Ryoichiro Kageyama

Control of endodermal endocrine development by Hes-1

Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.

Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain

Neurogenesis occurs continuously in the forebrain of adult mammals, but the functional importance of adult neurogenesis is still unclear. Here, using a genetic labeling method in adult mice, we found that continuous neurogenesis results in the replacement of the majority of granule neurons in the olfactory bulb and a substantial addition of granule neurons to the hippocampal dentate gyrus. Genetic ablation of newly formed neurons in adult mice led to a gradual decrease in the number of granule cells in the olfactory bulb, inhibition of increases in the granule cell number in the dentate gyrus and impairment of behaviors in contextual and spatial memory, which are known to depend on hippocampus. These results suggest that continuous neurogenesis is required for the maintenance and reorganization of the whole interneuron system in the olfactory bulb, the modulation and refinement of the existing neuronal circuits in the dentate gyrus and the normal behaviors involved in hippocampal-dependent memory.

Hes1 and Hes5 as Notch effectors in mammalian neuronal differentiation

While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and Enhancer‐of‐split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild‐type, Hes1‐null, Hes5‐null and Hes1‐Hes5 double‐null mouse embryos. We found that caNotch, which induced the endogenous Hes1 and Hes5 expression, inhibited neuronal differentiation in the wild‐type, Hes1‐null and Hes5‐null background, but not in the Hes1‐Hes5 double‐null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation.

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.

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.

The Hes gene family: repressors and oscillators that orchestrate embryogenesis

Embryogenesis involves orchestrated processes of cell proliferation and differentiation. The mammalian Hes basic helix-loop-helix repressor genes play central roles in these processes by maintaining progenitor cells in an undifferentiated state and by regulating binary cell fate decisions. Hes genes also display an oscillatory expression pattern and control the timing of biological events, such as somite segmentation. Many aspects of Hes expression are regulated by Notch signaling, which mediates cell-cell communication. This primer describes these pleiotropic roles of Hes genes in some developmental processes and aims to clarify the basic mechanism of how gene networks operate in vertebrate embryogenesis.

Essential Roles of Notch Signaling in Maintenance of Neural Stem Cells in Developing and Adult Brains

Activation of Notch signaling induces the expression of transcriptional repressor genes such as Hes1, leading to repression of proneural gene expression and maintenance of neural stem/progenitor cells. However, a requirement for Notch signaling in the telencephalon was not clear, because in Hes1;Hes3;Hes5 triple-mutant mice, neural stem/progenitor cells are depleted in most regions of the developing CNS, but not in the telencephalon. Here, we investigated a role for Notch signaling in the telencephalon by generating tamoxifen-inducible conditional knock-out mice that lack Rbpj, an intracellular signal mediator of all Notch receptors. When Rbpj was deleted in the embryonic brain, almost all telencephalic neural stem/progenitor cells prematurely differentiated into neurons and were depleted. When Rbpj was deleted in the adult brain, all neural stem cells differentiated into transit-amplifying cells and neurons. As a result, neurogenesis increased transiently, but 3 months later all neural stem cells were depleted and neurogenesis was totally lost. These results indicated an absolute requirement of Notch signaling for the maintenance of neural stem cells and a proper control of neurogenesis in both embryonic and adult brains.

Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system.

In the developing mammalian central nervous system, neural precursor cells present in the ventricular zone determine their fate to become neurons or glial cells, migrate towards the outer layers and undergo terminal differentiation. The transcriptional repressor HES‐1, a basic helix‐loop‐helix (bHLH) factor structurally related to the Drosophila hairy gene, is expressed at high levels throughout the ventricular zone, but the level decreases as neural differentiation proceeds. Because of this negative correlation, we tested whether continuous expression of HES‐1 inhibits neural differentiation. A HES‐1 and lacZ‐transducing retrovirus (SG‐HES1) and a control lacZ‐transducing retrovirus (SG) were injected into the lateral ventricles of mouse embryos, and the fate of the infected neural precursor cells was examined by X‐gal staining. The SG virus‐infected cells migrated and differentiated into neurons and glial cells. In contrast, the cells infected with SG‐HES1 virus remained in the ventricular/subventricular zone, decreased to approximately 10% in number as compared with that of the newborn during the postnatal 4‐5 weeks and, when they survived, were present exclusively in the ependymal layer. Furthermore, whereas cultured neural precursor cells infected with SG virus became immunoreactive for neuronal and glial markers, the cells infected with SG‐HES1 virus did not. These results show that persistent expression of HES‐1 severely perturbs neuronal and glial differentiation.

Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system.

Neural development involves the initial growth phase of dividing precursor cells and the subsequent differentiation phase of postmitotic cells. Recent studies indicate that the transition from the former phase to the latter is controlled antagonistically by multiple helix-loop-helix (HLH) genes. Cascades of neuronal HLH genes promote differentiation whereas anti-neuronal HLH genes repress them under the control of Notch and keep cells at the precursor stage. This antagonistic regulation may be essential for generation of the proper number of neurons and for morphogenesis of the nervous system.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-β, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix–loop–helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.