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The vertebrate segmentation clock: the tip of the iceberg
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The vertebrate segmentation clock: the tip of the iceberg Ertug˘rul M O¨zbudak1 and Olivier Pourquie´1,2 The vertebrate segmentation clock was identified 10 years ago as a molecular oscillator associated with the rhythmic production of embryonic somites. Since then, three major signaling pathways Notch, FGF, and Wnt have been shown to be activated periodically during segmentation and proposed to constitute the clockwork of the system. However, recent results from zebrafish embryonic studies demonstrate that Notch signaling is involved in the coupling of oscillations among cells rather than in the pacemaker of the oscillator. Furthermore, genetic analyses in mouse indicate that Wnt and FGF play only a permissive role in the control of the oscillations. Therefore, the nature of the segmentation clock pacemaker still remains elusive. Addresses 1 Stowers Institute for Medical Research, United States 2 Howard Hughes Medical Institute, United States Corresponding author: O¨zbudak, Ertug˘rul M ([email protected]) and Pourquie´, Olivier ([email protected])
Current Opinion in Genetics & Development 2008, 18:317–323 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Ottoline Leyser and Olivier Pourquie´ Available online 15th August 2008 0959-437X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2008.06.007
Introduction The vertebrate body can be subdivided along the anteroposterior (AP) axis into repeated structures called segments. This periodic pattern is established in the embryo during somitogenesis. Somites are epithelial blocks, generated in a rhythmic fashion from the paraxial mesoderm, which subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somite formation involves an oscillator called the segmentation clock that generates a periodic pulse of Notch, FGF, and Wnt signals that are converted into the periodic array of somite boundaries ([1] and references therein). The translation of this pulsation into the reiterated arrangement of segment boundaries along the AP axis involves dynamic gradients of FGF and Wnt signals that regress posteriorly as the embryonic axis elongates. Stripes of gene expression form rhythmically in the anterior presomitic mesoderm (PSM) at a defined level of the gradients (the wavefront) in response to the clock signal. This striped prepattern provides the blueprint from www.sciencedirect.com
which the morphological segment the somite will be formed. The rostrocaudal polarity of the future somite is established in the newly specified segment. This subdivision is also responsible for the definitive patterning of vertebrae that form when the posterior part of one somite fuses to the anterior part of the consecutive somite during a process termed re-segmentation. Finally, the formation of the morphological boundaries results in the separation of the epithelial somite from the PSM.
Notch signaling synchronizes oscillations among presomitic mesoderm cells Notch receptors and their ligands, Delta and Jagged/ Serrate, are transmembrane proteins and consequently, Notch signaling essentially takes place only among neighboring cells [2]. In zebrafish and mouse, several mutations in the genes involved in the Notch-signaling pathway specifically disrupt the segmentation clock and somite boundary formation [3]. In zebrafish, all of the cyclic genes identified to date are members of the Notchsignaling pathway. This led to the hypothesis that the Notch-signaling pathway is part of the segmentation clock [4–7]. However, when Notch signaling is disrupted in mouse and zebrafish, anterior somites are spared, and disruptions of somite boundary formation only appear posteriorly. This observation is difficult to reconcile with the hypothesis that Notch signaling acts as the pacemaker of the segmentation clock—unless the formation of the anterior somites is controlled by a Notch-independent clock. An alternative explanation was suggested by Lewis and colleagues, who proposed that the exclusive role of Notch signaling is to synchronize the cell-autonomous oscillations of neighboring cells in the PSM [8]. Thus, the delayed disruption in somite boundary formation evident in Notch mutants would result from the gradual desynchronization of oscillations in neighboring cells in the absence of cell-to-cell coupling [8]. In Notch mutants, oscillations of neighboring cells initially would begin as synchronized; however, in the absence of coupling, synchrony is progressively lost, resulting in a salt-and-pepper expression pattern of the cyclic genes in the PSM [8]. Oscillation of the Notch ligand deltaC in the PSM of wildtype embryos supports the hypothesis that the oscillations of a Notch-signaling ligand entrain neighboring cells [8]. Here, we discuss recent experiments, mostly in zebrafish embryos, which provide evidence for a role of Notch signaling in the synchronization of the oscillations among neighboring cells rather than as the clock pacemaker. In zebrafish, a simple oscillator model that essentially relies on the Her1/Her7 transcriptional repressors acting as a central pacemaker has been proposed Current Opinion in Genetics & Development 2008, 18:317–323
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[7,9–13,14,15,16]. In this model, oscillations are generated by a negative feedback loop in which the her genes are directly repressed by their own protein products (Figure 1a) [15]. To generate oscillations, the model takes into account a defined time delay in the autoinhibitory circuit that occurs from the beginning of the her RNA transcription until the Her protein binds to the her gene promoter [15]. Using plausible numerical values for the model parameters, oscillations exhibiting a period consistent with that observed in zebrafish have been obtained [15]. This Her1/Her7 intracellular oscillator was proposed to be linked to an intercellular oscillator involving the Notch-signaling pathway [15]. Her1/Her7 negatively
