A second focus of the lab is to understand how signaling specificity is achieved between the canonical Wnt/b-catenin pathway and Wnt/Fz-PCP signaling. The two pathways bifurcate at the level of Dishevelled (Dsh) and our efforts are currently concentrated on dissecting the pathway specific modifications of Dsh and the interaction(s) of Dsh with the Fz receptor complexes. Pathway specific Dsh membrane recruitment and Dsh phosphorylation are being studied in through functional genomics in cell culture assays, biochemical dissection, and in vivo studies. We have identified several genes that help in switching from on pathway to the other and that affect Dsh phosphorylation in distinct ways.

In parallel, we have identified a novel role for IFT-A ciliary proteins in the Wnt/b-catenin pathway acting downstream of b-catenin stabilization and upstream of transcriptional events. This IFT-A function is independent of the ciliary role of IFTs and is cytoplasmic and conserved in mammals. We have demonstrated that IFT-A and Kinesin 2 act together as a complex that binds β-catenin and microtubules and is required for nuclear translocation of b-catenin. Activation of Wnt-signaling in IFT-A or Kinesin 2 mutants fails to activate Wg/Wnt targets, due to markedly reduced nuclear β-catenin localization, although high levels of cytoplasmic β-catenin  are detected. Many questions remain however: for example (i) How is the Kinesin-2/IFT-A complex promoting nuclear localization? Preliminary data suggest it is by movement along microtubules, but this needs to be confirmed and the associated exciting cell biology dissected; and (ii) our preliminary data further suggest that there is an antagonistic factor that competes with IFT140 binding to β-catenin to ‘inhibit’ its nuclear translocation. We are addressing these exciting questions.

Figure: Nuclear localization of β-catenin (red) after Wnt-induction (left panel) or in axin-/- mutants (middle panel) is blocked in IFT140 mutants (right panel). E-cadherin is in green and DAPI in blue

Ommatidia are arranged with respect to the antero-posterior (A/P) and dorso-ventral (D/V) axes. While the A/P arrangement is established by progression of eye development, the D/V alignment is generated by Fz/PCP – and N and EGFR-signaling and their effectors via ommatidial rotation (OR). Preclusters emerge with a single symmetry axis, and the combination of Wnt/PCP-mediated cell fate induction and the subsequent OR realigns them at 90° from their original positio. Preclusters in dorsal and ventral halves rotate in opposite directions to create a line of mirror-symmetry across the D/V midline, the equator. Wnt/Fz-PCP mediated R3/R4 specification (defining ommatidial chirality) takes place just before OR initiates, determining direction of rotation. OR is a highly coordinated developmental cell motility process, entailing concerted regulation of force generation and associated cytoskeletal network. Its link to cell adhesion in particular E-cadherin complexes is essential.  Based on genetic requirements and effects of core Fz-PCP factors in OR, i

It is assumed that the R3/R4 cells are critical in asymmetrically regulating force generation, thus driving OR directionality. Other factors, incl. N-signaling, the Nmo kinase, and the EGFR-Ras pathways are thought to “execute” these inputs and affect cell adhesion and force generation. These observations raise three significant questions, which we study in this context:

(1) What is the nature of the mechanical forces generated by the core PCP factor-effector interactions and Notch or EGFR-signaling; (2) How are these mechanically transmitted to drive a coordinated process; and (3) What are the molecular connections between the signaling pathways and junctional and cytoskeletal components during OR?

Figure: Developing eye discs stained for photoreceptor markers Elav (all in red), Svp (R3/4 and R1/6 in green) and Boss (R8). Note change in angle (rotation) of ommatdial clusters by the Svp (green) staining. Young preculsters are up and more mature ommatidia are in the lower part of picture