Middelkoop, Teije et al. (2021) International Worm Meeting "The mitotic spindle and the cytokinetic furrow cooperatively align the dorsoventral axis with embryo geometry"

Cornell, Caitlin, Middelkoop, Teije, Grill, Stephan, Pimpale, Lokesh

[International Worm Meeting, 2021]

Establishment of the major body axes is essential for embryonic development. The C. elegans dorsoventral axis is established during the two-cell stage, and its orientation is defined by the division of the anterior AB blastomere. Earlier work in various holoblastic animals has shown that embryo geometry strongly affects early embryonic division patterns. However, whether and how embryo geometry affects the orientation of the dividing AB cell, and thereby the dorsoventral axis is unknown. To study this, we performed quantitative 3D live imaging of the cytoskeletal machinery in dividing AB blastomeres of embryos that are compressed perpendicular to the anteroposterior axis. In agreement with previous studies, we find that the AB cell division, and therefore the future dorsoventral axis, aligns parallel to the long cellular axis (which is perpendicular to the compression direction). Moreover, we show that, when viewed down the anteroposterior axis, the mitotic spindle of the AB blastomere is initially oriented randomly with respect to the long axis. During spindle elongation, it undergoes a rapid, large scale rotation to finally align parallel with the long axis. This mitotic spindle rotation is accompanied by a rotation of the cytokinetic furrow, ultimately resulting in the cell division axis to be aligned with the long cellular axis. Although the molecular mechanisms are still topic of debate, the mitotic spindle in many contexts is known to find the long axis of the cell. However, by performing conditional genetic perturbations, we find that force generation in the actomyosin layer is required and governs the kinetics of alignment of the mitotic spindle along the future DV axis. We speculate that the cooperative action of spindle and cortex promotes timely alignment of the cytokinetic machinery in fast-dividing cells.

Middelkoop, Teije C. et al. (2013) International Worm Meeting "dpy-19 and mig-21 control the persistent directionality of migrating Q neuroblasts in Caenorhabditis elegans."

Boxem, Mike, Middelkoop, Teije C., Koorman, Thijs, Korswagen, Hendrik C.

[International Worm Meeting, 2013]

During C. elegans development several cells migrate along the anteroposterior axis. We use the migration of Q neuroblasts as a model to study cell migration in vivo. At the time of hatching, two Q neuroblasts occupy equivalent left/right positions on the lateral sides of the animal. During larval development both Q neuroblasts polarize and migrate in opposite directions where the left Q neuroblast (QL) migrates posteriorly and the right Q neuroblast (QR) migrates anteriorly. After an initial short-range migration both Q neuroblasts undergo an identical pattern of division, generating Q descendants (Q.d) that migrate further along the anteroposterior axis. While Q.d migration is known to be controlled by Wnt signaling, the initial polarization process is Wnt signaling independent. Previously, two transmembrane proteins, MIG-21 and the conserved DPY-19 protein, were shown to control initial Q cell migration. Using high resolution time lapse imaging we have found that both genes are necessary for the formation of a protruding front with persistent directionality. In the absence of either dpy-19 or mig-21 the main protruding front changes its direction multiple times during migration resulting in a random migration direction. Upon loss of both genes we found that the direction of the main protruding front is persistent but is pointing towards the dorsal direction in both QL and QR. These results indicate that dpy-19 and mig-21 are both necessary for the persistent directionality of initial Q cell migration along the anteroposterior axis. In order to gain a functional understanding of this process we are now examining putative interactions of these genes with components involved in cytoskeletal remodeling. Furthermore, we are performing a genome-wide yeast-two-hybrid interaction screen in order to identify DPY-19 interactors.

