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Mechanism of Wnt signaling in development and disease

Wnt signaling plays a central role in embryonic development, adult tissue homeostasis and cancer. At the cellular level, Wnt proteins can trigger a wide variety of responses, ranging from cell fate specification and proliferation to cell polarization and migration. We are interested in the mechanism of the canonical and non-canonical Wnt signaling pathways that mediate these different responses. As a model we use the nematode C. elegans, which offers a powerful genetic system to study evolutionarily conserved Wnt signaling mechanisms at single cell resolution.

Among the cells that respond to Wnt ligands are the two Q neuroblasts and their descendants that migrate in opposite directions during early larval development. We and others have shown that the Q cell system provides a unique model to address questions on Wnt dependent cell migration, canonical and non-canonical Wnt signaling mechanisms and cross-talk between Wnt pathways. In our toolkit, we have methods for time-lapse spinning-disc confocal imaging of the migration process and single molecule mRNA in situ hybridization (smFISH) to quantitatively measure gene expression in vivo. In our recent work, we have concentrated on the following topics:

Regulation of canonical Wnt/beta-catenin signaling

Canonical Wnt/beta-catenin signaling controls the migration of the QL descendants by inducing expression of the target gene mab-5. In wild type animals, mab-5 shows very little variability in expression levels. To study how such low variability is achieved, we collaborated with the group of Alexander van Oudenaarden to combine smFISH with mutant analysis and mathematical modeling (Ji et al., Cell 2013). Interestingly, we found that when certain Frizzled Wnt receptors are mutated, the variability in mab-5 expression is strongly increased. Further examination of various Wnt pathway mutants led to the discovery that there is extensive cross-talk within the Wnt pathway, with embedded interlocked positive and negative feedback loops that dampen the variability in target gene expression. These results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness.

In a parallel line of research, we discovered that the HECT-domain containing ubiquitin ligase Huwe1 functions as an evolutionarily conserved negative regulator of Wnt/beta-catenin signaling (de Groot et al., Science Signal 2014). In collaboration with the group of Vita Bryja at Masaryk University in the Czech Republic, we found that Huwe1 binds to and ubiquitylates the cytoplasmic Wnt pathway component Dishevelled (Dvl). Importantly, instead of targeting Dvl for degradation, Huwe1 inhibits the multimerization of Dvl that is required for signaling activity.

Further reading:

Ji, N., Middelkoop, T.C., Mentink, R.A., Betist, M.C., Tonegawa, S., Mooijman, D., Korswagen, H.C.* and van Oudenaarden, A* (2013) Feedback control of gene expression in the Caenorhabditis elegans Wnt pathway. Cell, 155: 869-880. *Joint senior/corresponding authors. See also Cell Video abstract.

de Groot, R.E., Ganji, R.S., Bernatik, O., Lloyd-Lewis, B., Seipel, K., Sedova, K., Zdrahal, Z., Dhople, V.M., Date, T., Korswagen, H.C.* and Bryja, V.* (2014) Huwe1-mediated ubiquitylation of Dvl defines a novel negative feedback loop in the Wnt signaling pathway. Science Signal 7: ra26. *Joint senior/corresponding authors. See also Science Signaling Podcast on our paper.

Mechanism of Wnt dependent cell migration

Previous studies have shown that the migration of the QR descendants is dependent on multiple Wnt ligands and receptors. We found that the migration of the QR descendants can be divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism (Mentink et al., Dev Cell, in press). First, long-range anterior migration is mediated by parallel-acting MOM-5/Frizzled and CAM-1/Ror2 dependent non-canonical Wnt pathways. Second, once the QR descendants reach their final anterior position, migration is stopped by activation of canonical Wnt/beta-catenin signaling. Finally, the short-range dorsoventral migration that places the QR descendants at their ultimate positions is dependent on a planar cell polarity (PCP) related Wnt pathway. Interestingly, we found that Wnt ligands do not act instructively in this process. Instead, our results show that the QR descendants switch between these signaling pathways by temporally regulating the expression of a Frizzled Wnt receptor. Cell intrinsic timing of receptor expression is also an important guidance mechanism in mammalian nervous system development, but the mechanism of this temporal regulation is still unknown. We are currently investigating how temporal regulation of Wnt receptor expression is achieved and how Wnt pathway cross-talk controls QR descendant migration. Furthermore, we are interested in how signaling through CAM-1/Ror2 and MOM-5/Frizzled controls QR polarity and migration.

Further reading:

Mentink, R.A., Middelkoop, T.C., Rella, L., Ji, N., van Oudenaarden, A., and Korswagen, H.C. (2014) Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans. Dev Cell, in press.

The mechanism of Wnt secretion

Using a combination of genetic studies in C. elegans and cell biological experiments in mammalian tissue-culture cells, we have discovered that Wnt proteins require a specialized secretion pathway to be released from Wnt producing cells. A central player in this secretion pathway is the Wnt binding protein Wntless (Wls), which binds Wnt in the endoplasmic reticulum (ER) and escorts it through the Golgi network to the plasma membrane for release. We found that Wls is a limiting component in this pathway and that it needs to be recycled back to the trans-Golgi network (TGN) to take part in new rounds of Wnt secretion (Yang et al., Dev Cell 2008). This is mediated through a retrograde trafficking pathway that involves internalization of Wls from the plasma membrane and retrieval of Wls from endosomes to the TGN, a trafficking step that is mediated by the retromer complex. Loss of retromer function leads to a strong reduction in Wnt secretion and induces various Wnt signaling related defects (Coudreuse et al., Science 2006).

Further analysis of the retromer dependent endosome to TGN transport of Wls revealed that it is mediated by a novel retromer pathway (Silhankova et al., EMBO J 2010; Harterink et al., Nature Cell Biol 2011). In this pathway, the cargo-selective subunits of the retromer interact with the sorting nexin family member SNX3 and sort Wls into vesicular transport carriers that are morphologically distinct from the tubular transport carriers that are formed by the classical retromer complex.

Further reading:

de Groot, R.E., Rappel, S.B., Lorenowicz, M.J. and Korswagen H.C. (2014) Protein kinase CK2 is required for Wntless internalization and Wnt secretion. Cell Signal, in press.

Lorenowicz, M.J., Macurkova, M., Harterink, M., Middelkoop, T.C., de Groot, R., Betist, M.C. and Korswagen, H.C. (2013) Inhibition of late endosomal maturation restores Wnt secretion in Caenorhabditis elegans vps-29 retromer mutants. Cell Signal 26:19-31.

Harterink, M., Port, F., Lorenowicz, M., McGough, I., Silhankova, M., Betist, M., van Weering, J., van Heesbeen, R., Middelkoop, T., Basler, K., Cullen, P., Korswagen, H.C. (2011) A SNX3-dependent Retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biol, 13:914-923.

Silhankova, M., Port, F., Harterink, M., Basler, K., Korswagen, H.C. (2010) Wnt signaling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt producing cells. EMBO J, 15: 4098-105.

Yang, P.T., Lorenowicz, M., Silhankova, M., Coudreuse, D.Y.M., Betist, M.C. Korswagen, H.C. (2008) Wnt signaling requires retromer dependent recycling of MIG-14/Wls in Wnt producing cells. Dev Cell, 14:140-147.

Coudreuse, D.Y.M., Roel, G., Betist, M.C., Destrée, O. and Korswagen, H.C. (2006) Wnt gradient formation requires retromer function in Wnt producing cells. Science, 312:921-924.

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image description
Migrating QR neuroblast.
image description
smFISH staining of prkl-1 mRNA in the QR neuroblast descendant Each red dot represents a single prkl-1 mRNA molecule.