regulate deltaC, thus, triggering oscillations of this Notch ligand, which should, in turn, result in periodic Notch activation in neighboring cells (Figure 1a) [7,14,15,17]. Such a coupling could provide a basis for maintaining synchrony among the oscillations of neighboring cells [8]. By combining immunostaining and fluorescent in situ hybridization, the measured translational delay of DeltaC has been shown to be consistent with the constraint set by the delay model for DeltaC to entrain the oscillations of neighboring cells [14]. To test the role of Notch signaling in the coupling of the oscillations, Horikawa et al. transplanted zebrafish
Figure 1
The vertebrate somite segmentation clock. Left panels show the traveling stripes of the cyclic genes (in blue) in the zebrafish (top) and mouse (bottom) PSM. (a) In zebrafish, cell-autonomous oscillations of the her network are coupled between neighboring cells by Notch signaling. Notch signaling, together with FGF signaling, regulates the expression of her genes. Her genes regulate the expression of deltaC, which also oscillates and activates Notch signaling in the neighboring cells. (b) In mouse, oscillations of Notch, FGF, and Wnt networks run parallel to each other. Cross-regulation in between the three networks potentially entrains the subnetworks to oscillate in synchrony among the neighboring cells. Current Opinion in Genetics & Development 2008, 18:317–323
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PSM cells injected with morpholinos against her1 and her7 [18]. They observed that the oscillation phase of neighboring wild-type cells in the host zebrafish embryo was accelerated, resulting in an anterior shift of the somite boundaries. The transplanted cells show an elevated deltaC level owing to the release of the Her-mediated inhibition. The phenotype is rescued by co-injection of deltaC or deltaD morpholinos, together with her1/7 morpholinos, showing that the effect is due to interference with Notch signaling. However, the acceleration phenotype is not observed when transplanted cells are injected with deltaC or deltaD mRNAs, implying that other downstream targets of Her1/Her7 or complex modifications of endogenous Delta ligands are required. Also, when groups of PSM cells from donor embryos were transplanted into the PSM of host embryos, the transplanted cells synchronized their oscillations with the host cells within three clock cycles. The authors also reported variation in the oscillation phases of neighboring cells in wild-type embryos due to stochastic gene expression and cell division. Together, these data indicate that Notch provides a robust coupling system that ensures the synchronization of segmentation clock oscillations in the PSM despite this intrinsic noise. In three recent studies, the g-secretase inhibitor DAPT was used to block Notch signaling, which resulted in somite boundary defects after a long delay—regardless of the stage of development at which DAPT was applied [19,20,21]. Furthermore, a detailed analysis of early oscillations of the her genes in the zebrafish embryo showed that the initial oscillations begin around the time of gastrulation as in chick embryos [22]. These initial oscillations remain synchronized even if Notch signaling is blocked by DAPT treatment [20]. These results clearly argue against the proposal that Notch signaling is part of the segmentation clock pacemaker. DAPT pulse-chase experiments demonstrate that the posterior PSM has the capacity to recover and to form somite boundaries once the blockage of Notch signaling was alleviated [19,20,21]. The delay from the DAPT wash to the recovery of sharp somite boundaries is on the same timescale as the delay from the time DAPT is applied to the disruption of somite boundaries [20]. This latter result supports the hypothesis that Notch signaling operates only in the posterior PSM to maintain the synchronization of the cell-autonomous oscillations in zebrafish embryos. On the basis of these findings, Riedel-Kruse et al. developed an interesting model in which the balance between the strength of Notch signaling (synchronizing agent) and the noise in gene expression (desynchronizing agent) dictates whether the neighboring cells will or will not oscillate in synchrony [20]. Fitting the model to data, they predicted a short half-life for the active www.sciencedirect.com
version of the Notch receptor, the notch intracellular domain (NICD), which could be tested experimentally in the future. Mara and Holley analyzed the recovery of somite boundaries in DAPT-treated deltaC /+ or deltaD /+ embryos with or without injection of deltaC or deltaD mRNAs [19]. Injection of deltaC mRNA reduced the recovery percentage of somite boundaries (in both backgrounds); whereas, this percentage was unaffected when deltaD was injected and even increased in deltaD / + background. A similar result was obtained by Geffers et al. in mouse in regard to the functions of delta-like 1 (Dll1) and delta-like 3 (Dll3) in Notch signaling: expression of functional Dll3 from Dll1 locus does not rescue the Dll1 phenotype [23]. This result clearly shows that the different Delta ligands are not merely interchangeable—their contribution to Notch signaling is more complicated than a simple additive function. Further research is needed to unravel the contributions of deltaC and deltaD to Notch signaling. One could argue that treatment of embryos with DAPT might not eliminate all the Notch signaling and, hence, there may be just enough residual Notch oscillations remaining to support cell-autonomous oscillations. Therefore, the functions of Notch signaling might be to sustain cell-autonomous oscillations, as well