Middelkoop, Teije C et al. (2009) International Worm Meeting "Dissecting the spatiotemporal expression patterns of Wnt and Frizzled genes to obtain insight into Wnt-dependent processes in C. elegans."

van Oudenaarden, Alexander, Kim, Dong Hyun, Middelkoop, Teije C, Korswagen, Hendrik C

[International Worm Meeting, 2009]

During C. elegans development the Wnt signaling pathway is used repeatedly to dictate distinct developmental decisions. The C. elegans genome encodes five Wnt ligands and four Frizzled receptors that can function partially redundantly in a variety of processes. Although many events were shown to depend on Wnt signaling, little is known about the endogenous expression patterns of Wnts and Frizzleds throughout worm development. Therefore we sought to analyze the expression of Wnt ligands and their receptors using a novel single molecule fluorescence in situ hybridization method (FISH). Using this technique we are now analyzing endogenous expression of all the C. elegans Wnt and Fz homologs at the mRNA level. Ultimately, we will generate a detailed spatiotemporal map of Wnt and Fz expression throughout development. Furthermore, we are generating Wnt::protA fusion constructs to visualize the distribution of Wnt ligands at the protein level, as we have previously reported for EGL-20. Together, these data will give molecular insights into the complex signaling network of Wnt molecules and Frizzled receptors and may provide clues about the role of individual Wnt and Fz homologs in C. elegans development. In addition, we will extrapolate these data to Q neuroblast migration, a process known to depend on both canonical and non-canonical Wnt signaling. We will focus on the initial migration of Q neuroblasts since little is known about this process. By combining single molecule FISH, mutational analysis and cell biology we will provide molecular insights into the developmental control of Q neuroblast migration.

Harterink, Martin et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The Secreted-Frizzled Related Protein in C. elegans Wnt signaling"

Doan, Thang, Middelkoop, Teije, Korswagen, Hendrik C, Harterink, Martin

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The Wnt family of secreted signaling proteins is responsible for important developmental and homeostatic processes throughout the animal kingdom. Importantly, its deregulation is implicated in several human pathologies, most notably cancer. Therefore Wnt signaling has to be carefully regulated. Secreted Frizzled-related Proteins (SFRPs) are thought to negatively regulate Wnt signaling by binding to the Wnt ligand, although other functions in Wnt signaling have also been reported. Furthermore, changed SFRP expression patterns are associated with human pathologies (1). Recent evidence suggests that SFRPs can also enhance the diffusion of Wnt ligand and expand their signaling range, when ectopically expressed in the Xenopus embryo (2). Since C. elegans only has one sfrp gene, it represents the perfect model to carefully analyze SFRP function in Wnt signaling as well as its effect on the Wnt gradient formation. Here we will present our current results. 1. Bovolenta et al, JCS, 2008 2. Mii and Taira, Development, 2009

Ji, Ni et al. (2013) International Worm Meeting "Feedback Control of Gene Expression Variability in the Caenorhabditis elegans Wnt pathway."

Mentink, Remco, van Oudenaarden, Alexander, Middelkoop, Teije, Korswagen, Hendrik, Ji, Ni

[International Worm Meeting, 2013]

Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we use the stereotyped development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. Posterior migration of the left Q neuroblast (QL) depends on a canonical Wnt signaling pathway which functions cell-autonomously to activate the expression of mab-5/Hox. To identify the mechanism that ensures robust transcriptional activation, we used single molecule Fluorescent In Situ Hybridization (smFISH) to quantify the transcript levels of mab-5 and other genes of the Wnt pathway in QL in a series of wild type and Wnt signaling mutant strains. Interestingly, we found that mutants that perturb Wnt signaling frequently exhibited increased variability in mab-5 expression. Unexpectedly, these mutants also perturbed the levels of Frizzled (mig-1/Fz, lin-17/Fz and mom-5/Fz) expression in QL, indicative of feedback regulations within the Wnt pathway. Combining computational network inference with quantitative gene expression profiling, we deduced a most probable network topology consisting of interlocking positive and negative feedback loops targeting the Frizzled receptors and mab-5. Interestingly, model analysis suggests that positive feedback may cooperate with negative feedback to reduce variability, while keeping the expression of the target gene (mab-5) at elevated levels. A minimal model of this signaling network predicts changes in gene expression variability across various mutants. Our results challenge the conventional view of the Wnt signaling pathway as a feedforward cascade and implicate gene regulatory network as an effective mechanism to ensure developmental robustness. In an ongoing effort, we attempt to elucidate the biochemical nature of these genetic interactions.

