Modeling the segmentation clock as a network of coupled oscillations in the Notch, Wnt and FGF signaling pathways

The formation of somites in the course of vertebrate segmentation is governed by an oscillator known as the segmentation clock, which is characterized by a period ranging from 30 min to a few hours depending on the organism. This oscillator permits the synchronized activation of segmentation genes in successive cohorts of cells in the presomitic mesoderm in response to a periodic signal emitted by the segmentation clock, thereby defining the future segments. Recent microarray experiments [Dequeant, M.L., Glynn, E., Gaudenz, K., Wahl, M., Chen, J., Mushegian, A., Pourquie, O., 2006. A complex oscillating network of signaling genes underlies the mouse segmentation clock. Science 314, 1595-1598] indicate that the Notch, Wnt and Fibroblast Growth Factor (FGF) signaling pathways are involved in the mechanism of the segmentation clock. By means of computational modeling, we investigate the conditions in which sustained oscillations occur in these three signaling pathways. First we show that negative feedback mediated by the Lunatic Fringe protein on intracellular Notch activation can give rise to periodic behavior in the Notch pathway. We then show that negative feedback exerted by Axin2 on the degradation of β-catenin through formation of the Axin2 destruction complex can produce oscillations in the Wnt pathway. Likewise, negative feedback on FGF signaling mediated by the phosphatase product of the gene MKP3/Dusp6 can produce oscillatory gene expression in the FGF pathway. Coupling the Wnt, Notch and FGF oscillators through common intermediates can lead to synchronized oscillations in the three signaling pathways or to complex periodic behavior, depending on the relative periods of oscillations in the three pathways. The phase relationships between cycling genes in the three pathways depend on the nature of the coupling between the pathways and on their relative autonomous periods. The model provides a framework for analyzing the dynamics of the segmentation clock in terms of a network of oscillating modules involving the Wnt, Notch and FGF signaling pathways.     

Notch signalling synchronizes the zebrafish segmentation clock but is not needed to create somite boundaries

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish.     

Wnt3a plays a major role in the segmentation clock controlling somitogenesis

The vertebral column derives from somites generated by segmentation of presomitic mesoderm (PSM). Somitogenesis involves a molecular oscillator, the segmentation clock, controlling periodic Notch signaling in the PSM. Here, we establish a novel link between Wnt/beta-catenin signaling and the segmentation clock. Axin2, a negative regulator of the Wnt pathway, is directly controlled by Wnt/beta-catenin and shows oscillating expression in the PSM, even when Notch signaling is impaired, alternating with Lfng expression. Moreover, Wnt3a is required for oscillating Notch signaling activity in the PSM. We propose that the segmentation clock is established by Wnt/beta-catenin signaling via a negative-feedback mechanism and that Wnt3a controls the segmentation process in vertebrates.     

Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

Oscillators and the emergence of tissue organization during zebrafish somitogenesis

Genetic networks that include positive and negative feedback can exhibit oscillations. These oscillations are a form of emergence, which is when novel patterns or properties arise during self organization of complex systems. Within the extending trunk and tail of the developing vertebrate embryo, the somitogenesis oscillator governs the periodic formation of segments that ultimately become the vertebral column and musculature. These oscillations occur within the context of noise created by cell movement, mitosis, and stochastic gene expression. Here, we review recent progress in our understanding of the role of the Notch signaling pathway in the zebrafish segmentation oscillator and our appreciation of how the oscillator interfaces with different sources of noise.     

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.     

Priming, initiation and synchronization of the segmentation clock by deltaD and deltaC

Zebrafish somitogenesis is governed by a segmentation clock that generates oscillations in expression of several Notch pathway genes, including her1, her7 and deltaC. Using a combination of pharmacological inhibition and Mendelian genetics, we show that DeltaD and DeltaC, two Notch ligands, represent functionally distinct signals within the segmentation clock. Using high-resolution fluorescent in situ hybridization, the oscillations were divided into phases based on eight distinct subcellular patterns of mRNA localization for 140,000 cells. her1, her7 and deltaC expression was examined in wild-type, deltaD(-/-) and deltaC(-/-) embryos. We identified areas within the tailbud where the clock is set up in the progenitor cells (priming), where the clock starts running (initiation), and where the clocks of neighbouring cells are entrained (synchronization). We find that the clocks of motile cells are primed by deltaD in a progenitor zone in the posterior tailbud and that deltaD is required for cells to initiate oscillations on exiting this zone. Oscillations of adjacent cells are synchronized and amplified by deltaC in the posterior presomitic mesoderm as cell movement subsides and cells maintain stable neighbour relationships.     

Analysis of her1 and her7 mutants reveals a spatio temporal separation of the somite clock module

Somitogenesis is controlled by a genetic network consisting of an oscillator (clock) and a gradient (wavefront). The "hairy and Enhancer of Split"- related (her) genes act downstream of the Delta/Notch (D/N) signaling pathway, and are crucial components of the segmentation clock. Due to genome duplication events, the zebrafish genome, possesses two gene copies of the mouse Hes7 homologue: her1 and her7. To better understand the functional consequences of this gene duplication, and to determine possible independent roles for these two genes during segmentation, two zebrafish mutants her1(hu2124) and her7(hu2526) were analyzed. In the course of embryonic development, her1(hu2124) mutants exhibit disruption of the three anterior-most somite borders, whereas her7(hu2526) mutants display somite border defects restricted to somites 8 (+/-3) to 17 (+/-3) along the anterior-posterior axis. Analysis of the molecular defects in her1(hu2124) mutants reveals a her1 auto regulatory feedback loop during early somitogenesis that is crucial for correct patterning and independent of her7 oscillation. This feedback loop appears to be restricted to early segmentation, as cyclic her1 expression is restored in her1(hu2124) embryos at later stages of development. Moreover, only the anterior deltaC expression pattern is disrupted in the presomitic mesoderm of her1(hu2124) mutants, while the posterior expression pattern of deltaC remains unaltered. Together, this data indicates the existence of an independent and genetically separable anterior and posterior deltaC clock modules in the presomitic mesdorm (PSM).