Hiromi Shimojo

Oscillations in notch signaling regulate maintenance of neural progenitors.

Expression of the Notch effector gene Hes1 is required for maintenance of neural progenitors in the embryonic brain, but persistent and high levels of Hes1 expression inhibit proliferation and differentiation of these cells. Here, by using a real-time imaging method, we found that Hes1 expression dynamically oscillates in neural progenitors. Furthermore, sustained overexpression of Hes1 downregulates expression of proneural genes, Notch ligands, and cell cycle regulators, suggesting that their proper expression depends on Hes1 oscillation. Surprisingly, the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta-like1 (Dll1) are also expressed in an oscillatory manner by neural progenitors, and inhibition of Notch signaling, a condition known to induce neuronal differentiation, leads to downregulation of Hes1 and sustained upregulation of Ngn2 and Dll1. These results suggest that Hes1 oscillation regulates Ngn2 and Dll1 oscillations, which in turn lead to maintenance of neural progenitors by mutual activation of Notch signaling.

Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition

In the developing mammalian nervous system, neural progenitor cells first express the Notch effector Hes1 at variable levels and then proneural genes and Notch ligands in salt-and-pepper patterns. Recent real-time imaging analysis indicates that Hes1 expression in these cells oscillates with a period of about 2–3 h. Furthermore, the proneural gene Neurogenin-2 (Ngn2) and the Notch ligand gene Deltalike-1 (Dll1) are expressed cyclically in neural progenitor cells under the control of Hes1 oscillation but are expressed continuously in postmitotic neurons, which lose Hes1 expression. Hes1-driven Ngn2 and Dll1 oscillations seem to be advantageous for maintenance of a group of cells in an undifferentiated state by mutual activation of Notch signaling. This dynamic mode of gene expression would require a revision of the traditional view of how Notch-mediated lateral inhibition operates in the developing mammalian nervous system.

Dynamic regulation of Notch signaling in neural progenitor cells

In the developing nervous system, differentiating neurons express Delta and activate Notch signaling in their neighboring cells. As a result of Notch activation, neuronal differentiation is inhibited in neighboring cells and they remain neural progenitor cells. Thus, differentiation of neurons and maintenance of neural progenitor cells are well balanced owing to Notch signaling. Recent studies revealed that Notch signaling is under the control of more complex and dynamic regulation than previously thought, such as cell cycle dependent activation and oscillating gene expression. We discuss here recent advances in understanding how Notch signaling is regulated in the developing nervous system and what outcome each type of regulation of Notch signaling leads to. We highlight the role of Notch signaling in proliferation and differentiation of neural progenitor cells.

Visualization of embryonic neural stem cells using Hes promoters in transgenic mice.

In the central nervous system, neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner, suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist, we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice, GFP expression was restricted to undifferentiated cells in the VZ, which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture, indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development.

Dynamic expression of notch signaling genes in neural stem/progenitor cells.

In neural stem/progenitor cells, expression of the Notch effector Hes1, a transcriptional repressor, oscillates with a period of 2–3 h by negative feedback, and Hes1 oscillations induce the oscillatory expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand gene Delta-like1 (Dll1). Dll1 oscillation leads to the mutual activation of Notch signaling between neighboring cells, thereby maintaining a group of cells in the undifferentiated state. Not all cells express Hes1 in an oscillatory manner: cells in boundary regions such as the isthmus express Hes1 in a sustained manner, and these cells are rather dormant with regard to proliferation and differentiation. Thus, Hes1 allows cell proliferation and differentiation when its expression oscillates but induces dormancy when its expression is sustained. After Hes1 expression is repressed, Ngn2 is expressed in a sustained manner, promoting neuronal differentiation. Thus, Ngn2 leads to the maintenance of neural stem/progenitor cells by inducing Dll1 oscillation when its expression oscillates but to neuronal differentiation when its expression is sustained. These results indicate that the different dynamics of Hes1 and Ngn2 lead to different outcomes.

Ultradian Oscillations in Notch Signaling Regulate Dynamic Biological Events

Notch signaling regulates many dynamic processes; accordingly, expression of genes in this pathway is also dynamic. In mouse embryos, one dynamic process regulated by Notch is somite segmentation, which occurs with a 2-h periodicity. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the Notch effector gene Hes7. Loss of Hes7 expression and sustained expression of Hes7 result in identical and severe somite defects, suggesting that Hes7 oscillation is required for proper somite segmentation. Mathematical models of this oscillator have been used to generate and test hypothesis, helping to uncover the role of negative feedback in regulating the oscillator. Oscillations of another Notch effector gene, Hes1, plays an important role in maintenance of neural stem cells. Hes1 expression oscillates with a period of about 2-3h in neural stem cells, whereas sustained Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillations are important for their proper activities. Hes1 inhibits its own expression as well as the expression of the proneural gene Neurogenin2 and the Notch ligand Delta1, driving oscillations of these two genes. Delta1 oscillations in turn maintain neural stem cells by mutual activation of Notch signaling, which re-activates Hes1 to close the cycle. Hes1 expression also oscillates in embryonic stem (ES) cells. Cells expressing low and high levels of Hes1 tend to differentiate into neural and mesodermal cells, respectively. Furthermore, Hes1-null ES cells display early and uniform neural differentiation, indicating that Hes1 oscillations act to promote multipotency by generating heterogeneity in both the differentiation timing and the fate choice. Taken together, these results suggest that Notch signaling can drive short-period oscillatory expression of Hes7 and Hes1 (ultradian oscillation) and that ultradian oscillations are important for many biological events.

Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis

Notch signaling regulates tissue morphogenesis through cell-cell interactions. The Notch effectors Hes1 and Hes7 are expressed in an oscillatory manner and regulate developmental processes such as neurogenesis and somitogenesis, respectively. Expression of the mRNA for the mouse Notch ligand Delta-like1 (Dll1) is also oscillatory. However, the dynamics of Dll1 protein expression are controversial, and their functional significance is unknown. Here, we developed a live-imaging system and found that Dll1 protein expression oscillated in neural progenitors and presomitic mesoderm cells. Notably, when Dll1 expression was accelerated or delayed by shortening or elongating the Dll1 gene, Dll1 oscillations became severely dampened or quenched at intermediate levels, as modeled mathematically. Under this condition, Hes1 and Hes7 oscillations were also dampened. In the presomitic mesoderm, steady Dll1 expression led to severe fusion of somites and their derivatives, such as vertebrae and ribs. In the developing brain, steady Dll1 expression inhibited proliferation of neural progenitors and accelerated neurogenesis, whereas optogenetic induction of Dll1 oscillation efficiently maintained neural progenitors. These results indicate that the appropriate timing of Dll1 expression is critical for the oscillatory networks and suggest the functional significance of oscillatory cell-cell interactions in tissue morphogenesis.

Genetic visualization of notch signaling in mammalian neurogenesis

Notch signaling plays crucial roles in fate determination and the differentiation of neural stem cells in embryonic and adult brains. It is now clear that the notch pathway is under more complex and dynamic regulation than previously thought. To understand the functional details of notch signaling more precisely, it is important to reveal when, where, and how notch signaling is dynamically communicated between cells, for which the visualization of notch signaling is essential. In this review, we introduce recent technical advances in the visualization of notch signaling during neural development and in the adult brain, and we discuss the physiological significance of dynamic regulation of notch signaling.

Gene expression profiling of neural stem cells and identification of regulators of neural differentiation during cortical development.

During mammalian brain development, neural stem cells transform from neuroepithelial cells to radial glial cells and finally remain as astrocyte‐like cells in the postnatal and adult brain. Neuroepithelial cells divide symmetrically and expand the neural stem cell pool; after the onset of neurogenesis, radial glial cells sequentially produce deep layer neurons and then superficial layer neurons by asymmetric, self‐renewing divisions during cortical development. Thereafter, gliogenesis supersedes neurogenesis, while a subset of neural stem cells retain their stemness and lurk in the postnatal and adult brain. Thus, neural stem cells undergo alterations in morphology and the capacity to proliferate or give rise to various types of neural cells in a temporally regulated manner. To shed light on the temporal alterations of embryonic neural stem cells, we sorted the green fluorescent protein‐positive cells from the dorsolateral telencephalon (neocortical region) of pHes1‐d2EGFP transgenic mouse embryos at different developmental stages and performed gene expression profiling. Among dozens of transcription factors differentially expressed by cells in the ventricular zone during the course of development, several of them exhibited the activity to inhibit neuronal differentiation when overexpressed. Furthermore, knockdown of Tcf3 or Klf15 led to accelerated neuronal differentiation of neural stem cells in the developing cortex, and neurospheres originated from Klf15 knockdown cells mostly lacked neurogenic activities and only retained gliogenic activities. These results suggest that Tcf3 and Klf15 play critical roles in the maintenance of neural stem cells at early and late embryonic stages, respectively. STEM CELLS 2011;29:1817–1828

Rhythmic gene expression in somite formation and neural development

In mouse embryos, somite formation occurs every two hours, and 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 expression oscillates by negative feedback and is cooperatively regulated by Fgf and Notch signaling. Both loss of expression and sustained expression of Hes7 result in severe somite fusion, suggesting that Hes7 oscillation is required for proper somite segmentation. Expression of a related gene, Hes1, also oscillates by negative feedback with a period of about two hours in many cell types such as neural progenitor cells. Hes1 is required for maintenance of neural progenitor cells, but persistent Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillation is required for their proper activities. Hes1 oscillation regulates cyclic expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta1, which in turn lead to maintenance of neural progenitor cells by mutual activation of Notch signaling. Taken together, these results suggest that oscillatory expression with short periods (ultradian oscillation) plays an important role in many biological events.