Wnt/PCP Instructions for Cilia in Left-Right Asymmetry

Abstract

Wnt-Frizzled/planar cell polarity (PCP) signaling establishes cell orientation within the epithelial plane, but whether Wnts are instructive or permissive is debated. Reporting in Developmental Cell, Minegishi et al. (2017) uncover an instructive link from Wnt5a/b gradients to PCP-factor-regulated polarized cilia positioning that is essential to mouse embryo left-right asymmetry establishment.

Planar cell polarity (PCP) signaling controls cell orientation within the plane of an epithelium. PCP establishment is critical during many biological processes, including polarization of cytoskeletal elements within a single cell, coordination of cell movement during gastrulation and neurulation (such as conversion extension and neural tube closure), cell migration, and axon guidance, among many others (Singh and Mlodzik, 2012). Work in Drosophila has pioneered the mechanistic features of PCP establishment, along with the two molecular signaling systems that function in this context (Singh and Mlodzik, 2012). These systems, conserved in vertebrates, are the Frizzled (Fz)-PCP core group—which consists of Fz itself, Van Gogh (Vang; Vangl in vertebrates), Prickle (Pk), Flamingo (Fmi; Celsr in vertebrates), Disheveled (Dsh; Dvl in vertebrates), and Diego (Diversin and Inversin in vertebrates)—and the Ft-Ds system, which is centered around the protocadherins Fat (Ft) and Dachsous (Ds; Dchs in vertebrates) and the extracellular protein kinase Four jointed (Fj; Fjx in vertebrates) (Lawrence et al., 2007; Singh and Mlodzik, 2012).

Although the downstream molecular components are well characterized, the upstream global regulation of PCP axis determination remains debated, and polarity axis orientation of the core Fz/PCP system is thought to be determined by long-range global cues and local interactions between cells (Yang and Mlodzik, 2015). Multiple sources of directional cues for PCP axis establishment have been proposed, including cues from Wnt family proteins (Gros et al., 2009; Wu et al., 2013) and suggestions that the Fat-Ds system can act upstream of the core Fz/PCP pathway (Lawrence et al., 2007). Two previous studies have indicated that Wnt factors can instruct the axis orientation of PCP. One work used a gain-of-function assay to show that Wnt11 (and core Fz/PCP proteins) provides cues for muscle cell orientation in chicken somites (Gros et al., 2009). The other study showed in Drosophila wings that dWnt4 and Wg gradients are both necessary and sufficient to orient the PCP axis (Wu et al., 2013). The Drosophila experiments suggested that Wnts establish PCP axis orientation by modulating intercellular Fz-Vang interactions across cell membranes, which serve as directional cues for asymmetric core Fz/PCP factor localization. However, the number of studies documenting an instructive role of Wnts in PCP signaling remained limited and has not yet been defined via mouse genetics.

In this issue of Developmental Cell, Minegishi and colleagues (2017) address whether Wnt signaling gradients affect the positioning of basal bodies and assoiated cilia to the posterior region of node cells in the mouse. The precise posterior positioning of cilia in the node is critical for left-right (L/R) asymmetry establishment through coordinated beating (clockwise rotational motion) of properly aligned cilia and the associated directional flow of L/R determinants (Nonaka et al., 2005). This new study demonstrates that opposing gradients of Wnt5a, Wnt5b, and the Wnt inhibitor Sfrp proteins provide instructive directional cues for polarizing core PCP protein localization and the resulting positioning of basal bodies/primary cilia to the posterior region of node cells in mouse embryos, and hence L/R asymmetry.

The authors show that Wnt5a and Wnt5b are expressed locally posterior to the node, forming gradients with highest levels of Wnt5a/5b concentration at posterior node regions. An inverse gradient, with its peak at the anterior of the node, is formed by the Wnt inhibitors Sfrp1, Sfrp2, and Sfrp5. Because Sfrps are secreted proteins that bind and inhibit Wnt(s), the Sfrp expression pattern likely steepens the Wnt5a/5b activity gradient (Figure 1A). Strikingly, in Wnt5a^–/– Wnt5b^–/– double mutant embryos, localization of basal bodies/cilia to the posterior of nodal cells is significantly reduced, suggesting that the Wnts instruct the posterior localization. Similarly, basal body/cilia localizations are largely randomized in node cells of Sfrp1^–/– Sfrp2^–/– Sfrp5^–/– triple mutant embryos, indiating that Sfrp1, Sfrp2, and Sfrp5 gradients are functionally important (Figure 1A). Consistently, the left-right asymmetry marker Nodal is mis-regulated in Wnt5a^–/– Wnt5b^–/– or Sfrp1^–/– Sfrp2^–/– Sfrp5^–/– embryos. The authors further find that reduction in basal body/cilia localization to the posterior of cells of the node is more severe in Sfrp1^–/– Sfrp2^–/– Sfrp5^–/– embryos, as compared to Wnt5a^–/– Wnt5b^–/– embryos, thus suggesting that other, evenly expressed, Wnts, such as Wnt11, also contribute to and require the Sfrp proteins for the formation of an activity gradient.