as to synchronize the oscillations of neighboring cells. This residual Notch signal could also be sufficient to contribute to the formation of sharp somite boundaries in the anterior PSM [24]. To address these possibilities further, ¨ zbudak and Lewis overexpressed the cleaved Notch O intracellular domain (NICD) of the Notch receptor, which acts as a constitutively active receptor [21]. Expression of NICD in transgenic zebrafish embryos resulted in major morphological defects in many embryonic tissues, and it also increased expression of her4, a direct Notch target, shortly after application of a heat shock pulse. However, as observed in the DAPT experiments, many somites formed normally until the boundary disruption began. Then, a transgenic animal was generated in which the her1 promoter drives the expression of a GFP reporter. Blockage of Notch signaling by DAPT reduced the expression of her1-GFP by only 25%, suggesting that her1 expression is only partly controlled by Notch signaling (Figure 1a). Transient disruption of the oscillatory expression of her1 and her7 by overexpression of either protein in zebrafish transgenic embryos did not prevent the formation of the next fourto-five somite boundaries [14], implying that the cells located in the prospective four-to-five somite region in the anterior PSM are already time-stamped, and information from the somite clock has already been passed to downstream mediators at this spatial location. The authors developed a modified version of the delay model, which explains both the results of their time-perturbation Current Opinion in Genetics & Development 2008, 18:317–323
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experiments and the mutant and wild-type phenotypes [21]. These results further support the idea that, at least in zebrafish, the role of Notch signaling is to synchronize cell-autonomous oscillations. A direct demonstration using real-time imaging of the role of Notch signaling in the synchronization of the oscillations among the PSM cells, however, is still lacking. Desynchronization was, nevertheless, observed by in situ hybridization in dissociated PSM cells in the chick embryo [25]. In mouse, single-cell oscillations in dissociated PSM cells were demonstrated using a real-time Hes7-luciferase transgenic reporter [26]. Unlike in the intact PSM, the period and the amplitude of single-cell oscillations in cell culture were quite noisy [26], consistent with the model that cell–cell interactions mediated by Notch signaling are required for the coupling and the synchronization of the oscillations in the PSM. These data provide strong evidence supporting the existence of cell-autonomous oscillations, at least in mouse. The amniote cyclic gene network is more complex than in zebrafish as it involves also the Wnt-signaling and FGF-signaling pathways (Figure 1b) [27]. Hence, the simple Her-based delay model is unlikely to apply to amniotes. In mouse, both Wnt and FGF pathways have been shown to act upstream of the oscillations of the Notch target genes, suggesting that Hes genes are unlikely to act as the pacemaker for the segmentation clock [28,29,30]. Lunatic fringe (Lfng) is a Notch target that shows cyclic expression in amniotes but is not expressed in zebrafish PSM. Lfng codes for a glycosyltransferase that can inhibit Notch signaling and, thus, act as a negative feedback inhibitor in the PSM [31,32]. In contrast to the zebrafish cyclic genes that show a salt-and-pepper expression pattern in the PSM of Notch mutants, in mouse Notch mutants or in chick embryos treated with DAPT, Lfng expression is completely downregulated [31,33]. A mutation in Lfng was identified in a human patient with a severe disruption of the segmental organization of the spine called spondylocostal dysostosis (SCOD3) [34]. In mouse, Lfng mutants are viable and also demonstrate very severe anomalies of spine segmentation [35,36]. Interestingly, selective deletion of the regulatory sequence driving the oscillatory expression of Lfng in the posterior PSM results in disruption of the vertebral column all the way to the lumbar region but leaves the sacral and caudal vertebrae intact [37]. Therefore, this indicates that the segmentation clock might exhibit different regional requirements along the AP axis. Although oscillations of the Notch ligand Dll1 have been reported in mouse [38], thus far, no evidence has been provided to support the role of Notch signaling in the synchronization of the oscillations in any amniote species. Thus, in amniotes such as chick or mouse, the role(s) of Notch signaling in the segmentation clock appears different than in zebrafish. Current Opinion in Genetics & Development 2008, 18:317–323
Wnt and FGF signaling plays a permissive role in the control of segmentation clock oscillations FGF signaling has been shown, initially, to establish a posterior-to-anterior gradient in the PSM [39,40]. This gradient was proposed to define a threshold (the wavefront or determination front) at which PSM cells become competent to respond to the segmentation clock [39,40]. At the wavefront level, a stripe of gene expression is activated in response to the clock signal, thus, establishing the segmental prepattern ([1,41] and references therein). The recent observation that many oscillating genes, including Snail1/2, Spry2, Dusp6, and Dusp4, are targets of the FGF pathway [27,30,42] has forced the role of FGF signaling in somitogenesis to be reconsidered. These oscillations suggest that periodic activation of the FGF pathway in the posterior PSM occurs in synchrony with the Notch oscillations. These findings are supported by the dynamic expression of ERK phosphorylation in the mouse PSM [30]. Many of the newly discovered oscillating genes in the FGF-signaling pathway are negative regulators of the FGF pathway and, hence, have the potential to drive or pace the