The inter-follicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells, label retaining cells (LRCs), are stem cells, while non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1CreER and Slc1a3CreER genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation, and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. Upon wounding or selective killing, they can temporarily replenish each other’s territory. These two discrete inter-follicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

Maintaining Germline Stem Cells Spermatogonial stem cell pools in postnatal testes have to be maintained to continuously generate spermatozoa. It has been difficult to identify these stem cells in vivo, because of their small numbers and lack of appropriate molecular markers, but now Sada et al. (p. 1394 ) show that the RNA-binding protein NANOS2 is expressed in a small subset of spermatogonia that behave as self-renewing stem cells in intact testes. By a combinatorial use of loss- and gain-of-function studies, NANOS2 was found to be essential for the maintenance of the immature state of spermatogonial stem cells by supporting their self-renewing properties and by suppressing differentiation.

Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A(s) to A(pr) cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A(s) to A(al)) and differentiating A(1) spermatogonia. In our present study, we find that A(s) and A(pr) can be basically classified into three categories: (1) GFRalpha1(+)Nanos2(+)Nanos3(-)Ngn3(-), (2) GFRalpha1(+)Nanos2(+)Nanos3(+)Ngn3(-), and (3) GFRalpha1(-)Nanos2(+/-)Nanos3(+)Ngn3(+). We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.

The expression of C/EBP␣, which may govern transcription of mature hepatocyte marker genes, was suppressed in periportal hepatoblasts in mouse liver development, leading to biliary cell differentiation. This study was undertaken to analyze how inactivation of the Cebpa gene affects biliary cell differentiation and gene expression of the regulatory genes for that differentiation, including Hnf1b and Hnf6. In the knockout mouse liver at midgestation stages, pseudoglandular structures were abundantly induced in the parenchyma with elevated expression of Hnf6 and Hnf1b mRNAs. The wild-type liver parenchyma expressed mRNAs of these transcription factors at low levels, though periportal biliary progenitors had strong expression of them. These results suggest that expression of Hnf6 and Hnf1b is downstream of C/EBP␣ action in fetal liver development, and that the suppression of C/EBP␣ expression in periportal hepatoblasts may lead to expression of Hnf6 and Hnf1b mRNAs. Immunohistochemical studies with biliary cell markers in knockout livers demonstrated that differentiated biliary epithelial cells were confined to around the portal veins. The suppression of C/EBP␣ expression may result in upregulation of Hnf6 and Hnf1b gene expression, but be insufficient for biliary cell differentiation. When liver fragments of Cebpa-knockout fetuses, in which hepatoblasts were contained as an endodermal component, were transplanted in the testis of Scid (Prkdc) male mice, almost all hepatoblasts gave rise to biliary epithelial cells. Wild-type hepatoblasts constructed mature hepatic tissue accompanied by biliary cell differentiation. These results also demonstrate that the suppression of C/EBP␣ expression may stimulate biliary cell differentiation. Development 133, 4233-4243 (2006) DEVELOPMENT 4234 their up-or downstream relationships with the action of C/EBP␣ in biliary cell differentiation. The knockout mice are neonatal lethal because of hypoglycemia accompanied by hyperammonemia (Flodby et al., 1996; Kimura et al., 1998;Wang et al., 1995). Thus, it is intriguing to study what type of histology the knockout liver exhibits and how biliary epithelial tissue is induced when the knockouts survive. In vitro studies have demonstrated that knockout hepatocytes can immortalize at a higher frequency (Soriano et al., 1998). KEY WORDS: C/EBP␣ ␣, Knockout, Hepatoblasts, Biliary epithelial cells, Bile ducts, Morphogenesis, MouseIn the present study, we demonstrate the expression of biliary cell markers in pseudoglandular cells of Cebpa-knockout livers. Inactivation of the Cebpa gene not only suppresses hepatocyte maturation, but also upregulates regulatory genes for biliary cell differentiation such as Hnf6 and Hnf1b genes in the liver parenchyma, suggesting that the absence of C/EBP␣ in normal biliary cells induces Hnf6 and Hnf1b expression, leading to biliary cell differentiation. Jag1 and Notch2 mRNAs were also upregulated in knockout livers, but not to the same extent as Hnf6 and Hnf1b mRNAs. Testicular transplants of the knockout livers...

In many adult tissues, homeostasis relies on self-renewing stem cells that are primed for differentiation. The reconciliation mechanisms of these characteristics remain a fundamental question in stem cell biology. We propose that regulation at the post-transcriptional level is essential for homeostasis in murine spermatogonial stem cells (SSCs). Here, we show that Nanos2, an evolutionarily conserved RNA-binding protein, works with other cellular messenger ribonucleoprotein (mRNP) components to ensure the primitive status of SSCs through a dual mechanism that involves (1) direct recruitment and translational repression of genes that promote spermatogonial differentiation and (2) repression of the target of rapamycin complex 1 (mTORC1), a well-known negative pathway for SSC self-renewal, by sequestration of the core factor mTOR in mRNPs. This mechanism links mRNA turnover to mTORC1 signaling through Nanos2-containing mRNPs and establishes a post-transcriptional buffering system to facilitate SSC homeostasis in the fluctuating environment within the seminiferous tubule.

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cellintrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. STEM CELLS 2012;30:280-291 Disclosure of potential conflicts of interest is found at the end of this article.

Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying (TA) matrix cells. EPs can revert to SCs upon injury, but whether this de-differentiation occurs in normal HF homeostasis (hair cycle), and the mechanisms regulating both differentiation and de-differentiation are unclear. Here we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome towards a cell-state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and de-differentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche.

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