Further support for the hierarchical two

Further support for the hierarchical two-stem-cell model came with the discovery of an additional reserve ISC marker locus, Hopx, encoding an atypical homeodomain protein with functions in early heart development (Chen et al., 2002). An IRES-CreERT2 cassette inserted into the endogenous Hopx locus revealed that, like Bmi1-CreERT2 cells, Hopx-CreERT2 cells are capable of giving rise to Lgr5 cells (Takeda et al., 2011). Thus, reserve ISCs give rise to progeny including active Lgr5 CBCs that become dependent on canonical Wnt activity. The precise relationship between Hopx-CreERT2- and Bmi1-CreERT2-marked cells remains unexamined. Despite the elegant genetic evidence supporting the existence of a two-stem-cell system (active CBCs and reserve ISCs), considerable controversy exists regarding the identity of these stem cells and their relationship to one another. Specifically, the messenger RNAs emanating from the endogenous loci used in generation of marker notch signaling pathway including Bmi1, Tert, and Hopx exist at higher levels in the Lgr5-EGFP-high population in comparison to the Lgr5-EGFP-low population (Muñoz et al., 2012), and the endogenous Bmi1 and Tert transcripts can be detected throughout almost all cells of the crypt below the transit-amplifying (T/A) zone (Itzkovitz et al., 2012). These findings led to suggestions that the marked stem cells may represent a single population or that they exist in a continuum, not discernible as distinct populations. Many of these discrepancies could be accounted for if, in fact, these reporter alleles mark heterogeneous populations that are mistakenly assumed to be homogenous in population-based analyses and/or if the presence of endogenous mRNAs does not correlate with reporter activity emanating from a single locus. Further complexities in our understanding of ISC biology arose in recent reports describing the existence of secretory precursor cells of the intestine. One report described these secretory precursors as long-lived LRCs that express high levels of Lgr5 and resist intermediate doses of γ-IR (6 Gy; Buczacki et al., 2013). This finding was particularly curious in light of classic studies describing the intestinal LRC as being exquisitely radiosensitive (undergoing apoptosis in response to as little as 1 Gy γ-IR; Potten et al., 2002), and studies using highly sensitive multi-isotope imaging mass spectrometry suggest that there are no LRCs in the intestinal epithelium (Steinhauser et al., 2012). Thus, the existence and identity of LRCs of the intestinal epithelium remains controversial, and how these cells relate to the reserve ISCs marked by Bmi1- or Hopx-CreERT2 activity is entirely unknown. In a separate study, another group identified secretory precursor cells as a proliferative population marked by expression of the Notch ligand Dll1 (van Es et al., 2012). These cells have a very specific gene-expression pattern with high expression of Notch ligands (Dll), low levels of Notch receptors and target genes (Hes), and high levels of Atoh1 (Math1), which is suppressed by Notch signaling and promotes differentiation into the secretory lineage (van Es et al., 2012). In contrast to the secretory LRCs, the Dll1+ secretory precursors do not express Wnt target genes including Lgr5 (van Es et al., 2012). Interestingly, both the Dll1+ and label-retaining secretory precursor cells exhibited broad stem cell activity (generating not only secretory lineages) in response to epithelial damage, although these were rare events (Buczacki et al., 2013; van Es et al., 2012). In an attempt to reconcile conflicting reports in the literature and provide a foundation for understanding intestinal stem cell dynamics and hierarchy moving forward, we undertook a comprehensive comparative analysis of established ISC knockin reporter alleles including Lgr5-EGFP-IRES-CreERT2, Bmi1-EGFP, Bmi1-CreERT2, Hopx-CreERT2, and Hopx-EGFP. We apply single-cell analyses to address the heterogeneity inherent in these populations, whether they exist as molecularly distinct stem cell pools, and how they differ in their proliferative output. Through the analysis of Wnt, Notch, proliferation, differentiation, and stem-cell-related transcripts along with lineage tracing and cell cycle analyses, we place these marked populations in a model hierarchy. Our findings begin to reconcile the contrasting literature regarding the identity of ISC populations marked by proxy reporter alleles and support the existence of a two-stem-cell model.