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. 2017 Mar 13;26(11):2053–2061. doi: 10.1093/hmg/ddx095

Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis

Hwa-Seon Seo 1, Raymond Habas 2, Chenbei Chang 1,*, Jianbo Wang 1,*
PMCID: PMC6075608  PMID: 28334810

Abstract

Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl → Daam1 cascade during CE. Our finding uncovers Vangl2–Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.

Introduction

The ability of cells to coordinate their individual polarity with the body axis is a fundamental character of higher order animals, including humans. This is commonly observed in static epithelial tissues that display polarized orientation of special structures on the surface, such as hair follicles, stereociliary bundles in the inner ear, and Drosophila wing hairs and bristles, and is referred to as planar cell polarity (PCP) (1–3). During the dynamic process of convergent extension (CE) morphogenesis in vertebrates, cells also intercalate, rearrange, migrate or divide in a polarized fashion to reshape tissues and extend body axis (4,5). Molecularly, both epithelial PCP and CE are regulated by a group of core proteins of the PCP pathway, a branch of the β-catenin independent non-canonical Wnt pathway (1,2,6–8). Disruption of PCP gene-mediated morphogenesis contributes to various congenital disorders, including neural tube closure defects (9–12), skeletal malformation in Robinow syndrome and Brachydactyly Type B (13,14), and cardiac anomalies in 22q11.2 deletion syndrome (15).

The fact that epithelial PCP and vertebrate CE share molecular components clearly indicates that the two processes evolve from a common ancestral mechanism, but a number of studies have pointed to potential divergent actions of PCP proteins in these two processes. Elegant fly genetic studies have led to a feedback competition model to explain epithelial PCP. In this model, intracellular antagonism between the core proteins Vang Gogh (Vang)/Prickle (Pk) and Frizzled (Fz)/Dishevelled (Dsh/Dvl), coupled with intercellular interaction between Vang and Fz, results in asymmetric distribution of Vang/Pk and Fz/Dsh complexes on opposing cell cortices to coordinate and propagate cell polarity (16–19). During CE, however, asymmetric distribution of PCP proteins has not been consistently observed [reviewed in (8)]. In addition, while it is clear that non-canonical Wnt ligand and Fz receptor signal through Dvl to activate PCP signaling during CE, the role of Vangl2 (Vang-like 2, an ortholog of Vang) in this process appears unorthodox. In contrast to its proposed antagonistic role in fly epithelial PCP, existing genetic studies suggest that Vangl2 interacts synergistically with Wnt5a/Fz/Dvl during CE in the mouse (20–23). Similar synergistic interaction was also found between Vangl2 and Ror2, a co-receptor of Fz, and led to a proposal that Vangl2 forms a receptor complex with Ror2 in response to non-canonical Wnt ligand (14). Furthermore, direct binding between Vangl2 and Dvl has been reported by several groups (24–27), even though they partition into separate cortical localization in epithelial PCP (8,18,24).

Given these discrepancies in the current literature, understanding the mechanism of Vangl2 action is crucial in resolving the puzzle of PCP signaling during morphogenesis. In our present study, we address this issue by first delineating the functional relationship between Vangl2 and Dvl during CE. We then use imaging and biochemical approaches to define the mechanistic basis underlying their functional interaction. Based on our findings, we propose a simple and coherent model to explain the logic of PCP signaling during CE and provide a framework for future study of the molecular mechanism underlying PCP-mediated tissue morphogenesis.

