Prickle and Ror modulate Dishevelled-Vangl interaction to regulate non-canonical Wnt signaling during convergent extension

Reviewer #1 (Public Review):

Summary: Planar cell polarity core proteins Frizzled (Fz)/Dishevelled (Dvl) and Van Gogh-like (Vangl)/Prickle (Pk) are localized on opposite sides of the cell and engage in reciprocal repression to modulate cellular polarity within the plane of static epithelium. In this interesting manuscript, the authors explore how the anterior core proteins (Vangl/Pk) inhibit the posterior core protein (Dvl). The authors propose that Pk assists Vangl2 in sequestering both Dvl2 and Ror2, while Ror2 is essential for Dvl to transition from Vangl to Fz in response to non-canonical Wnt signaling. There are several points that affect the strength of the author's conclusions.

Strengths: The strengths of the manuscript are in the very interesting and new concept for a model of how non-canonical Wnt induces Dvl to transition from Vangl to Fz. Prickle and Vangl2 are proposed to play an opposing role to suppress Dvl activity during convergent extension movements, whereas Ror antagonizes Vangl and may be required for the transition.

Weaknesses: The weaknesses are in the clarity and resolution of the data that forms the basis of the model. In addition to whole embryo morphology that is used as evidence for convergent extension (CE) defects, two forms of data are presented, co-expression and IP, as well as a strong reliance on IF of exogenously expressed proteins. Thus, it is critical that both forms of evidence be very strong and clear, and this is where there are deficiencies; 1) For vast majority of experiments general morphology and LWR was used as evidence of effects on convergent extension movements rather than Keller explants or actual cell movements in the embryo. 2) The study would benefit from high or super resolution microscopy, since in many cases the differences in protein localization are not very pronounced. 3) The IP and Western analysis data often show subtle differences, and not apparent in some cases. 4) It is not clear how many biological repeats were performed or how and whether statistical analyses were performed.

Reviewer #2 (Public Review):

The authors use Xenopus embryos to study feedback interactions between the planar cell polarity (PCP) proteins in the context of convergence and extension. They show that binding of the cytoplasmic polarity protein Pk2 to Vangl2 is needed for them to synergistically suppress defects in convergence and extension caused by Dvl overexpression. They then examine protein localizations in animal cap cells, and show that Wnt11-induced accumulation of Fzd7, Ror2 and Dvl into plasma membrane patches is disrupted by the functional Vangl2/Pk complex. This disperses Fzd and causes its endocytosis, while Dvl remains at the plasma membrane.

This is a potentially interesting paper, showing mechanisms by which Vangl2/Pk can functionally antagonize Fzd/Dvl during planar cell polarity.

The protein localization experiments in animal cap assays are for the most part convincing, but with the caveat that the authors assume that the proteins are acting within the same cell. As Fzd and Vangl2 are thought to localize to opposite cell ends in many contexts, can the authors be sure that the effects they observe are not due to trans interactions?

The authors propose a model whereby Vangl2 acts as an adaptor between Dvl and Ror, to first prevent ectopic activation of signaling, and then to relay Dvl to Fzd upon Wnt stimulation. This is based on the observation that Ror2 can be co-IPed with Vangl2 but not Dvl; and secondly that the distribution of Ror2 in membrane patches after Wnt11 stimulation is broader than that of Fzd7/Dvl, while Vangl2 localizes to the edges of these patches. The data for both these points is not wholly convincing. The co-IP of Ror2 and Vangl2 is very weak, and the input of Dvl into the same experiment is very low, so any direct interaction could have been missed. Secondly, the broader distribution of Ror2 in membrane patches is very subtle, and further analysis would be needed to firm up this conclusion.

A final caveat to these experiments is that in the animal cap assays, loss of function and gain of function both cause convergence and extension defects, so any genetic interactions need to be treated with caution i.e. two injected factors enhancing a phenotype does not imply they act in the same direction in a pathway, in particular as there are both cis/trans and positive/negative feedbacks between the PCP proteins.