Taeko Kobayashi

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.

Roles of Hes genes in neural development

Hes genes are mammalian homologues of Drosophila hairy and Enhancer of split, which encode basic helix‐loop‐helix (bHLH) transcriptional repressors. In the developing central nervous system, Hes1, Hes3 and Hes5 are highly expressed by neural stem cells. Inactivation of these Hes genes leads to upregulation of proneural genes, acceleration of neurogenesis and premature depletion of neural stem cells. Conversely, overexpression of Hes genes leads to inhibition of neurogenesis and maintenance of neural stem cells. At later stages of development, Hes genes promote gliogenesis. Furthermore, Hes genes regulate maintenance of boundaries, which partition the nervous system into many compartments and endow the neighboring compartments with regional identities by secreting morphogens. Boundary cells usually proliferate slowly and do not give rise to neurons, unlike neural stem cells in compartments. Interestingly, these different characteristics between boundary cells and compartmental neural stem cells are regulated by different modes of Hes1 expression, which is variable in neural stem cells in compartments and persistent and high in boundary cells. Thus, Hes genes play an essential role in neural development by regulating proliferation, differentiation and specification of neural stem cells.

The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells

Stem cells do not all respond the same way, but the mechanisms underlying this heterogeneity are not well understood. Here, we found that expression of Hes1 and its downstream genes oscillate in mouse embryonic stem (ES) cells. Those expressing low and high levels of Hes1 tended to differentiate into neural and mesodermal cells, respectively. Furthermore, inactivation of Hes1 facilitated neural differentiation more uniformly at earlier time. Thus, Hes1-null ES cells display less heterogeneity in both the differentiation timing and fate choice, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells even under the same environmental conditions.

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104–Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration‐defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41–Cys44. Remarkably, the Cys41–Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41–Cys44 became sensitive to the reducing agent when membrane was prepared from quinone‐ or heme‐depleted cells or when a membrane sample was deaerated. Thus, the Cys41–Val–Leu–Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.

Involvement of valosin-containing protein (VCP)/p97 in the formation and clearance of abnormal protein aggregates

Abnormal protein aggregates are commonly observed in affected neurons in many neurodegenerative disorders. We have reported that valosin‐containing protein (VCP) co‐localizes with protein aggregates in patients’ neurons and in cultured cells expressing diseased proteins. However, the significance of such co‐localization remains elucidated. Here we report the involvement of VCP in the re‐solubilization process of abnormal protein aggregates. VCP recognized and accumulated onto pre‐formed protein aggregates created by proteasome inhibition. VCP knockdown or the expression of dominant‐negative VCP both significantly delayed the elimination of ubiquitin‐positive aggregates. VCP was involved in the clearance of pre‐formed polyglutamine aggregates as well. Paradoxically, VCP knockdown also diminished polyglutamine aggregate formation. Furthermore, its ATPase activity was required for the re‐solubilization and re‐activation of heat‐denatured proteins, such as luciferase, from insoluble aggregates. We thus propose that VCP functions as a mediator for both aggregate formation and clearance depending upon the concentration of soluble aggregate‐prone proteins, indicating dual VCP functions as an aggregate formase and an unfoldase.

The role of Hes genes in intestinal development, homeostasis and tumor formation.

Notch signaling regulates intestinal development, homeostasis and tumorigenesis, but its precise downstream mechanism remains largely unknown. Here we found that inactivation of the Notch effectors Hes1, Hes3 and Hes5, but not Hes1 alone, led to reduced cell proliferation, increased secretory cell formation and altered intestinal structures in adult mice. However, in Apc mutation-induced intestinal tumors, inactivation of Hes1 alone was sufficient for reducing tumor cell proliferation and inducing differentiation of tumor cells into all types of intestinal epithelial cells, but without affecting the homeostasis of normal crypts owing to genetic redundancy. These results indicated that Hes genes cooperatively regulate intestinal development and homeostasis and raised the possibility that Hes1 is a promising target to induce the differentiation of tumor cells.

Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3–5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1‐high ES cells are prone to the mesodermal fate, while Hes1‐low ES cells are prone to the neural fate. These outcomes of Hes1‐high and Hes1‐low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.

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.

Expression Dynamics and Functions of Hes Factors in Development and Diseases

Hes genes, encoding basic helix-loop-helix (HLH) transcriptional repressors, are mammalian homologues of Drosophila hairy and Enhancer of split genes, both of which are required for normal neurogenesis in Drosophila. There are seven members in the human Hes family, Hes1-7, which are expressed in many tissues and play various roles mainly in development. All Hes proteins have three conserved domains: basic HLH (bHLH), Orange, and WRPW domains. The basic region binds to target DNA sequences, while the HLH region forms homo- and heterodimers with other bHLH proteins, the Orange domain is responsible for the selection of partners during heterodimer formation, and the WRPW domain recruits corepressors. Hes1, Hes5, and Hes7 are known as downstream effectors of canonical Notch signaling, which regulates cell differentiation via cell-cell interaction. Hes factors regulate many events in development by repressing the expression of target genes, many of which encode transcriptional activators that promote cell differentiation. For example, Hes1, Hes3, and Hes5 are highly expressed by neural stem cells, and inactivation of these genes results in insufficient maintenance of stem cell proliferation and prematurely promotes neuronal differentiation. Recently, it was shown that the expression dynamics of Hes1 plays crucial roles in proper developmental timings and fate-determination steps of embryonic stem cells and neural progenitor cells. Here, we discuss some key features of Hes factors in development and diseases.

Identification of a segment of DsbB essential for its respiration‐coupled oxidation

In the Escherichia coli protein disulphide bond formation pathway, membrane‐bound DsbB oxidizes periplasmic DsbA, the disulphide bond‐introducing enzyme. The Cys‐41–Val–Leu–Cys‐44 motif in the first periplasmic domain of DsbB is kept strongly oxidized by the respiratory function of the cell. We now show that the characteristic dithiothreitol resistance of the Cys‐41–Cys‐44 bond was retained even when the flanked Val–Leu combination was replaced by XX sequences from other oxidoreductases. Results of insertion mutagenesis showed that only the insertions (1–31 amino acids) in the region C‐terminally adjacent to the CXXC motif impaired the oxidized state of DsbB. Deletion of a single amino acid from this region also rendered DsbB reduced and inactive. However, single amino acid substitutions of the four residues flanked by CXXC and the transmembrane segment did not abolish the oxidation of DsbB. These results suggest that some physical property, such as distance of the CXXC motif from the membrane, is important for the respiration‐coupled oxidation of DsbB.