Results and Discussion

Vangl2 and Dvl interact both antagonistically and synergistically during neurulation in mice

In mice, loss of different combinations of the Dvl orthologs Dvl1, 2 and 3, or both Fz3 and 6, perturbs CE-like morphogenesis during neurulation and causes craniorachischisis, a severe neural tube defect in which the entire neural tube from the midbrain to the tail fails to close (20,21,28). Genetic and functional studies indicate that Vangl is also indispensable for CE: disruption of Vangl2 similarly causes craniorachischisis (25,29–34). Looptail (Vangl2Lp), a semi-dominant loss-of-function mutation in Vangl2 (29,30,32,35), provides a sensitized genetic background to study PCP signaling in mice. To determine the functional relationship between Vangl2 and Dvl during neural tube closure, we utilized Vangl2Lp heterozygous mutant, and a Dvl2-EGFP BAC (bacterial artificial chromosome) transgene that functions similarly to endogenous Dvl2 gene (21). We found that homozygozing Dvl2-EGPF, which increased total Dvl2 expression levels, did not cause any detectable defects in the wild-type background. Homozygozing Dvl2-EGPF in Vangl2Lp/+ heterozygous background, however, caused craniorachischisis with 42% penetrance (5/12 embryos, Supplementary Material, Fig. S1). The data indicate that increased Dvl2 exacerbates the phenotype associated with reduced Vangl2, a result consistent with an antagonistic mode of interaction and suggesting that sufficient level of Vangl2 is required to suppress Dvl function during neurulation. This result contrasts with our prior mouse genetic studies showing synergistic interaction between Vangl2 and Dvl, in which case reducing the dosage of Dvl2 or Dvl3 in Vangl2Lp/+ heterozygous background results in craniorachischisis (20,21). The combined results imply that Vangl2 is required to both constrain and promote Dvl function, such that when Vangl2 level is reduced, either increasing or decreasing the total amount of Dvl can readily interfere with proper CE-like morphogenesis required for neurulation.

Vangl2 exerts dual negative and positive regulation on Dvl → Daam1 branch of PCP signaling during CE in Xenopus

To understand how Vangl2 can exert such complex regulation on Dvl during morphogenesis, we utilized the Xenopus model, in which PCP signaling in CE has been well characterized and multiple gene activities can be quantitatively manipulated simultaneously. We first injected mouse Vangl2, tagged with tdTomato or EGFP (tdT-mVangl2/EGFP-mVangl2) (36), into the dorsal marginal zone (DMZ) of Xenopus embryos. Their over-expression can disrupt CE, producing shortened anteroposterior (A–P) axis and open neural tube (Fig. 1A and C), similar to over-expression of Xenopus Vangl2 (XVangl2) (31,37). Conversely, moderate expression of mVangl2 can rescue the CE defects caused by anti-sense morpholino knockdown of endogenous XVangl2 (XV-MO) (37) in either the DMZ or activin-treated animal cap explant. These results indicate that the fluorescently tagged mVangl2 can function like XVangl2 in Xenopus (Supplementary Material, Fig. S2).

Figure 1.

Figure 1

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Vangl2 antagonizes Dvl during CE in Xenopus. (A) Over-expression of either tdTomato-tagged mouse Vangl2 (tdT-mVangl2, 200 pg RNA) or EGFP-tagged mouse Dvl2 (Dvl2-EGFP, 500 pg RNA) alone in the DMZ induces CE defects. Co-expressing Dvl2-EGFP together tdT-mVangl2, however, can suppress Vangl2 over-expression induced CE defects to different degrees, with 100 pg Dvl2-EGFP partially, and 250 pg Dvl2-EGFP more significantly, neutralized he CE defect. (B) Similarly, in activin-treated animal cap explants, individually over-expressing either tdT-mVangl2 or Dvl2-EGFP inhibits CE, while co-expressing Dvl2-EGFP can rescue tdT-Vangl2 induced CE defects. (C) Quantification of CE defects in (A). Injected embryos were divided into three categories based on the severity of CE defects: light gray bar (wild-type), dark gray bar (mild defect) and black bar (severe defect). (D) Quantification of CE phenotype in (B). The length of the injected explants in each group was measured, and the mean and standard deviation were determined for statistical analyses. Quantity of injected mRNA is indicated above each panel in (A) and (B), or below each column in (C) and (D). The number of scored embryos or explants is indicated above each column in (C) and (D).

