The initiation and propagation of Hes7 oscillation are cooperatively regulated by Fgf and notch signaling in the somite segmentation clock

Periodic formation of somites is controlled by the segmentation clock, where the oscillator Hes7 regulates cyclic expression of the Notch modulator Lunatic fringe. Here, we show that Hes7 also regulates cyclic expression of the Fgf signaling inhibitor Dusp4 and links Notch and Fgf oscillations in phase. Strikingly, inactivation of Notch signaling abolishes the propagation but allows the initiation of Hes7 oscillation. By contrast, transient inactivation of Fgf signaling abolishes the initiation, whereas sustained inactivation abolishes both the initiation and propagation of Hes7 oscillation. We thus propose that Hes7 oscillation is initiated by Fgf signaling and propagated/maintained anteriorly by Notch signaling.     

The vertebrate segmentation clock and its role in skeletal birth defects

The segmental structure of the vertebrate body plan is most evident in the axial skeleton. The regulated generation of somites, a process called somitogenesis, underlies the vertebrate body plan and is crucial for proper skeletal development. A genetic clock regulates this process, controlling the timing of somite development. Molecular evidence for the existence of the segmentation clock was first described in the expression of Notch signaling pathway members, several of which are expressed in a cyclic fashion in the presomitic mesoderm (PSM). The Wnt and fibroblast growth factor (FGF) pathways have also recently been linked to the segmentation clock, suggesting that a complex, interconnected network of three signaling pathways regulates the timing of somitogenesis. Mutations in genes that have been linked to the clock frequently cause abnormal segmentation in model organisms. Additionally, at least two human disorders, spondylocostal dysostosis (SCDO) and Alagille syndrome (AGS), are caused by mutations in Notch pathway genes and exhibit vertebral column defects, suggesting that mutations that disrupt segmentation clock function in humans can cause congenital skeletal defects. Thus, it is clear that the correct, cyclic function of the Notch pathway within the vertebrate segmentation clock is essential for proper somitogenesis. In the future, with a large number of additional cyclic genes recently identified, the complex interactions between the various signaling pathways making up the segmentation clock will be elucidated and refined.     

Hes1 and Hes5 regulate vascular remodeling and arterial specification of endothelial cells in brain vascular development

The vascular system is the first organ to form in the developing mammalian embryo. The Notch signaling pathway is an evolutionarily conserved signaling mechanism essential for proper embryonic development in almost all vertebrate organs. The analysis of targeted mouse mutants has demonstrated essential roles of the Notch signaling pathway in embryonic vascular development. However, Notch signaling-deficient mice have so far not been examined in detail in the head region. The bHLH genes Hes1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs. Here, we report that endothelial-specific Hes1 and Hes5 mutant embryos exhibited defective vascular remodeling in the brain. In addition, arterial identity of endothelial cells was partially lost in the brain of these mutant mice. These data suggest that Hes1 and Hes5 regulate vascular remodeling and arterial fate specification of endothelial cells in the development of the brain. Hes1 and Hes5 represent critical transducers of Notch signals in brain vascular development.     

A proposed mechanism for the interaction of the segmentation clock and the determination front in somitogenesis

Background

Recent discoveries in the field of somitogenesis have confirmed, for the most part, the feasibility of the clock and wavefront model. There are good candidates for both the clock (various genes expressed cyclically in the tail bud of vertebrate embryos have been discovered) and the wavefront (there are at least three different substances, whose expression levels vary along the presomitic mesoderm [PSM], that have important effects on the formation of somites). Nevertheless, the mechanisms through which the wavefront interacts with the clock to arrest the oscillations and induce somite formation have not yet been fully elucidated.

Principal Findings

In this work, we propose a gene regulatory network which is consistent with all known experimental facts in embryonic mice, and whose dynamic behaviour provides a potential explanation for the periodic aggregation of PSM cells into blocks: the first step leading to the formation of somites.

Significance

To our knowledge, this is the first proposed mechanism that fully explains how a block of PSM cells can stop oscillating simultaneously, and how this process is repeated periodically, via the interaction of the segmentation clock and the determination front.     