oscillations. Niwa et al. reported that neither mutations in the Notch pathway nor DAPT treatment abolishes Hes7 oscillations in the posterior PSM, while they affect Hes7 expression in the anterior PSM [30]. In the posterior PSM, Hes7 oscillations act downstream of FGF signaling to control the periodic expression of the FGF inhibitor Dusp4, which in turn can drive the periodic inhibition of this pathway. Wahl et al. and Niwa et al. demonstrated that Cre-mediated, tissue-specific deletion of the unique FGF receptor expressed in the PSM FGFR1 abolishes oscillations of Hes7, Lfng, Axin2, and Spry2 in the posterior PSM [29,30]. Niwa et al. proposed a model in which FGF signaling initiates the Hes7 oscillations in the tail bud, while Notch signaling maintains the oscillations in the anterior PSM. They identified a feedback loop between Hes7 and Dusp4, resulting in a coupling between Notch and FGF signals in the PSM. In vitro treatment of mouse embryos with the FGF inhibitor SU5402 leads to a rapid arrest of Axin2 and Spry2 oscillations; whereas, Hes7 and Lfng oscillations cease only after a delay of more than one cycle [29,30]. These experiments are consistent with a role for FGF signaling upstream of the Notch oscillations. However, introducing a constitutively stable version of b-catenin in mutant mouse embryos, in which the FGFR1 is conditionally deleted in the PSM, restores the formation of the Lfng stripes [43]. These embryos show constitutive nuclear b-catenin expression and lack FGF signaling in the PSM; yet, they appear to be capable of producing Lfng oscillations, therefore, arguing against a role for FGF or Wnt signaling as a pacemaker for the segmentation clock. In zebrafish, FGF signaling regulates her1, her7, and deltaC oscillations by regulating expression of the Her family member Her13.2 (Figure 1a), which then works together with Her1 to repress target genes www.sciencedirect.com
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[44]. However, the exact role of Her13.2 expression alone on somite segmentation requires further analysis [45]. Therefore, FGF signaling appears to play a permissive role in the control of the segmentation clock oscillations. Oscillations of Axin2, a target of the Wnt canonical pathway in the mouse embryo PSM, led to the suggestion that Wnt signaling was also involved in the amniote segmentation clock [23]. A recent microarray screen, aimed at identifying all the cyclic genes in the mouse transcriptome, identified many Wnt target genes, including several negative feedback inhibitors such as dickkopf homolog 1 (Dkk1) or dapper homolog 1 (Dact1) [27]. These Wnt cyclic genes exhibit oscillations in opposite phase to the Notch and FGF target genes [27]. In mouse, Lfng oscillations are disrupted in the Wnt pathway mutant vestigial tail (vt); whereas, Axin2 oscillations are retained in the RBPjk and Hes7 mutants [28,46]. This argues that Wnt signaling acts upstream of the Notch oscillations. Strikingly, Wnt signaling, like FGF signaling, also establishes a posterior-to-anterior signaling gradient along the PSM [28,39,40,43,47]. In mouse, nuclear b-catenin exhibits a clear gradient running from the tail bud to the determination front level [43]. Results from two recent studies, in which transgenic animals were used in conditional gain-of-function and loss-of-function of Wnt/ b-catenin signaling, address the role of Wnt signaling in mouse somite segmentation [43,48]. Gain-of-function of Wnt/b-catenin signaling expands the PSM as judged by the increased size of the posterior expression domain of genes such as Brachyury or T-box 6 (Tbx6). In the bcatenin gain-of-function experiments, Dkk1 [43] and Lfng still were expressed in a dynamic fashion in the posterior PSM [43,48]. Using a transgenic reporter mouse in which the Lfng promoter is fused to the Venus YFP, Aulehla et al. demonstrated that Lfng continues to oscillate with the same period in the b-catenin gain-offunction mutants [43]. The most striking effect in these mutants is an AP extension of the oscillatory domain, which results in a multi-stripe, oscillatory expression pattern in the enlarged PSM [43,48]. These results imply that whereas Wnt/b-catenin and FGF pathways are required for positioning the determination front and controling the size of the oscillatory domain in the PSM, neither pathway controls the rhythmicity of Lfng expression [43].
Conclusion These recent data discussed in this review argue that Notch acts as a coupling device that synchronizes oscillations among PSM cells. Furthermore, Wnt cyclic and FGF cyclic genes oscillate independently of Notch, and oscillations of the Notch target Lfng are still observed in mutants exhibiting constitutive b-catenin activation and no FGF signaling in the PSM. Thus, none of the three signaling pathways periodically activated in the PSM individually appear to act as a global clock pacemaker. www.sciencedirect.com
Therefore, this raises doubts about the current models in which the periodic gene expression associated to the segmentation clock is usually presented as resulting from the dynamic properties of the individual cyclic gene networks. It is possible that each subnetwork has the capacity to pace its own oscillations independent of the oscillations of the other subnetworks while couplings among the subnetworks entrain them to each other in amniotes [49]. Alternatively, it could be that the network of cyclic genes that underlies the segmentation clock, in fact, is entrained by an outside pacemaker to the system that remains to be identified.
Acknowledgements The authors thank C Gomez, A Aulehla, B Bernazeraf, and G Neto for critical reading of the manuscript. They also thank S Esteban for artwork and J Chatfield for editorial assistance. This work is supported by Stowers Institute for Medical Research. O Pourquie´ is a Howard Hughes Medical Institute Investigator.