To investigate how Vangl2 may regulate Dvl, we co-injected EGFP tagged mouse Dvl2 (Dvl2-EGFP) with tdT-mVangl2. We found that Dvl2-EGFP dose-dependently rescued the CE defects induced by tdT-mVangl2 over-expression in either the DMZ or the animal cap explant (Fig. 1), indicating that Vangl2 and Dvl antagonize each other and that Vangl2 over-expression likely disrupts CE by suppressing Dvl function.

To confirm this finding, we examined the functional interaction between Vangl2 and Daam1 (Dishevelled associated activator of morphogenesis 1), a formin protein that functions as a PCP effector downstream of Dvl during CE (38,39). Consistent with the prior findings, full length Daam1 over-expression alone has minimal effect on CE, but can strongly synergize with a small amount of Dvl2 to disrupt CE (Supplementary Material, Fig. S3). The synergistic interaction between Dvl2 and Daam1 can be blocked efficiently by mVangl2 (Supplementary Material, Fig. S3). On the other hand, C-Daam1, a truncated Daam1 that functions in a constitutively active fashion (38), can rescue the CE defects induced by mVangl2 over-expression (Supplementary Material, Fig. S4A, B, E and f). Taken together, these data clearly indicate that Vangl2 over-expression functionally inhibits the Dvl → Daam1 branch of PCP signaling during CE.

To test whether endogenous Vangl2 also antagonizes Dvl/Daam1 function, we examined XVangl2 morphant Xenopus embryos. Indeed, CE defects caused by knocking-down of endogenous XVangl2 with XV-MO can be rescued by co-injecting N-Daam1, a dominant negative mutant that inhibits Daam1 activity (38) (Supplementary Material, Fig. S4C, D, G and H). Similarly, CE defects in XVangl2 morphants can also be rescued by inhibiting Dvl activity with a small amount of Xdd1 (10 pg), a dominant negative mutant of Dvl (40,41) (Fig. 2A, B, D and E). Therefore, these epistasis experiments indicate that endogenous Vangl2 also negatively regulates Dvl → Daam1 branch of PCP signaling cascade during CE.

Figure 2.

Figure 2

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Bi-modal interactions between Vangl2 and Dvl during Xenopus CE. (A) Severe CE defects induced by knocking-down endogenous XVangl2 with 50 ng XV-MO can be suppressed by co-injecting 10 pg RNA encoding Xdd1, a dominant negative mutant of Dvl. However, co-injecting higher level of Xdd1 (50 pg) could no longer rescue the CE defect in XVangl2 morphants, but instead slightly enhanced their CE defects. Injecting 50 pg Xdd1 alone has minimal effect on CE. (B) Milder CE defects induced by partial XVangl2 knock-down (25 ng XV-MO) can also be rescued by co-injecting 10 pg Xdd1, but significantly enhanced by co-injecting 50 pg Xdd1. (C) Similarly, CE defects induced by partial XVangl2 knock-down (25 ng XV-MO) can also be rescued by co-injecting 1 pg RNA encoding Dsh-MA, a mitochondria-tethered Dvl that specifically inhibits Dvl function in PCP signaling through sequestering endogenous Dvl to the mitochondria. Co-injecting 25 pg Dsh-MA into XVangl2 morphants; however, enhances CE defects. Injecting 25 pg Dsh-MA alone can lead to mild CE defects. (D), (E) and (F) Quantification of CE defects in (A), (B) and (C), respectively.

