Frizzled-dependent Planar Cell Polarity without Wnt Ligands
Preprint posted on May 24, 2020 https://www.biorxiv.org/content/10.1101/2020.05.23.108977v1
Article now published in Developmental Cell at http://dx.doi.org/10.1016/j.devcel.2020.08.004
Wnt ligands are not required for planar cell polarity in the Drosophila wing or notum
Preprint posted on June 10, 2020 https://www.biorxiv.org/content/10.1101/2020.06.05.137182v2
Building complex organisms requires precise organisation. A key component of this organisation is setting up polarity within both cells and tissues. Polarity can come in many different flavours, but one element is planar cell polarity (PCP) which runs orthogonal to the ‘better known’ apical-basal polarity. It is essential to many morphogenetic processes, as well as the formation of cellular protrusions such as hairs. In addition to causing gross defects during development, mutations in PCP genes are also linked with deafness and cancer. A set of core PCP components, including (using the Drosophila names) Frizzled receptor (Ftz), Dishevelled (Dsh), Van Gogh and Prickle, has been identified. The inhibitory relationship between the core PCP proteins makes it easy to understand how PCP can be set up on an intracellular and a local level, but organisation at a global level has not been well described. A morphogen gradient has been proposed to set up the required asymmetry at that global tissue level. Given the involvement of Frizzled and Dishevelled, unsurprisingly Wnt ligands have been proposed as potential secreted factors. The role of Wnts has been studied already and the results are mixed. In zebrafish, Xenopus and mouse, mutation or deletion of Wnt causes defects in axis and tissue morphogenesis due to aberrant PCP. Further, it is known that ectopic expression of Wnts (Wingless/DWnt4) disrupts PCP in the forming Drosophila wing, but contrary, loss-of-function clones show no defects. The authors in these highlighted preprints set out to perform experiments with a complete loss-of-function throughout the whole tissue and do so with some elegant genetic tricks in Drosophila.
Together these preprints find that Wnts are not required for setting up PCP in the Drosophila wing, or the notum. Their experiments allow for the temporal distinction between the well described role of Wg in growth and specification and a potential role in PCP. The preprint from the Perrimon lab uses somatic CRISPR to make pairwise mutant combinations in the all Wnts identified to be expressed in the wing primordia. They back this up with complementary RNAi experiments. The preprint from the McGough and Vincent labs inhibits the Wg gradient by expressing a tissue-specific non-diffusible Wg and combining this with mutants in a further four Wnt genes, to generate an impressive quintuple mutant, thus removing all diffusible Wnts from the developing wing. They also remove diffusible Wnts by disrupting the trafficking of Wntless (Wls), the adaptor required for the progress of Wnts through the secretory pathway. They control this temporally and specifically in the wing primordia using nubbin-Gal4 to express a GFP nanobody with a KDEL motif to trap the GFP-tagged WIs, and consequently all Wnt proteins, in the endoplasmic reticulum.
From all of these comprehensive experiments the authors see no defects in PCP due to the inhibition of Wnts, and only see incorrectly specified PCP when removing components of the core PCP machinery including Fzl and Dsh. Both papers also show that any diffusible factor that may be required for PCP patterning cannot be solely produced by the cells in the wing margin. This is by destroying the wing margin by expressing pro-apoptotic genes (Yu et al.) or as a consequence of wg mutation (Ewen-Campen et al.) without any consequences for wing hair orientation.
Questions for the authors
- Is a global cue required to set up PCP, or could local organisation spread throughout a tissue?
- If a global cue is required, where is it likely to originate from as both papers exclude the wing margin?
- Why is there more variability in somatic CRISPR phenotypes than RNAi, when both are driven by the same Gal4?
We’re very grateful for your interest in our research, and for these questions. We are also glad to see that Yu, Vincent, McGough and colleagues have come to very similar conclusions using independent methods – it is always edifying to see independent labs make similar observations.
Let’s begin with Question #3, a technical question on why there is variability with somatic CRISPR compared to RNAi. When we use the Gal4-UAS system to express Cas9 and an sgRNA in a tissue, we are essentially asking each and every cell in that tissue to edit the target sequence independently. The variability we see within and between individuals is likely the combination of two factors. First, Cas9 cleavage is not 100% effective in every cell within a tissue – some cells may escape cleavage altogether, and remain wildtype. Second, when Cas9 does successfully cleave its target, the response within each cell is stochastic, leading to different outcomes in the sequence of the repaired DNA after editing. While we may wish that loss-of-function mutations are the dominant outcome, in fact any number of insertions, deletions, frame-shifts, or other repair outcomes are possible, and some subset of these outcomes (for example, a three- or six-basepair frameshift) may lead to functional wildtype protein still being produced.
