br Results br Discussion The

Results
Discussion The availability of Lgr5 as a specific marker for stem A-1210477 in the intestine and other tissues has allowed the unequivocal identification of intestinal stem cells, as well as molecular profiling (Muñoz et al., 2012), the establishment of culture methods (Sato et al., 2009), and the identification of signaling cascades involved in stem cell homeostasis (de Lau et al., 2011). Although the identification of Lgr5 and the generation of the Lgr5-GFP-IRES-CreERT2 KI mouse were pivotal for the identification of intestinal stem cells (Barker et al., 2007), the low expression levels of Lgr5 and the mosaic expression of the first KI allele have limited Lgr5’s use as a marker for stem cells. For example, cell ablation studies are not possible due to the suboptimal penetrance in this particular model. Olfm4 was reported to be a highly specific stem cell marker (Itzkovitz et al., 2012; Muñoz et al., 2012; van der Flier et al., 2009a) and noted for the high levels of RNA in the stem cells (Muñoz et al., 2012). Recently, a pool of noncycling stem cells was identified in the pool of Lgr5+ crypt cells (Buczacki et al., 2013). Because of the highly similar expression profiles of Olfm4 and Lgr5, it is likely that these cells are also marked by Olfm4. Here, we have described the generation of an Olfm4-IRES-eGFPCreERT2 allele that allows the visualization of stem cells of the small intestine, as well as the genetic manipulation of these cells. In contrast to some of the previously described mouse models (Barker et al., 2007), the Olfm4-IRES-eGFPCreERT2 allele is expressed in all crypts of the intestine and is not silenced. In individual crypts, Olfm4-IRES-eGFPCreERT2 is expressed in all of the stem cells that are also marked by Lgr5, including the label-retaining stem cells (Buczacki et al., 2013) and the so-called “border cells” (Ritsma et al., 2014). This allows for the quantitative manipulation of the entire stem cell pool. Replenishment of the intestinal epithelium occurs via a pattern of neutral drift dynamics (Lopez-Garcia et al., 2010; Ritsma et al., 2014; Snippert et al., 2010) in which “unhealthy” stem cell clones are rapidly lost. In previous studies, competition of wild-type stem cells with genetically altered stem cells made it difficult to discern phenotypes (van der Flier et al., 2009b). The complete penetrance of the Olfm4-IRES-eGFPCreERT2 allele circumvents this problem by allowing the simultaneous alteration of a large majority of the stem cells, favoring the new genotype. In our analysis of the Olfm4-IRES-eGFPCreERT2 allele, we found a limited activation of the Rosa-LacZ reporter in the absence of tamoxifen; however, this does not influence the usefulness of this model for cell ablation studies. We also show the Olfm4-driven expression of GFP in organoid cultures derived from Olfm4-IRES-eGPCreERT2 animals, where it is observed exclusively in the slender cells between the Paneth cells at the bottom of the crypt-like buds of the cultures. Due to the rapid expansion of the organoids in this culture model, the Paneth and stem cell domain is enlarged, and Olfm4-driven GFP expression marks the complete stem cell pool in these cultures. In contrast to Lgr5, the expression of the introduced eGFPCreERT2 fusion gene was limited to cells of the small intestine only. This restricted expression pattern has some potential advantages, such as the possibility of targeting intestinal stem cells without altering stem cell pools in other tissues. OLFM4 was identified in cells of the myeloid lineage, and OLFM4 RNA was observed in human colon, stomach, and bone marrow in addition to the small intestine (Zhang et al., 2002). No LacZ reporter gene expression was detected in these organs in our mouse model. The restricted expression pattern may reflect a more limited function of Olfm4 in the mouse, raising the possibility that other Olfactomedin family members are coexpressed with Lgr5 in other tissues.