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Current Opinion in Genetics & Development 2008, 18:317–323
The vertebrate segmentation clock: the tip of the iceberg Ertug˘rul M O¨zbudak1 and Olivier Pourquie´1,2 The vertebrate segmentation clock was identified 10 years ago as a molecular oscillator associated with the rhythmic production of embryonic somites. Since then, three major signaling pathways Notch, FGF, and Wnt have been shown to be activated periodically during segmentation and proposed to constitute the clockwork of the system. However, recent results from zebrafish embryonic studies demonstrate that Notch signaling is involved in the coupling of oscillations among cells rather than in the pacemaker of the oscillator. Furthermore, genetic analyses in mouse indicate that Wnt and FGF play only a permissive role in the control of the oscillations. Therefore, the nature of the segmentation clock pacemaker still remains elusive. Addresses 1 Stowers Institute for Medical Research, United States 2 Howard Hughes Medical Institute, United States Corresponding author: O¨zbudak, Ertug˘rul M ([email protected]) and Pourquie´, Olivier ([email protected])
Current Opinion in Genetics & Development 2008, 18:317–323 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Ottoline Leyser and Olivier Pourquie´ Available online 15th August 2008 0959-437X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2008.06.007
Introduction The vertebrate body can be subdivided along the anteroposterior (AP) axis into repeated structures called segments. This periodic pattern is established in the embryo during somitogenesis. Somites are epithelial blocks, generated in a rhythmic fashion from the paraxial mesoderm, which subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somite formation involves an oscillator called the segmentation clock that generates a periodic pulse of Notch, FGF, and Wnt signals that are converted into the periodic array of somite boundaries ([1] and references therein). The translation of this pulsation into the reiterated arrangement of segment boundaries along the AP axis involves dynamic gradients of FGF and Wnt signals that regress posteriorly as the embryonic axis elongates. Stripes of gene expression form rhythmically in the anterior presomitic mesoderm (PSM) at a defined level of the gradients (the wavefront) in response to the clock signal. This striped prepattern provides the blueprint from www.sciencedirect.com
which the morphological segment the somite will be formed. The rostrocaudal polarity of the future somite is established in the newly specified segment. This subdivision is also responsible for the definitive patterning of vertebrae that form when the posterior part of one somite fuses to the anterior part of the consecutive somite during a process termed re-segmentation. Finally, the formation of the morphological boundaries results in the separation of the epithelial somite from the PSM.
Notch signaling synchronizes oscillations among presomitic mesoderm cells Notch receptors and their ligands, Delta and Jagged/ Serrate, are transmembrane proteins and consequently, Notch signaling essentially takes place only among neighboring cells [2]. In zebrafish and mouse, several mutations in the genes involved in the Notch-signaling pathway specifically disrupt the segmentation clock and somite boundary formation [3]. In zebrafish, all of the cyclic genes identified to date are members of the Notchsignaling pathway. This led to the hypothesis that the Notch-signaling pathway is part of the segmentation clock [4–7]. However, when Notch signaling is disrupted in mouse and zebrafish, anterior somites are spared, and disruptions of somite boundary formation only appear posteriorly. This observation is difficult to reconcile with the hypothesis that Notch signaling acts as the pacemaker of the segmentation clock—unless the formation of the anterior somites is controlled by a Notch-independent clock. An alternative explanation was suggested by Lewis and colleagues, who proposed that the exclusive role of Notch signaling is to synchronize the cell-autonomous oscillations of neighboring cells in the PSM [8]. Thus, the delayed disruption in somite boundary formation evident in Notch mutants would result from the gradual desynchronization of oscillations in neighboring cells in the absence of cell-to-cell coupling [8]. In Notch mutants, oscillations of neighboring cells initially would begin as synchronized; however, in the absence of coupling, synchrony is progressively lost, resulting in a salt-and-pepper expression pattern of the cyclic genes in the PSM [8]. Oscillation of the Notch ligand deltaC in the PSM of wildtype embryos supports the hypothesis that the oscillations of a Notch-signaling ligand entrain neighboring cells [8]. Here, we discuss recent experiments, mostly in zebrafish embryos, which provide evidence for a role of Notch signaling in the synchronization of the oscillations among neighboring cells rather than as the clock pacemaker. In zebrafish, a simple oscillator model that essentially relies on the Her1/Her7 transcriptional repressors acting as a central pacemaker has been proposed Current Opinion in Genetics & Development 2008, 18:317–323
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[7,9–13,14,15,16]. In this model, oscillations are generated by a negative feedback loop in which the her genes are directly repressed by their own protein products (Figure 1a) [15]. To generate oscillations, the model takes into account a defined time delay in the autoinhibitory circuit that occurs from the beginning of the her RNA transcription until the Her protein binds to the her gene promoter [15]. Using plausible numerical values for the model parameters, oscillations exhibiting a period consistent with that observed in zebrafish have been obtained [15]. This Her1/Her7 intracellular oscillator was proposed to be linked to an intercellular oscillator involving the Notch-signaling pathway [15]. Her1/Her7 negatively
