[Mechanism for the molecular clock controlling somite segmentation].
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Animals↗
Biomedical subjects
Yasumasa Bessho
Publications and source records attributed to 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.
Animals↗
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 (approximately 30 min compared with approximately 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.
Animals↗
Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification.
Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loop-helix (bHLH) genes. However, the precise mechanism for specification of retinal neuronal subtypes, particularly horizontal neurons and photoreceptors, remains to be determined. Here, we examined retinas with three different combinations of triple bHLH gene mutations. In retinas lacking the bHLH genes Ngn2, Math3, and NeuroD, horizontal neurons as well as other neurons such as bipolar cells were severely decreased in number. In the retina lacking the bHLH genes Mash1, Ngn2, and Math3, horizontal and other neurons were severely decreased, whereas ganglion cells were increased. In the retina lacking the bHLH genes Mash1, Math3, and NeuroD, photoreceptors were severely decreased, whereas ganglion cells were increased. In all cases, glial cells were increased. The increase and decrease of these cells were the result of cell fate changes and cell death and seem to be partly attributable to the remaining bHLH gene expression, which also changes because of triple bHLH gene mutations. These results indicate that multiple bHLH genes cross-regulate each other, cooperatively specify neuronal subtypes, and regulate neuronal survival in the developing retina.
Animals↗
Roles of the bHLH gene Hes1 in retinal morphogenesis.
During retinal development, common precursors give rise to various types of cells in a time course specific to each cell type. Previously, we demonstrated that the bHLH gene Hes1 inhibits neuronal differentiation whereas, in Hes1-null retina, precursors prematurely differentiate into neurons and form abnormal rosette-like structures. Thus, Hes1 is essential for maintenance of precursors and morphogenesis of the neural retina. However, the precise causal link between premature differentiation and abnormal structures remains to be determined. Here, we found that misexpression of Hes1 in the developing retina promotes formation of undifferentiated precursor-like cells, whereas in Hes1-null retina, precursors are not properly maintained and prematurely differentiate into ganglion cells. Strikingly, those prematurely differentiated ganglion cells erupt into the subretinal space through the regions where precursors and the outer limiting membrane are lost. These results indicate that Hes1 maintains precursors and the outer limiting membrane and thereby regulates retinal morphogenesis.
Animals↗
Identification of a novel basic helix-loop-helix gene, Heslike, and its role in GABAergic neurogenesis.
Neuronal subtype specification depends on multiple transcription factors such as basic helix-loop-helix (bHLH) factors. However, transcription factor codes for most neurons remain to be determined. Here, we report identification of a novel mouse bHLH factor, termed Heslike, that has Hes1-like bHLH domain and transcriptional repressor activity. Heslike is coexpressed with the bHLH factor Mash1 in brain regions that give rise to GABAergic neurons. In the mesencephalon and the caudal diencephalon, coexpression of Heslike and Mash1 is initially restricted to small regions but expanded dorsally from embryonic day 9.5 onward, and this expansion of coexpression is followed by GABAergic neurogenesis. Misexpression of Heslike in mouse embryos generates ectopic GABAergic neurons only from the Mash1(+) region. In contrast, in the mesencephalon and the caudal diencephalon of Mash1-null mice, GABAergic neurons are almost completely missing and, instead, other neurons are generated, although Heslike is still expressed. Furthermore, coexpression of Heslike and Mash1 significantly promotes formation of GABAergic neurons, compared with each gene alone, in neural precursor cell culture. Thus, Heslike or Mash1 alone is not sufficient, but their coexpression may be important for generation of GABAergic neurons. These results suggest that combinations of distinct bHLH factors promote formation of distinct neuronal subtypes, thereby increasing neuronal diversity.
Amino Acid Sequence↗
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.
Animals↗
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.
Animals↗
Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock.
Hes7, a bHLH gene essential for somitogenesis, displays cyclic expression of mRNA in the presomitic mesoderm (PSM). Here, we show that Hes7 protein is also expressed in a dynamic manner, which depends on proteasome-mediated degradation. Spatial comparison revealed that Hes7 and Lunatic fringe (Lfng) transcription occurs in the Hes7 protein-negative domains. Furthermore, Hes7 and Lfng transcription is constitutively up-regulated in the absence of Hes7 protein and down-regulated by stabilization of Hes7 protein. Thus, periodic repression by Hes7 protein is critical for the cyclic transcription of Hes7 and Lfng, and this negative feedback represents a molecular basis for the segmentation clock.
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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.
Animals↗
Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop.
Transcription of messenger RNAs (mRNAs) for Notch signaling molecules oscillates with 2-hour cycles, and this oscillation is important for coordinated somite segmentation. However, the molecular mechanism of such oscillation remains to be determined. Here, we show that serum treatment of cultured cells induces cyclic expression of both mRNA and protein of the Notch effector Hes1, a basic helix-loop-helix (bHLH) factor, with 2-hour periodicity. Cycling is cell-autonomous and depends on negative autoregulation of hes1 transcription and ubiquitin-proteasome-mediated degradation of Hes1 protein. Because Hes1 oscillation can be seen in many cell types, this clock may regulate timing in many biological systems.
Acetylcysteine↗
Math3 and NeuroD regulate amacrine cell fate specification in the retina.
The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NEUROD: We have found that, in Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells did not die but adopted the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures showed that, although Math3 and NeuroD alone only promoted rod genesis, they significantly increased the population of amacrine cells when the homeobox gene Pax6 or Six3 was co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype.
Amacrine Cells↗