Figure 1. Wnt Gradients Regulate PCP Orientation and Basal Body/Centriole Positioning.

Conserved alignment of Wnt gradients, core Fz/PCP factor complexes, and basal body/centriole positioning in the mouse node (A) and in the Drosophila wings (B). In each panel, the schematic of cellular arrangements is reflected in the position of the centrioles/basal bodies in red (with a magenta line to schematize cilia in A). The side of each cell that displays enriched Vang/Vangl-Pk complexes is blue; the Fz-Dsh complexes are indicated in dark yellow.

Because Wnt5/11 family members have been strongly linked to core Fz/PCP signaling, the authors examined the localization of core PCP factors focusing on Vangl1, Vangl2, and Pk2. These are in wild-type node cells localized at the anterior cellular membrane, with Fzds and Dvls presumably on the posterior side (analogous to other mouse tissues and Drosophila). In Wnt5a^–/– Wnt5b^–/– embryos, Vangl1/2 and Pk2 are localized largely uniformly. To confirm the requirement of core Fz/PCP factors as effectors of the Wnt gradients, the authors demonstrate that pk1^–/– pk2^–/– double mutants also display randomized ciliary positioning. These data suggest that the Wnt activity gradients instruct basal body/cilia positioning via polarization of the core Fz/PCP proteins. Importantly, Minegishi and colleagues (2017) also demonstrate that Wnt5a is sufficient to redirect PCP orientation and basal body/cilia positioning when mis-expressed. They find that ectopic single-cell expression of Wnt5a redirects core PCP asymmetry and basal body/cilia positioning toward the Wnt5-expressing cell and that uniform Wnt5a expression in the node causes randomization of basal body/cilia positioning.

Taken together, the above data establish that Wnt gradients are both necessary and sufficient to (re)polarize basal body/cilia positioning in cells of the mouse node. As such, this study provides strong genetic evidence to now link instructive Wnt/PCP signaling to basal body/ciliary positioning, and it expands the genetic systems where gradients of Wnt proteins instruct PCP orientation. In Drosophila, Wnts have been shown to be a key cue for long-range core Fz/PCP protein polarization via core Fz/PCP signaling (Wu et al., 2013). This new work establishes similar PCP protein polarization patterns related to Wnt gradients (Figure 1), with both systems demonstrating that Wnt gradients are both necessary and sufficient to instruct PCP-mediated cellular orientation.

From the work of Minegishi et al. (2017), a second conserved link emerges with regard to centriole positioning. Basal bodies are specialized centrioles, and their positioning dependence on Wnt/PCP echoes the recent demonstration that centriole positioning is regulated in response to asymmetric localization of core Fz/PCP complexes in Drosophila wings (Carvajal-Gonzalez et al., 2016a), where centrioles are located to the side of cells that is enriched in the Fz-Dsh complex and away from the Vang-Pk complex (Figure 1B). This effect represents essentially the same type of regulation as that observed by Minegishi et al. (2017) in the mouse node (Figure 1). In addition, previous work in zebrafish also suggested a link between core Fz/PCP factors and basal body/cilia positioning along the anteroposterior axis in the cells of the neural tube (Borovina et al., 2010), where the basal body/cilium localized to the posterior side of each cell, away from the Vang-Pk complexes, like in Drosophila and the current mouse study. Taken together with Minegishi et al. (2017), these observations indicate that ciliary/basal body positioning, and similarly centriole localization in Drosophila, is a key evolutionarily conserved readout of Wnt-Fz/PCP signaling, as was recently proposed (Carvajal-Gonzalez et al., 2016b).