Cell-cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis.

Whereas stem and progenitor cells proliferate to maintain tissue homeostasis, fully differentiated cells exit the cell cycle. How cell identity and cell-cycle state are coordinated during differentiation is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs). CySCs give rise to post-mitotic cyst cells, providing a tractable model to study the links between stem cell identity and proliferation. We show that, while cell-cycle progression is required for CySC self-renewal, the E2f1/Dp transcription factor is dispensable for self-renewal but instead must be silenced by the Drosophila retinoblastoma homolog, Rbf, to permit differentiation. Continued E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial biogenesis rescues the differentiation of CySCs with ectopic E2f1/Dp activity but not their cell-cycle exit. In sum, E2f1/Dp coordinates cell-cycle progression with stem cell identity by regulating the metabolic state of CySCs.

Proliferative stem cells maintain quiescence of their niche by secreting the Activin inhibitor Follistatin.

Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent "hub" cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.

Inhibition of PKCε induces primordial germ cell reprogramming into pluripotency by HIF1&2 upregulation and histone acetylation.

Historically, primordial germ cells (PGCs) have been a good model to study pluripotency. Despite their low numbers and limited accessibility in the mouse embryo, they can be easily and rapidly reprogrammed at high efficiency with external physicochemical factors and do not require transcription factor transfection. Employing this model to deepen our understanding of cell reprogramming, we specifically aimed to determine the relevance of Ca2+ signal transduction pathway components in the reprogramming process. Our results showed that PGC reprogramming requires a normal extracellular [Ca2+] range, in contrast to neoplastic or transformed cells, which can continue to proliferate in Ca2+-deficient media, differentiating normal reprogramming from neoplastic transformation. Our results also showed that a spike in extracellular [Ca2+] of 1-3 mM can directly reprogram PGC. Intracellular manipulation of Ca2+ signal transduction pathway components revealed that inhibition of classical Ca2+ and diacylglycerol (DAG)-dependent PKCs, or intriguingly, of only the novel DAG-dependent PKC, PKCε, were able to induce reprogramming. PKCε inhibition changed the metabolism of PGCs toward glycolysis, increasing the proportion of inactive mitochondria. This metabolic switch from oxidative phosphorylation to glycolysis is mediated by hypoxia-inducible factors (HIFs), given we found upregulation of both HIF1α and HIF2α in the first 48 hours of culturing. PKCε inhibition did not change the classical pluripotency gene expression of PGCs, Oct4, or Nanog. PKCε inhibition changed the histone acetylation of PGCs, with histones H2B, H3, and H4 becoming acetylated in PKCε-inhibited cultures (markers were H2BacK20, H3acK9, and H4acK5K8, K12, K16), suggesting that reprogramming by PKCε inhibition is mediated by histone acetylation.

Hypoxia induces pluripotency in primordial germ cells by HIF1α stabilization and Oct4 deregulation.

AIMS: To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). RESULTS: We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified was the Yamanaka genes but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. Hypoxia induced a glycolytic program in PGCs in detriment of mitochondrial oxidative phosphorylation. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGLs), which differentiate into cells of the three germ layers in vitro and contribute to the internal cell mass of the blastocyst in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1α stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to the lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signaling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. INNOVATION AND CONCLUSION: Our data suggest that the two main properties of stemness, pluripotency and self-renewal, are differentially regulated in PGC reprogramming induced by hypoxia.