regulate deltaC, thus, triggering oscillations of this Notch ligand, which should, in turn, result in periodic Notch activation in neighboring cells (Figure 1a) [7,14,15,17]. Such a coupling could provide a basis for maintaining synchrony among the oscillations of neighboring cells [8]. By combining immunostaining and fluorescent in situ hybridization, the measured translational delay of DeltaC has been shown to be consistent with the constraint set by the delay model for DeltaC to entrain the oscillations of neighboring cells [14]. To test the role of Notch signaling in the coupling of the oscillations, Horikawa et al. transplanted zebrafish
Figure 1
The vertebrate somite segmentation clock. Left panels show the traveling stripes of the cyclic genes (in blue) in the zebrafish (top) and mouse (bottom) PSM. (a) In zebrafish, cell-autonomous oscillations of the her network are coupled between neighboring cells by Notch signaling. Notch signaling, together with FGF signaling, regulates the expression of her genes. Her genes regulate the expression of deltaC, which also oscillates and activates Notch signaling in the neighboring cells. (b) In mouse, oscillations of Notch, FGF, and Wnt networks run parallel to each other. Cross-regulation in between the three networks potentially entrains the subnetworks to oscillate in synchrony among the neighboring cells. Current Opinion in Genetics & Development 2008, 18:317–323
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PSM cells injected with morpholinos against her1 and her7 [18]. They observed that the oscillation phase of neighboring wild-type cells in the host zebrafish embryo was accelerated, resulting in an anterior shift of the somite boundaries. The transplanted cells show an elevated deltaC level owing to the release of the Her-mediated inhibition. The phenotype is rescued by co-injection of deltaC or deltaD morpholinos, together with her1/7 morpholinos, showing that the effect is due to interference with Notch signaling. However, the acceleration phenotype is not observed when transplanted cells are injected with deltaC or deltaD mRNAs, implying that other downstream targets of Her1/Her7 or complex modifications of endogenous Delta ligands are required. Also, when groups of PSM cells from donor embryos were transplanted into the PSM of host embryos, the transplanted cells synchronized their oscillations with the host cells within three clock cycles. The authors also reported variation in the oscillation phases of neighboring cells in wild-type embryos due to stochastic gene expression and cell division. Together, these data indicate that Notch provides a robust coupling system that ensures the synchronization of segmentation clock oscillations in the PSM despite this intrinsic noise. In three recent studies, the g-secretase inhibitor DAPT was used to block Notch signaling, which resulted in somite boundary defects after a long delay—regardless of the stage of development at which DAPT was applied [19,20,21]. Furthermore, a detailed analysis of early oscillations of the her genes in the zebrafish embryo showed that the initial oscillations begin around the time of gastrulation as in chick embryos [22]. These initial oscillations remain synchronized even if Notch signaling is blocked by DAPT treatment [20]. These results clearly argue against the proposal that Notch signaling is part of the segmentation clock pacemaker. DAPT pulse-chase experiments demonstrate that the posterior PSM has the capacity to recover and to form somite boundaries once the blockage of Notch signaling was alleviated [19,20,21]. The delay from the DAPT wash to the recovery of sharp somite boundaries is on the same timescale as the delay from the time DAPT is applied to the disruption of somite boundaries [20]. This latter result supports the hypothesis that Notch signaling operates only in the posterior PSM to maintain the synchronization of the cell-autonomous oscillations in zebrafish embryos. On the basis of these findings, Riedel-Kruse et al. developed an interesting model in which the balance between the strength of Notch signaling (synchronizing agent) and the noise in gene expression (desynchronizing agent) dictates whether the neighboring cells will or will not oscillate in synchrony [20]. Fitting the model to data, they predicted a short half-life for the active www.sciencedirect.com
version of the Notch receptor, the notch intracellular domain (NICD), which could be tested experimentally in the future. Mara and Holley analyzed the recovery of somite boundaries in DAPT-treated deltaC /+ or deltaD /+ embryos with or without injection of deltaC or deltaD mRNAs [19]. Injection of deltaC mRNA reduced the recovery percentage of somite boundaries (in both backgrounds); whereas, this percentage was unaffected when deltaD was injected and even increased in deltaD / + background. A similar result was obtained by Geffers et al. in mouse in regard to the functions of delta-like 1 (Dll1) and delta-like 3 (Dll3) in Notch signaling: expression of functional Dll3 from Dll1 locus does not rescue the Dll1 phenotype [23]. This result clearly shows that the different Delta ligands are not merely interchangeable—their contribution to Notch signaling is more complicated than a simple additive function. Further research is needed to unravel the contributions of deltaC and deltaD to Notch signaling. One could argue that treatment of embryos with DAPT might not eliminate all the Notch signaling and, hence, there may be just enough residual Notch oscillations remaining to support cell-autonomous oscillations. Therefore, the functions of Notch signaling might be to sustain cell-autonomous oscillations, as well as to synchronize the oscillations of neighboring cells. This residual Notch