Cell-Cell Communication:  Does Wnt act as a positional cue?

Establishing cell polarity is essential to generate cellular diversity. Cell polarity requires intricate cell-cell signalling and remodeling of the underlying cytoskeleton - a phenomenon that remains under-explored in developmental systems.

To explore this intricate process, we are studying the four-cell stage C. elegans embryo. At this stage, signals from one cell result in polarization of it’s neighbouring responding cell to generate daughter cells with distinct developmental fates. The signalling cell produces two signals, MES-1 (a transmembrane protein) and a Wnt homolog, MOM-2, that polarize the responding cell, aligning the mitotic spindle as well as regulating gene expression. It is known that both pathways are required for polarized division, and cell manipulation experiments suggest that it is the position of the Wnt signal alone that determines spindle orientation (Goldstein et al., 2006). However, it is unknown whether this is a direct effect of Wnt, or an indirect downstream effect.

To address this issue, we are using a novel approach in which beads coated with purified Wnt proteins are used to manipulate the position of the Wnt source on an isolated responding cell. We have also constructed a strain in which GFP-tagged tubulin is expressed in a Wnt background to examine the effects of a local Wnt signal on spindle dynamics in real-time. Our studies suggest that Wnt may act as an instructive cue at high levels, but as a permissive cue at low levels, and that there is another, perhaps weaker, instructive cue in Wnt mutant signalling cells. We are also genetically altering the levels of Wnt to further test whether this is true in vivo. Combining these cell manipulation experiments with imaging, we hope to extend this approach to investigate the effects of Wnt signalling on the localization of downstream cell polarity proteins by high resolution microscopy in C. elegans.

The Arp2/3 complex is important for morphogenesis in various developmental systems, but specific in vivo roles for this complex in cells that move during morphogenesis are not well understood. We have examined cellular roles for Arp2/3 in the C. elegans embryo.

In C. elegans, the first morphogenetic movement, gastrulation, is initiated by the internalization of two endodermal precursor cells. These cells undergo a myosin-dependent apical constriction, pulling a ring of six neighboring cells into a gap left behind on the ventral surface of the embryo. In agreement with a previous report (Severson et al., 2002), we found that in Arp2/3-depleted C. elegans embryos, membrane blebs form and the endodermal precursor cells fail to fully internalize. We show that these cells are normal with respect to several key requirements for gastrulation: cell cycle timing, endodermal fate, apicobasal cell polarity, and apical accumulation and activation of myosin II. To further understand Arp2/3’s function in gastrulation, we examined F-actin dynamics in wild-type embryos. We found that three cells of the ring of six neighboring cells extend short, dynamic, F-actin-rich processes at their apical borders with the internalizing cells. These processes failed to form in embryos that were depleted of Arp2/3, or of the apical protein PAR-3.

Our results identify an in vivo role for Arp2/3 in the formation of subcellular structures during morphogenesis. The results also suggest a new layer to the model of C. elegans gastrulation: in addition to apical constriction, internalization of the endoderm may involve dynamic, Arp2/3-dependent, F-actin-rich extensions on one side of a ring of neighboring cells.