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.     

A Wnt Oscillator Model for Somitogenesis

We propose a model for the segmentation clock in vertebrate somitogenesis, based on the Wnt signaling pathway. The core of the model is a negative feedback loop centered around the Axin2 protein. Axin2 is activated by β-catenin, which in turn is degraded by a complex of GSK3β and Axin2. The model produces oscillatory states of the involved constituents with typical time periods of a few hours (ultradian oscillations). The oscillations are robust to changes in parameter values and are often spiky, where low concentration values of β-catenin are interrupted by sharp peaks. Necessary for the oscillations is the saturated degradation of Axin2. Somite formation in chick and mouse embryos is controlled by a spatial Wnt gradient which we introduce in the model through a time-dependent decrease in Wnt3a ligand level. We find that the oscillations disappear as the ligand concentration decreases, in agreement with observations on embryos.     

The segmentation clock in mice: interaction between the Wnt and Notch signalling pathways

In the last few years, the efforts to elucidate the mechanisms underlying the segmentation clock in various vertebrate species have multiplied. Early evidence suggested that oscillations are caused by one of the genes under the Notch signalling pathway (like those of the her or Hes families). Recently, Aulehla et al. [Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395-406] discovered that Axin2 (a gene under the Wnt3a signalling pathway) also oscillates in the presomitic mesoderm (PSM) of mice embryos and proposed some mechanisms through which the Notch and Wnt3a pathways may interact. They further suggested that a decreasing concentration of Wnt3a along the PSM may be the gradient the segmentation clock interacts with to form somites. These results were reviewed by Rida et al. [A notch feeling of somite segmentation and beyond. Dev. Biol. 265, 2-22], who introduced a complex clockwork comprising genes Hes1, Lfng (under the Notch pathway), and Axin2, as well as their multiple interactions. In the present work we develop a mathematical model based on the Rida et al. review and use it to tackle some of the questions raided by the Aulehla et al. paper: can the Axin2 feedback loop constitute a clock? Could a decreasing Wnt3a signaling constitute the wavefront, where phase is recorded and the spatial pattern laid down? What is the master oscillator?     

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.     

Wnt ligand–dependent activation of the negative feedback regulator Nkd1

Misregulation of Wnt signaling is at the root of many diseases, most notably colorectal cancer, and although we understand the activation of the pathway, we have a very poor understanding of the circumstances under which Wnt signaling turns itself off. There are numerous negative feedback regulators of Wnt signaling, but two stand out as constitutive and obligate Wnt-induced regulators: Axin2 and Nkd1. Whereas Axin2 behaves similarly to Axin in the destruction complex, Nkd1 is more enigmatic. Here we use zebrafish blastula cells that are responsive Wnt signaling to demonstrate that Nkd1 activity is specifically dependent on Wnt ligand activation of the receptor. Furthermore, our results support the hypothesis that Nkd1 is recruited to the Wnt signalosome with Dvl2, where it becomes activated to move into the cytoplasm to interact with β-catenin, inhibiting its nuclear accumulation. Comparison of these results with Nkd function in Drosophila generates a unified and conserved model for the role of this negative feedback regulator in the modulation of Wnt signaling.     

Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.

Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of beta-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by beta-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.     

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/beta-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.     

Axin: a master scaffold for multiple signaling pathways

Axin was originally identified from the characterization of the Fused locus, the disruption of which leads to duplication of axis and embryonic lethality. It is a multidomain protein that interacts with multiple proteins and functions as a negative regulator of Wnt signaling by downregulating the beta-catenin levels. Recently, it was demonstrated that Axin also plays an important role in a JNK signaling pathway. Axin utilizes discriminatory domains for its distinct roles in the Wnt pathway and in the Axin/JNK pathway. Here we review the data that show how Axin regulates multiple signaling pathways by serving as a scaffold protein, controlling diverse cellular functions in proliferation, fate determination, and suppression of tumorigenesis.     

Differential molecular assemblies underlie the dual function of Axin in modulating the WNT and JNK pathways.

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.     

Expression of somite segmentation genes in amphioxus: a clock without a wavefront?