signal could also be sufficient to contribute to the formation of sharp somite boundaries in the anterior PSM [24]. To address these possibilities further, ¨ zbudak and Lewis overexpressed the cleaved Notch O intracellular domain (NICD) of the Notch receptor, which acts as a constitutively active receptor [21]. Expression of NICD in transgenic zebrafish embryos resulted in major morphological defects in many embryonic tissues, and it also increased expression of her4, a direct Notch target, shortly after application of a heat shock pulse. However, as observed in the DAPT experiments, many somites formed normally until the boundary disruption began. Then, a transgenic animal was generated in which the her1 promoter drives the expression of a GFP reporter. Blockage of Notch signaling by DAPT reduced the expression of her1-GFP by only 25%, suggesting that her1 expression is only partly controlled by Notch signaling (Figure 1a). Transient disruption of the oscillatory expression of her1 and her7 by overexpression of either protein in zebrafish transgenic embryos did not prevent the formation of the next fourto-five somite boundaries [14], implying that the cells located in the prospective four-to-five somite region in the anterior PSM are already time-stamped, and information from the somite clock has already been passed to downstream mediators at this spatial location. The authors developed a modified version of the delay model, which explains both the results of their time-perturbation Current Opinion in Genetics & Development 2008, 18:317–323
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experiments and the mutant and wild-type phenotypes [21]. These results further support the idea that, at least in zebrafish, the role of Notch signaling is to synchronize cell-autonomous oscillations. A direct demonstration using real-time imaging of the role of Notch signaling in the synchronization of the oscillations among the PSM cells, however, is still lacking. Desynchronization was, nevertheless, observed by in situ hybridization in dissociated PSM cells in the chick embryo [25]. In mouse, single-cell oscillations in dissociated PSM cells were demonstrated using a real-time Hes7-luciferase transgenic reporter [26]. Unlike in the intact PSM, the period and the amplitude of single-cell oscillations in cell culture were quite noisy [26], consistent with the model that cell–cell interactions mediated by Notch signaling are required for the coupling and the synchronization of the oscillations in the PSM. These data provide strong evidence supporting the existence of cell-autonomous oscillations, at least in mouse. The amniote cyclic gene network is more complex than in zebrafish as it involves also the Wnt-signaling and FGF-signaling pathways (Figure 1b) [27]. Hence, the simple Her-based delay model is unlikely to apply to amniotes. In mouse, both Wnt and FGF pathways have been shown to act upstream of the oscillations of the Notch target genes, suggesting that Hes genes are unlikely to act as the pacemaker for the segmentation clock [28,29,30]. Lunatic fringe (Lfng) is a Notch target that shows cyclic expression in amniotes but is not expressed in zebrafish PSM. Lfng codes for a glycosyltransferase that can inhibit Notch signaling and, thus, act as a negative feedback inhibitor in the PSM [31,32]. In contrast to the zebrafish cyclic genes that show a salt-and-pepper expression pattern in the PSM of Notch mutants, in mouse Notch mutants or in chick embryos treated with DAPT, Lfng expression is completely downregulated [31,33]. A mutation in Lfng was identified in a human patient with a severe disruption of the segmental organization of the spine called spondylocostal dysostosis (SCOD3) [34]. In mouse, Lfng mutants are viable and also demonstrate very severe anomalies of spine segmentation [35,36]. Interestingly, selective deletion of the regulatory sequence driving the oscillatory expression of Lfng in the posterior PSM results in disruption of the vertebral column all the way to the lumbar region but leaves the sacral and caudal vertebrae intact [37]. Therefore, this indicates that the segmentation clock might exhibit different regional requirements along the AP axis. Although oscillations of the Notch ligand Dll1 have been reported in mouse [38], thus far, no evidence has been provided to support the role of Notch signaling in the synchronization of the oscillations in any amniote species. Thus, in amniotes such as chick or mouse, the role(s) of Notch signaling in the segmentation clock appears different than in zebrafish. Current Opinion in Genetics & Development 2008, 18:317–323
Wnt and FGF signaling plays a permissive role in the control of segmentation clock oscillations FGF signaling has been shown, initially, to establish a posterior-to-anterior gradient in the PSM [39,40]. This gradient was proposed to define a threshold (the wavefront or determination front) at which PSM cells become competent to respond to the segmentation clock [39,40]. At the wavefront level, a stripe of gene expression is activated in response to the clock signal, thus, establishing the segmental prepattern ([1,41] and references therein). The recent observation that many oscillating genes, including Snail1/2, Spry2, Dusp6, and Dusp4, are targets of the FGF pathway [27,30,42] has forced the role of FGF signaling in somitogenesis to be reconsidered. These oscillations suggest that periodic activation of the FGF pathway in the posterior PSM occurs in synchrony with the Notch oscillations. These findings are supported by the dynamic expression of ERK phosphorylation in the mouse PSM [30]. Many of the newly discovered oscillating genes in the FGF-signaling pathway are negative regulators of the FGF pathway and, hence, have the potential to drive or pace the oscillations. Niwa et al. reported that neither mutations in the Notch pathway nor DAPT treatment