dazl expression results in infertility in both sexes in mice, with germ cell loss during development and a final block at meiosis (Ruggiu et al., 1997; Schrans-Stassen et al., 2001). In the C57Bl/6 background, apoptosis of Dazl knockout germ cells is observed at the early gonocyte stage (Lin and Page 2005). Given the importance of DAZ family proteins in germ cells, much effort has been invested in elucidating the molecular mechanisms of Dazl function. Lin and colleagues demonstrated that DAZL is a meiosis-promoting factor in developing germ cells (Lin et al., 2008). Indeed, in the absence of DAZL, the germ cells fail to develop beyond the PGC stage as shown by continued expression of pluripotency markers. These findings gave rise to the idea that DAZL is a “licensing factor” that is required for PGC sexual differentiation (Gill et al., 2011). However, the mechanism by which DAZL promotes meiotic entry remains unclear. To elucidate the function of DAZL in germ cell development, several groups have identified mRNA binding partners in coimmunoprecipitation experiments (Fox et al., 2005; Reynolds et al., 2005; Zeng et al., 2008) and yeast three-hybrid assays (Venables et al., 2001). Potential mRNA targets include Mvh (Reynolds et al., 2005), Scp3 (Reynolds et al., 2007), and Tex19.1 (Zeng et al., 2009). In most of these studies, DAZL was shown to function as a translational enhancer. Yet, the ablation of Tex19, Mvh, or Scp3 in mice results in fertility phenotypes that are patently less severe and arise much later in development than the Dazl knockout phenotypes, suggesting that DAZL may have additional roles during the PGC stage of mammalian gametogenesis.