Lorenowicz, Magdalena et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The role of the Wnt sorting receptor Wls in the regulation of Wnt secretion"

Silhankova, Marie, Middelkoop, Teije, Lorenowicz, Magdalena, Harterink, Martin, Korswagen, Hendrik C

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Wnts are highly conserved secreted proteins that participate in multiple developmental events during embryogenesis and control adult tissue homeostasis. Importantly, deregulation of Wnt signaling is frequently associated with cancer. Although signaling cascades initiated by Wnt proteins have been extensively studied, little is known about how Wnt proteins are produced and secreted. Recent studies have identified the retromer, an intracellular protein-sorting complex, and Wntless (Wls), a multipass transmembrane protein, as new factors important in the regulation of Wnt secretion. Wls binds to Wnt and growing evidence suggests that it functions as a Wnt sorting receptor. Moreover, our recent data demonstrate tha t Wls is a target of the retromer complex and that retromer-dependent trafficking is part of a transport pathway that retrieves Wls from the plasma membrane. Here we search for new components of the specialized Wnt secretory pathway and test at which step of the Wnt secretory route these new components function. In addition, we study the role of established regulators of vesicle trafficking such as Rab GTPases in Wls trafficking and Wnt secretion. We found that Rab7 is one of the small GTPases important in the control of Wls trafficking. In the absence of Rab7, Wls accumulates in large vesicular structures in HeLa cells that are positive for the late endosome marker CD63. Moreover, knock down of rab-7 or expression of dominant negative rab-7 in Wnt producing cells in C. elegans rescues the Wnt signaling defect of the weak retromer mutant vps-29. The depletion of rab-7 results also in rescue of egl-20/Wnt gradient formation in this mutant. In contrast, overexpression of rab- 7 in Wnt producing cells leads to an increase in the Wnt signaling defect of vps-29 mutant. Altgether, these data indicate that Rab7 may function at an important crossroads in the choice between Wls recycling and degradation.

Ebbing, Annabel L.P. et al. (2015) International Worm Meeting "Directional persistence of initial Q neuroblast migration in Caenorhabditis elegans requires both Fat-like cadherins CDH-3 and CDH-4."

F. Povoa, Euclides, Ebbing, Annabel L.P., Ji, Ni, Betist, Marco C., Korswagen, Hendrik C., Rella, Lorenzo, Middelkoop, Teije C.

[International Worm Meeting, 2015]

Q neuroblasts have been frequently used as a model to study directional cell migration in Caenorhabditis elegans. After hatching of the larvae, the Q neuroblasts are situated in two bilateral rows of hypodermal seam cells, in-between seam cells V4 and V5. From this point onward the Q neuroblasts migrate in a left-right asymmetric manner. QL migrates posteriorly, whereas QR migrates anteriorly. During this migration both QL and QR will undergo an identical pattern of cell divisions. Q daughter cell migration has been well studied, and a clear involvement for canonical as well as non-canonical Wnt signaling has been found. Initial Q neuroblast migration has been less well studied, however, it is clear that it is Wnt independent.Several mutations causing defects in initial migration have been found, such as mutations in mig-15, mig-21, dpy-19, and unc-40. Here, we studied the involvement of the fat-like cadherin encoding genes cdh-3 and cdh-4 in initial Q neuroblast migration. Fat-like cadherins form a subclass of the cadherin-superfamily, consisting of large transmembrane proteins with many cadherin repeats in their extracellular domain. They were first identified in Drosophila, where unlike a classical cadherin adhesive function, a role in planar cell polarity, Hippo signaling and border cell migration was discovered. In vertebrates, the Fat-like cadherins play an important role in the development of the nervous system.Our research shows that both C. elegans fat-like cadherin orthologs, cdh-3 and cdh-4, are required for initial Q neuroblast migration. More specifically, in vivo imaging shows that they are involved in the persistence and directionality of the protrusive fronts of Q neuroblasts. This directionality is required to ensure proper migration of Q daughter cells. Moreover, both fat-like cadherins are shown to act in a linear genetic pathway. Quantitative gene expression analysis using single molecule mRNA FISH shows that the expression levels of both genes are upregulated during initial Q neuroblast migration. The dynamic expression indicates a possible cell-autonomous function; however, we are currently doing research to ascertain this. Taken together, the requirement of the Fat-like cadherins in initial Q neuroblast migration in C. elegans strengthens the notion of a role for Fat-like cadherins in directional migration.