It is puzzling to observe in this study that the

It is puzzling to observe in this study that the stabilizing β-catenin mutation, which acts dominantly in many other tissues, affected the GFRα1+ cell pool only when introduced homozygously, and that heterozygous deletion of β-catenin caused affected spermatogenesis. These findings imply that Wnt/β-catenin signal is strongly suppressed in stem spermatogonia. Shisa6 appears to be involved in this suppression; however, a lack of an apparent phenotype in Shisa6 mice suggests that multiple mechanisms are involved. Other cell-autonomous Wnt inhibitor(s) could also be involved, such as Shisa2, whose expression was also enriched in the GFRα1+ fraction (Figure S7B). We also speculate that the nuclear accumulation of β-catenin could be prevented by sequestration by E-cadherin, as proposed in the colon epithelium, which also requires homozygous introduction of the stabilizing β-catenin mutation to transform (Huels et al., 2015). In accordance with this, undifferentiated spermatogonia express high levels of E-cadherin (Nakagawa et al., 2010; Tokuda et al., 2007), and showed prominent cytoplasmic β-catenin staining in GFRα1+ CX-5461 cost (Figure S7C). This study illustrates a unique mode of Wnt inhibitor function. Many Wnt inhibitors are secreted proteins, act in a non-cell-autonomous manner, and tune the spatial pattern of Wnt activity. These include Dickkopf proteins (Dkks), secreted Frizzled-related proteins (Sfrps), and Wnt inhibitory factor 1 (Wif1). Similarly, cell-autonomous Wnt inhibitors identified so far (xshisa1, xshisa2, xshisa3, Apcdd1, Tiki1, and flop1/2) also shape the spatial patterns of Wnt activity (Cruciat and Niehrs, 2013; Miyagi et al., 2015). In contrast, this study suggests that heterogeneous Shisa6 expression variegates the stem/progenitor spermatogonia in terms of their sensitivity to Wnt/β-catenin signaling in a spatially uniform facultative microenvironment. Given that SHISA6 confers resistance to differentiation-inducing Wnt/β-catenin signaling, SHISA6+ cells should be a key population to understand this stem cell system. Although their in-depth characterization was difficult, we could analyze T (Brachyury)+ cells that showed a major overlap with Shisa6+ cells, taking advantage of an engineered allele. T+/GFRα1+ cells are morphologically more biased to As cells, compared with the T–/GFRα1+ cells (Figure 6E), and persist throughout the cycle of the seminiferous epithelium (Figure 7G). Furthermore, pulse-labeled T+ cells showed a long-term contribution to spermatogenesis (Figures 7G–7L). These features of T+ cells are postulated for the stem cells. Echoing with this is the suggestion that T is crucial for the self-renewing potential of cultured spermatogonia (Wu et al., 2011). We assume that Shisa6+ cells also showed similar stem cell-related characteristics, although we cannot formally exclude the possibility that only the T+/SHISA6– cells exhibited the long-term stem cell functions. It is not easy, however, to be conclusive with the identity of stem cells (Yoshida, 2017). A number of genes (e.g., Id4, Pax7, Erbb3, and Bmi1) have been reported to delineate subsets of GFRα1+ cells showing stem cell-related characteristics, like what has been shown for T+ (and probably SHISA6+) cells in this study. Understandably, it is proposed that cells expressing these genes are the bona fide stem cells. It is puzzling, however, that these genes appear to delineate different populations. For example, the observed frequencies were very different, namely, 1.1 ± 0.1 Id4-GFP+ cells/tubule section (Chan et al., 2014) and about one PAX7+ cell in an entire testis cross-section (which usually contains >100 tubules) (Aloisio et al., 2014); these genes show very different degrees of enrichment to the GFRα1+ fraction (Figure S7D). Furthermore, intravital live-imaging studies demonstrated that GFRα1+ cells continually interconvert between the states of As, Apr, and Aal through incomplete division and intercellular bridge breakdown (Hara et al., 2014). Combined with clonal fate analysis and mathematical modeling, it is proposed that the entire GFRα1+ population may comprise a single stem cell pool. This stem cell system, therefore, should be more complex and dynamic than has been considered, where SHISA6+/T+ cells (or states) should play some key roles. A considerable hypothesis is that GFRα1+ cells may interconvert between SHISA6/T-positive and -negative states, which show high and low potentials of self-renewal, respectively.