Intracellular pH controls WNT downstream of glycolysis in amniote embryos.

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic ß-catenin acetylation downstream of WNT signalling. As acetylated ß-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.

High Hes1 expression and resultant Ascl1 suppression regulate quiescent vs. active neural stem cells in the adult mouse brain.

Somatic stem/progenitor cells are active in embryonic tissues but quiescent in many adult tissues. The detailed mechanisms that regulate active versus quiescent stem cell states are largely unknown. In active neural stem cells, Hes1 expression oscillates and drives cyclic expression of the proneural gene Ascl1, which activates cell proliferation. Here, we found that in quiescent neural stem cells in the adult mouse brain, Hes1 levels are oscillatory, although the peaks and troughs are higher than those in active neural stem cells, causing Ascl1 expression to be continuously suppressed. Inactivation of Hes1 and its related genes up-regulates Ascl1 expression and increases neurogenesis. This causes rapid depletion of neural stem cells and premature termination of neurogenesis. Conversely, sustained Hes1 expression represses Ascl1, inhibits neurogenesis, and maintains quiescent neural stem cells. In contrast, induction of Ascl1 oscillations activates neural stem cells and increases neurogenesis in the adult mouse brain. Thus, Ascl1 oscillations, which normally depend on Hes1 oscillations, regulate the active state, while high Hes1 expression and resultant Ascl1 suppression promote quiescence in neural stem cells.

Visualization of Notch signaling oscillation in cells and tissues.

The Notch signaling effectors Hes1 and Hes7 exhibit oscillatory expression with a period of about 2-3 h during embryogenesis. Hes1 oscillation is important for proliferation and differentiation of neural stem cells, whereas Hes7 oscillation regulates periodic formation of somites. Continuous expression of Hes1 and Hes7 inhibits these developmental processes. Thus, expression dynamics are very important for gene functions, but it is difficult to distinguish between oscillatory and persistent expression by conventional methods such as in situ hybridization and immunostaining. Here, we describe time-lapse imaging methods using destabilized luciferase reporters and a highly sensitive cooled charge-coupled device camera, which can monitor dynamic gene expression. Furthermore, the expression of two genes can be examined simultaneously by a dual reporter system using two-color luciferase reporters. Time-lapse imaging analyses reveal how dynamically gene expression changes in many biological events.

The roles and mechanism of ultradian oscillatory expression of the mouse Hes genes.

Somites, metameric structures, give rise to the vertebral column, ribs, skeletal muscles and subcutaneous tissues. In mouse embryos, a pair of somites is formed every 2h by segmentation of the anterior parts of the presomitic mesoderm. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the basic helix-loop-helix gene Hes7. Hes7 oscillation is regulated by negative feedback with a delayed timing. This process has been mathematically simulated by differential-delay equations, which predict that negative feedback with shorter delays would abolish oscillations or produce dampened but more rapid oscillations. We found that reducing the number of introns within the Hes7 gene shortens the delay and abolishes Hes7 oscillation or results in a more rapid tempo of Hes7 oscillation, increasing the number of somites and vertebrae in the cervical and upper thoracic region. We also found that Hes1, a Hes7-related gene, is expressed in an oscillatory manner by many cell types, including fibroblasts and neural stem cells. In these cells, Hes1 expression oscillates with a period of about 2-3h, and this oscillation is important for cell cycle progression. Furthermore, in neural stem cells, Hes1 oscillation drives cyclic expression of the proneural genes Ascl1 and Neurogenin2 and regulates multipotency. Hes1 expression oscillates more slowly in embryonic stem cells, and Hes1 oscillation regulates their fate preferences. Taken together, these results suggest that oscillatory expression with short periods (ultradian oscillation) is important for many biological events.

Oscillatory control of factors determining multipotency and fate in mouse neural progenitors.

The basic helix-loop-helix transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate fate choice of neurons, astrocytes, and oligodendrocytes, respectively. These same factors are coexpressed by neural progenitor cells. Here, we found by time-lapse imaging that these factors are expressed in an oscillatory manner by mouse neural progenitor cells. In each differentiation lineage, one of the factors becomes dominant. We used optogenetics to control expression of Ascl1 and found that, although sustained Ascl1 expression promotes neuronal fate determination, oscillatory Ascl1 expression maintains proliferating neural progenitor cells. Thus, the multipotent state correlates with oscillatory expression of several fate-determination factors, whereas the differentiated state correlates with sustained expression of a single factor.

Oscillatory links of Fgf signaling and Hes7 in the segmentation clock.

Somitogenesis is controlled by the segmentation clock, where the oscillatory expression of cyclic genes such as Hes7 leads to the periodic expression of Mesp2, a master gene for somite formation. Fgf signaling induces the oscillatory expression of Hes7 while Hes7 drives coupled oscillations in Fgf and Notch signaling, which inhibits and activates Mesp2 expression, respectively. Because of different oscillatory dynamics, oscillation in Fgf signaling dissociates from oscillation in Notch signaling in S-1, a prospective somite region, where Notch signaling induces Mesp2 expression when Fgf signaling becomes off. Thus, oscillation in Fgf signaling regulates the timing of Mesp2 expression and the pace of somitogenesis. In addition, Fgf signaling was found to be a primary target for hypoxia, which causes phenotypic variations of heterozygous mutations in Hes7 or Mesp2, suggesting gene-environment interaction through this signaling.

Accelerating the tempo of the segmentation clock by reducing the number of introns in the Hes7 gene.

Periodic somite segmentation is controlled by the cyclic gene Hes7, whose oscillatory expression depends upon negative feedback with a delayed timing. The mechanism that regulates the pace of segmentation remains to be determined, but mathematical modeling has predicted that negative feedback with shorter delays would give rise to dampened but more rapid oscillations. Here, we show that reducing the number of introns within the Hes7 gene shortens the delay and results in a more rapid tempo of both Hes7 oscillation and somite segmentation, increasing the number of somites and vertebrae in the cervical and upper thoracic region. These results suggest that the number of introns is important for the appropriate tempo of oscillatory expression and that Hes7 is a key regulator of the pace of the segmentation clock.

Oscillatory gene expression and somitogenesis.

A bilateral pair of somites forms periodically by segmentation of the anterior ends of the presomitic mesoderm (PSM). This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic gene expression. Expression of her1 and her7 in zebrafish and Hes7 in mice oscillates by negative feedback, and mathematical models have been used to generate and test hypotheses to aide elucidation of the role of negative feedback in regulating oscillatory expression. her/Hes genes induce oscillatory expression of the Notch ligand deltaC in zebrafish and the Notch modulator Lunatic fringe in mice, which lead to synchronization of oscillatory gene expression between neighboring PSM cells. In the mouse PSM, Hes7 induces coupled oscillations of Notch and Fgf signaling, while Notch and Fgf signaling cooperatively regulate Hes7 oscillation, indicating that Hes7 and Notch and Fgf signaling form the oscillator networks. Notch signaling activates, but Fgf signaling represses, expression of the master regulator for somitogenesis Mesp2, and coupled oscillations in Notch and Fgf signaling dissociate in the anterior PSM, which allows Notch signaling-induced synchronized cells to express Mesp2 after these cells are freed from Fgf signaling. These results together suggest that Notch signaling defines the prospective somite region, while Fgf signaling regulates the pace of segmentation. It is likely that these oscillator networks constitute the core of the segmentation clock, but it remains to be determined whether as yet unknown oscillators function behind the scenes.