In the basal chordate amphioxus (Branchiostoma), somites extend the full length of the body. The anteriormost somites segment during the gastrula and neurula stages from dorsolateral grooves of the archenteron. The remaining ones pinch off, one at a time, from the tail bud. These posterior somites appear to be homologous to those of vertebrates, even though the latter pinch off from the anterior end of bands of presomitic mesoderm rather than directly from the tail bud. To gain insights into the evolution of mesodermal segmentation in chordates, we determined the expression of ten genes in nascent amphioxus somites. Five (Uncx4.1, NeuroD/atonal-related, IrxA, Pcdhdelta2-17/18, and Hey1) are expressed in stripes in the dorsolateral mesoderm at the gastrula stage and in the tail bud while three (Paraxis, Lcx, and Axin) are expressed in the posterior mesendoderm at the gastrula and neurula stages and in the tail bud at later stages. Expression of two genes (Pbx and OligA) suggests roles in the anterior somites that may be unrelated to initial segmentation. Together with previous data, our results indicate that, with the exception that Engrailed is only segmentally expressed in the anterior somites, the genetic mechanisms controlling formation of both the anterior and posterior somites are probably largely identical. Thus, the fundamental pathways for mesodermal segmentation involving Notch-Delta, Wnt/beta-catenin, and Fgf signaling were already in place in the common ancestor of amphioxus and vertebrates although budding of somites from bands of presomitic mesoderm exhibiting waves of expression of Notch, Wnt, and Fgf target genes was likely a vertebrate novelty. Given the conservation of segmentation gene expression between amphioxus and vertebrate somites, we propose that the clock mechanism may have been established in the basal chordate, while the wavefront evolved later in the vertebrate lineage.     

A clock-work somite

Somites are transient structures which represent the most overt segmental feature of the vertebrate embryo. The strict temporal regulation of somitogenesis is of critical developmental importance since many segmental structures adopt a periodicity based on that of the somites. Until recently, the mechanisms underlying the periodicity of somitogenesis were largely unknown. Based on the oscillations of c-hairy1 and lunatic fringe RNA, we now have evidence for an intrinsic segmentation clock in presomitic cells. Translation of this temporal periodicity into a spatial periodicity, through somite formation, requires Notch signaling. While the Hox genes are certainly involved, it remains unknown how the metameric vertebrate axis becomes regionalized along the antero-posterior (AP) dimension into the occipital, cervical, thoracic, lumbar, and sacral domains. We discuss the implications of cell division as a clock mechanism underlying the regionalization of somites and their derivatives along the AP axis. Possible links between the segmentation clock and axial regionalization are also discussed. BioEssays 22:72-83, 2000.     

Inhibition of Wnt signaling pathway by a novel axin-binding protein

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.     

The vertebrate segmentation clock: the tip of the iceberg

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.     

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

Large‐scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi‐dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA‐mediated gene knockdown on Wnt signaling. Meta‐analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates β‐catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.     

Dimerization choices control the ability of axin and dishevelled to activate c-Jun N-terminal kinase/stress-activated protein kinase

Axin and Dishevelled are two downstream components of the Wnt signaling pathway. Dishevelled is a positive regulator and is placed genetically between Frizzled and glycogen synthase kinase-3beta, whereas Axin is a negative regulator that acts downstream of glycogen synthase kinase-3beta. It is intriguing that they each can activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) when expressed in the cell. We set out to address if Axin and Dishevelled are functionally cooperative, antagonistic, or entirely independent, in terms of the JNK activation event. We found that in contrast to Axin, Dvl2 activation of JNK does not require MEKK1, and complex formation between Dvl2 and Axin is independent of Axin-MEKK1 binding. Furthermore, Dvl2-DIX and Dvl2-DeltaDEP proteins deficient for JNK activation can attenuate Axin-activated JNK activity by disrupting Axin dimerization. However, Axin-DeltaMID, Axin-DeltaC, and Axin-CT proteins deficient for JNK activation cannot interfere with Dvl2-activated JNK activity. These results indicate that unlike the strict requirement of homodimerization for Axin function, Dvl2 can activate JNK either as a monomer or homodimer/heterodimer. We suggest that there may be a switch mechanism based on dimerization combinations, that commands cells to activate Wnt signaling or JNK activation, and to turn on specific activators of JNK in response to various environmental cues.