Yasumasa Bessho

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.

Mouse Fbw7/Sel-10/Cdc4 Is Required for Notch Degradation during Vascular Development

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development.

Instability of Hes7 protein is crucial for the somite segmentation clock

During somitogenesis, a pair of somites buds off from the presomitic mesoderm every 2 hours in mouse embryos, suggesting that somite segmentation is controlled by a biological clock with a 2-hour cycle. Expression of the basic helix-loop-helix factor Hes7, an effector of Notch signaling, follows a 2-hour oscillatory cycle controlled by negative feedback; this is proposed to be the molecular basis for the somite segmentation clock. If the proposal is correct, this clock should depend crucially on the short lifetime of Hes7. To address the biological importance of Hes7 instability, we generated mice expressing mutant Hes7 with a longer half-life (∼30 min compared with ∼22 min for wild-type Hes7) but normal repressor activity. In these mice, somite segmentation and oscillatory expression became severely disorganized after a few normal cycles of segmentation. We simulated this effect mathematically using a direct autorepression model. Thus, instability of Hes7 is essential for sustained oscillation and for its function as a segmentation clock.

Regulation of Neuropeptide Expression in Cultured Cerebral Cortical Neurons by Brain‐Derived Neurotrophic Factor

Abstract: The neuropeptide‐inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain‐derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15‐fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin‐3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival‐promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.

Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm

Whereas Notch signalling is essential for somitogenesis, mice deficient for the basic helix‐loop‐helix (bHLH) genes Hes1 and Hes5, downstream Notch effectors, display normal somite formation, indicating that there may be an as‐yet unidentified Hes1‐related bHLH gene.

Selective up-regulation of an NMDA receptor subunit mRNA in cultured cerebellar granule cells by K(+)-induced depolarization and NMDA treatment.

High KCI or NMDA treatment promotes the survival of cultured neonatal cerebellar granule cells, and these cells become sensitive to NMDA toxicity after prolonged K+ depolarization. Following both treatments, the NMDA receptor increases, as assessed by fura-2 fluorescence analysis of NMDA receptor-mediated intracellular Ca2+ increase. Northern analysis indicates that both treatments specifically up-regulate NMDAR2A subunit mRNA through an increase in resting intracellular Ca2+ concentration. Antisense oligonucleotide analysis further indicates that NMDAR2A mRNA up-regulation is responsible for NMDA receptor induction. Our results demonstrate that regulation of a specific NMDA receptor subunit mRNA governs NMDA receptor induction, which is thought to play an important role in granule cell survival and death.

Hes1 and Hes3 regulate maintenance of the isthmic organizer and development of the mid/hindbrain

The isthmic organizer, which is located at the midbrain–hindbrain boundary, plays an essential role in development of the midbrain and anterior hindbrain. It has been shown that homeobox genes regulate establishment of the isthmic organizer, but the mechanism by which the organizer is maintained is not well understood. Here, we found that, in mice doubly mutant for the basic helix–loop–helix genes Hes1 and Hes3, the midbrain and anterior hindbrain structures are missing without any significant cell death. In these mutants, the isthmic organizer cells prematurely differentiate into neurons and terminate expression of secreting molecules such as Fgf8 and Wnt1 and the paired box genes Pax2/5, all of which are essential for the isthmic organizer function. These results indicate that Hes1 and Hes3 prevent premature differentiation and maintain the organizer activity of the isthmic cells, thereby regulating the development of the midbrain and anterior hindbrain.

The basic helix-loop-helix genes Hesr1/Hey1 and Hesr2/Hey2 regulate maintenance of neural precursor cells in the brain.

Neural precursor cells proliferate in the ventricular zone while giving rise to neurons of deep layers first, then those of the superficial layers, and lastly, glial cells in the brain. Thus, it is essential to maintain neural precursor cells until late stages of neural development for generation of a wide variety of cell types. Here, we found that the Hes-related basic helix-loop-helix (bHLH) genes Hesr1/Hey1 and Hesr2/Hey2 are expressed in the ventricular zone, which contains neural precursor cells. Misexpression of Hesr1 and Hesr2 by electroporation in mouse brain at embryonic day 13.5 transiently maintains neural precursor cells and thereby increases late-born neurons, which are located in the superficial layers. In contrast, misexpression of the genes at later stages inhibits neurogenesis and promotes generation of astroglial cells. In transient transfection assay with cultured cells, both Hesr1 and Hesr2 inhibit transcription induced by the neuronal bHLH genes Mash1 and Math3. These results indicate that Hesr1 and Hesr2 negatively regulate neuronal bHLH genes, promote maintenance of neural precursor cells, and increase late-born cell types in the developing brain.

The Basic Helix-Loop-Helix Gene hesr2 Promotes Gliogenesis in Mouse Retina

Members of a subclass of hairy/Enhancer of split [E(spl)] homologs, calledhesr genes, are structurally related to another subclass of hairy/E(spl) homologs,Hes genes, which play an important role in neural development. To characterize the roles of hesr genes in neural development, we used the retina as a model system. In situ hybridization analysis indicated that allhesr genes are expressed in the developing retina, but only hesr2 expression is associated spatially with gliogenesis. Each member was then misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates, which well mimic in vivo retinal development. Interestingly,hesr2 but not hesr1 orhesr3 promoted gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopted the glial fate by misexpression of hesr2. The gliogenic activity ofhesr2 was more profound when it was misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genes Mash1 andMath3, which increases Müller glia at the expense of bipolar cells, upregulated hesr2 expression. These results indicate that, among structurally related hesrgenes, only hesr2 promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation between hesr2 and Mash1–Math3 may determine the ratios of neurons and glia.

Oscillations, clocks and segmentation.

Notch signalling molecules, such as the basic helix-loop-helix factors Hes1 and Hes7, periodically change their expression in the presomitic mesoderm, and each cycle of gene expression is associated with somite formation (every two hours in mouse). This cyclic expression is the manifestation of an intrinsic mechanism, called the segmentation clock, which is essential for coordinated somite segmentation. Interestingly, the oscillatory expression of Hes1 is observed in many cell types after serum stimulation, suggesting that this ultradian clock is not unique to presomitic mesoderm cells but widely distributed. This oscillation depends on the negative feedback loop, and once its promoter is constitutively activated, Hes1 seems to start oscillatory gene expression autonomously. Thus, Hes1 acts as a device that transduces a direct current of input into an alternating current, which ticks the hours in many biological systems.