Roles of the basic helix-loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain

Neural stem cells, which differentiate into neurons and glia, are present in the ventricular zone of the embryonal brain. The precise mechanism by which neural stem cells are maintained during embryogenesis remains to be determined. Here, we found that transient misexpression of the basic helix-loop-helix genes Hes1 and Hes5 keeps embryonal telencephalic cells undifferentiated although they have been shown to induce gliogenesis in the retina. These telencephalic cells later differentiate into neurons and astroglia when Hes expression is down-regulated, suggesting that Hes1- and Hes5- expressing cells are maintained as neural stem cells during embryogenesis. Conversely, in the absence of Hes1 and Hes5, neural stem cells are not properly maintained, generating fewer and smaller neurospheres than the wild type. These results indicate that Hes1 and Hes5 play an important role in the maintenance of neural stem cells but not in gliogenesis in the embryonal telencephalon.     

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.     

A beta-catenin gradient links the clock and wavefront systems in mouse embryo segmentation

Rhythmic production of vertebral precursors, the somites, causes bilateral columns of embryonic segments to form. This process involves a molecular oscillator--the segmentation clock--whose signal is translated into a spatial, periodic pattern by a complex signalling gradient system within the presomitic mesoderm (PSM). In mouse embryos, Wnt signalling has been implicated in both the clock and gradient mechanisms, but how the Wnt pathway can perform these two functions simultaneously remains unclear. Here, we use a yellow fluorescent protein (YFP)-based, real-time imaging system in mouse embryos to demonstrate that clock oscillations are independent of beta-catenin protein levels. In contrast, we show that the Wnt-signalling gradient is established through a nuclear beta-catenin protein gradient in the posterior PSM. This gradient of nuclear beta-catenin defines the size of the oscillatory field and controls key aspects of PSM maturation and segment formation, emphasizing the central role of Wnt signalling in this process.     

A segmentation clock patterns cellular differentiation in a bacterial biofilm

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Hey2 functions in parallel with Hes1 and Hes5 for mammalian auditory sensory organ development

Background  

During mouse development, the precursor cells that give rise to the auditory sensory organ, the organ of Corti, are specified prior to embryonic day 14.5 (E14.5). Subsequently, the sensory domain is patterned precisely into one row of inner and three rows of outer sensory hair cells interdigitated with supporting cells. Both the restriction of the sensory domain and the patterning of the sensory mosaic of the organ of Corti involve Notch-mediated lateral inhibition and cellular rearrangement characteristic of convergent extension. This study explores the expression and function of a putative Notch target gene.     

Griseofulvin: A potential agent of chromosomal segregation in cultured cells

The fungicidal compound griseofulvin (GF) induces abnormalities in nuclear division in mammalian cells cultured in vitro. For these properties it has been studied as a potential agent of chromosomal segregation. A marked effect on the dynamics of chromosomal complements was observed both on diploid and heteroploid cell lines, including hybrids produced by fusion. After treatment for three days with doses ranging from 40 to 60 μg/ml, according to the cell type, a tendency to a doubling of the chromosomal set was evident. When cells were allowed to recover in normal medium in the absence of GF a scattering of the distribution of the chromosomal numbers occured. After removal of the drug a selective advantage of the double chromosomal complements was observed on prolonged cultures. The possibility of using GF to induce chromosomal segregation for linkage studies and for chromosomal assignment is discussed.     

Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine.     

Synchronized oscillation of the segmentation clock gene in vertebrate development

In vertebrate somitogenesis, “segmentation clock” genes (her in zebrafish, hes in mouse, and hairy in chick) show oscillation, synchronized over nearby cells through intercellular interaction. In zebrafish, neighboring cells interact by Delta-Notch signaling to realize synchronization. Under Delta-Notch, however, a cell with a high expression of the segmentation clock gene tends to suppress its expression in adjacent cells, which might produce spatial heterogeneity instead of synchronized oscillation. Here we studied the conditions under which pre-somitic mesoderm cells show synchronized oscillation of gene expression mathematically. We adopted a model that explicitly considers the kinetics of the mRNA and proteins of the segmentation clock gene and cell–cell interaction via Delta-Notch signaling. From statistical study of a model with randomly generated parameters, we revealed how the likelihood that the system generates stable synchronized oscillation depends on the rate of each reaction in the gene–protein kinetics.