abolishes Hes7 oscillations in the posterior PSM, while they affect Hes7 expression in the anterior PSM [30]. In the posterior PSM, Hes7 oscillations act downstream of FGF signaling to control the periodic expression of the FGF inhibitor Dusp4, which in turn can drive the periodic inhibition of this pathway. Wahl et al. and Niwa et al. demonstrated that Cre-mediated, tissue-specific deletion of the unique FGF receptor expressed in the PSM FGFR1 abolishes oscillations of Hes7, Lfng, Axin2, and Spry2 in the posterior PSM [29,30]. Niwa et al. proposed a model in which FGF signaling initiates the Hes7 oscillations in the tail bud, while Notch signaling maintains the oscillations in the anterior PSM. They identified a feedback loop between Hes7 and Dusp4, resulting in a coupling between Notch and FGF signals in the PSM. In vitro treatment of mouse embryos with the FGF inhibitor SU5402 leads to a rapid arrest of Axin2 and Spry2 oscillations; whereas, Hes7 and Lfng oscillations cease only after a delay of more than one cycle [29,30]. These experiments are consistent with a role for FGF signaling upstream of the Notch oscillations. However, introducing a constitutively stable version of b-catenin in mutant mouse embryos, in which the FGFR1 is conditionally deleted in the PSM, restores the formation of the Lfng stripes [43]. These embryos show constitutive nuclear b-catenin expression and lack FGF signaling in the PSM; yet, they appear to be capable of producing Lfng oscillations, therefore, arguing against a role for FGF or Wnt signaling as a pacemaker for the segmentation clock. In zebrafish, FGF signaling regulates her1, her7, and deltaC oscillations by regulating expression of the Her family member Her13.2 (Figure 1a), which then works together with Her1 to repress target genes www.sciencedirect.com
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[44]. However, the exact role of Her13.2 expression alone on somite segmentation requires further analysis [45]. Therefore, FGF signaling appears to play a permissive role in the control of the segmentation clock oscillations. Oscillations of Axin2, a target of the Wnt canonical pathway in the mouse embryo PSM, led to the suggestion that Wnt signaling was also involved in the amniote segmentation clock [23]. A recent microarray screen, aimed at identifying all the cyclic genes in the mouse transcriptome, identified many Wnt target genes, including several negative feedback inhibitors such as dickkopf homolog 1 (Dkk1) or dapper homolog 1 (Dact1) [27]. These Wnt cyclic genes exhibit oscillations in opposite phase to the Notch and FGF target genes [27]. In mouse, Lfng oscillations are disrupted in the Wnt pathway mutant vestigial tail (vt); whereas, Axin2 oscillations are retained in the RBPjk and Hes7 mutants [28,46]. This argues that Wnt signaling acts upstream of the Notch oscillations. Strikingly, Wnt signaling, like FGF signaling, also establishes a posterior-to-anterior signaling gradient along the PSM [28,39,40,43,47]. In mouse, nuclear b-catenin exhibits a clear gradient running from the tail bud to the determination front level [43]. Results from two recent studies, in which transgenic animals were used in conditional gain-of-function and loss-of-function of Wnt/ b-catenin signaling, address the role of Wnt signaling in mouse somite segmentation [43,48]. Gain-of-function of Wnt/b-catenin signaling expands the PSM as judged by the increased size of the posterior expression domain of genes such as Brachyury or T-box 6 (Tbx6). In the bcatenin gain-of-function experiments, Dkk1 [43] and Lfng still were expressed in a dynamic fashion in the posterior PSM [43,48]. Using a transgenic reporter mouse in which the Lfng promoter is fused to the Venus YFP, Aulehla et al. demonstrated that Lfng continues to oscillate with the same period in the b-catenin gain-offunction mutants [43]. The most striking effect in these mutants is an AP extension of the oscillatory domain, which results in a multi-stripe, oscillatory expression pattern in the enlarged PSM [43,48]. These results imply that whereas Wnt/b-catenin and FGF pathways are required for positioning the determination front and controling the size of the oscillatory domain in the PSM, neither pathway controls the rhythmicity of Lfng expression [43].
Conclusion These recent data discussed in this review argue that Notch acts as a coupling device that synchronizes oscillations among PSM cells. Furthermore, Wnt cyclic and FGF cyclic genes oscillate independently of Notch, and oscillations of the Notch target Lfng are still observed in mutants exhibiting constitutive b-catenin activation and no FGF signaling in the PSM. Thus, none of the three signaling pathways periodically activated in the PSM individually appear to act as a global clock pacemaker. www.sciencedirect.com
Therefore, this raises doubts about the current models in which the periodic gene expression associated to the segmentation clock is usually presented as resulting from the dynamic properties of the individual cyclic gene networks. It is possible that each subnetwork has the capacity to pace its own oscillations independent of the oscillations of the other subnetworks while couplings among the subnetworks entrain them to each other in amniotes [49]. Alternatively, it could be that the network of cyclic genes that underlies the segmentation clock, in fact, is entrained by an outside pacemaker to the system that remains to be identified.
Acknowledgements The authors thank C Gomez, A Aulehla, B Bernazeraf, and G Neto for critical reading of the manuscript. They also thank S Esteban for artwork and J Chatfield for editorial assistance. This work is supported by Stowers Institute for Medical Research. O Pourquie´ is a Howard Hughes Medical Institute Investigator.
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Current Opinion in Genetics & Development 2008, 18:317–323