A pioneering study on somatic CRISPR in Drosophila by Fillip Port & Simon Bullock (PMID 27595403) has shown that one technical approach to address these issues is to use two sgRNAs against each target gene, which both increases the chance of a cleavage event, and also increase the chance that at least one of these two events will cause a loss-of-function phenotype. While this approach does improve CRISPR-based knock-outs, it does not make them 100% effective. In our study – and this is consistent with results presented by Port & Bullock – we do still see mosaicism within individuals, and variability between individuals. This is likely an inherent issue with somatic CRISPR. This is probably particularly relevant with secreted proteins such as Wnts, as the wildtype protein from even a few wildtype cells may still function across larger regions of a tissue. While there is also variability between individuals with UAS:RNAi, it tends to be less pronounced.
To address the questions of whether there is a global cue to set up PCP, and if so where it originates, the simple answer is that we don’t know. In fact, that’s one of the important conclusions of our paper: we believe this remains an important open question. One motivation for publishing this manuscript is to note that we could not independently confirm a 2013 study (PMID 23912125) that found Wg + Wnt4 are redundantly required to serve as a global PCP cue, and thus that we consider it relevant to continue studying this question. As for speculating how a global PCP cue could work, previous studies have suggested roles for a gradient of Fat/Daschous and also tissue flows, but this is far from established. When I was a first-year graduate student, I was taught that a good threshold for whether or not to include a speculation in the Discussion of a manuscript is whether you’d be willing to make a $500 bet on it*. With that in mind, I’ll refrain from speculating on how global PCP is established in the fly wing, and leave it to future empirical studies.
-Ben Ewen-Campen (on behalf of authors)
*Thanks to Dr. Andrew Murray for this valuable lesson.
PCP is an important area of research, yet there are still many unsolved or controversial questions in the field. One of the long-debated topics is the role of Wnts in PCP. One of the difficulties of assaying the contribution of Wnts to the establishment of PCP is the potential redundancy between family members. The advent of CRISPR-Cas9 allowed us to generate reporters of all the Drosophila Wnts to determine which are expressed in the Drosophila wing disc, and then via iterative rounds of CRISPR-Cas9 mutate each of the Wnts expressed in the wing disc. To reinforce our conclusion, we used two completely different approaches to inhibit Wnt function. In one we devised a way to trap Wnts in the endoplasmic reticulum and in the other we ablated all the Wnt secreting cells. PCP was unaffected in both these conditions.
Questions 1 and 2 raised at the end of the article touch upon the most fundamental, yet unresolved element in PCP – what is the global cue that sets up PCP, if such a cue is required at all? At this point, we can only speculate.
Various studies have shown that clones of frizzled or dishevelled mutants, which perturb local organisation of PCP, only lead to localised hair misorientation. No PCP defect propagates far from these clones. This indicates that the amplification of asymmetric localisation of PCP proteins is insufficient to uniformly align cell polarity across the tissue plane, and that one or multiple inputs must organize PCP globally. However, the identification of the global PCP cue has been challenging. The various candidates proposed include the Ft/Ds pathway and the mechanical forces associated with tissue morphogenesis.
Inputs from both the Ft/Ds system and mechanical forces could suffice without an input from the margin. Ds is expressed in a graded manner with higher levels in the proximal part of the wing, while contraction of the hinge could initiate any mechanical forces that input into the PCP pathway. However, there is evidence suggesting that uniform expression of Ds is sufficient to establish PCP correctly. Perhaps other morphogenic organizers such as Hedgehog and Dpp should be considered since they influence growth and morphogenesis and would not be affected by ablation of the wing margin, where all the Wnts are expressed. Ultimately, it is likely that redundant mechanisms, local and global, are at work, Thus, it is possible that multiple mechanisms (even perhaps in some cases involving Wnts) would act together to establish PCP. Depending on the developmental time and tissue context, some cues can have more influence than others.
Posted on: 10th August 2020 , updated on: 11th August 2020