Interestingly, in the above experiments, when we used a slightly higher dose of Xdd1 (50 pg) to inhibit Dvl function, we suddenly lost the rescue of the CE defects in XVangl2 morphants. Instead, 50 pg Xdd1 slightly enhanced the severity of CE defects in XVangl2 morphants (Fig. 2A, B, D and E). The result suggests that similarly to that observed in mice, there is also a synergistic interaction between endogenous Vangl2 and Dvl in Xenopus. This synergy could be observed most clearly in embryos with partial knockdown of XVangl2 activity (25 ng XV-MO), in which co-injection of 10 pg Xdd1 rescued, but 50 pg Xdd1 significantly enhanced, the CE defects (Fig. 2B and E). In light of a recent report that suggested a potential gain-of-function activity for Xdd1 (42), we also confirmed the bi-modal interaction between Vangl2 and Dvl using a different Dvl dominant-negative mutant, Dsh-MA, which has been shown to specifically inhibit Dvl function in PCP signaling by dimerizing with and sequestering endogenous Dvl to the mitochondria (43) (Supplementary Material, Fig. S5). Similarly to Xdd1, whereas co-injecting small dose (1 pg) of Dsh-MA partially rescued the CE defects induced by 25 ng XV-MO, co-injecting a higher dose (25 pg) of Dsh-MA exacerbated the CE defects induced by XV-MO (Fig. 2C and F). Together, these simultaneous gain- and loss-of-function experiments show that Vangl2 has both positive and negative roles in regulating Dvl function during CE.

Vangl2 cell-autonomously promotes efficient plasma membrane recruitment of Dvl during CE

To understand mechanistically how Vangl2 may regulate Dvl → Daam1 branch of PCP signaling during CE, we first investigated how Vangl2 affects Dvl/Daam1 subcellular localization because Dvl plasma recruitment is a pre-requisite for PCP signaling (43,44) and Dvl is thought to activate Daam1 near the plasma membrane (38,45). We first injected 200 pg of GFP-Daam1 alone, and discovered that Daam1 was associated primarily in a uniform fashion with the plasma membrane in either the animal cap or the DMZ explant cells. Co-injecting varying levels of tdT-mVangl2 does not alter Daam1 localization (Supplementary Material, Fig. S6A–D2). We next injected 100 pg of Dvl2-EGFP or Dvl2-mCh (Fig. 3A and data not shown) into the DMZ of early embryos. At this dose, EGFP or mCh tagged Dvl2 does not affect CE. In DMZ cells undergoing active CE, Dvl2-EGFP is recruited to the inner surface of the plasma membrane [Fig. 3A and (46)]. Co-injecting 400 pg Vangl2, a dose two times as much as required to inhibit Dvl function during CE (Fig. 1A), did not disrupt plasma membrane recruitment of Dvl2-EGFP (Fig. 3B1 and B2). Intriguingly, Vangl2 co-expression appeared to increase Dvl2-EGFP membrane recruitment slightly. To test this further, we increased Dvl2 injection to 500 pg. At this high dose, much of the over-expressed Dvl2 was distributed diffusely in the cytoplasm (Fig. 3C). Vangl2 co-injection, however, markedly promoted over-expressed Dvl2 to localize to the plasma membrane in either the DMZ (Fig. 3D1 and D2) or the animal cap explants (Supplementary Material, Fig. S6E–F2). Based on these data, we conclude that Vangl2 over-expression does not inhibit plasma membrane distribution of either Dvl or Daam1, but instead can promote plasma membrane recruitment of Dvl.

Figure 3.

Figure 3

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Vangl2 cell-autonomously promotes plasma membrane recruitment of Dvl. (A) Low level expression of Dvl2-EGFP (100 pg) in the DMZ cells undergoing active CE results in plasma membrane enriched localization of Dvl. (B1, B2) Co-expression of high level tdT-mVangl2 (400 pg) does not diminish Dvl2-EGFP enrichment at the plasma membrane. (C) High level expression of Dvl2-mCherry (Dvl2-mCh, 500 pg) in the DMZ leads to diffused distribution of Dvl throughout the cytoplasm. (D1, D2) Interestingly, co-expression of EGFP-mVangl2 (400 pg) efficiently recruits Dvl2-mCh to the plasma membrane. (E and F) To test whether Vangl2 promotes Dvl plasma membrane recruitment cell-autonomously or cell-non-autonomously, we separately injected Dvl2-mCh and EGFP-mVangl2 into the DMZ of two adjacent blastomeres. In this case, EGFP-mVangl2 remains localized at the plasma membrane, but fails recruit Dvl2-mCh to the plasma membrane in neighboring cells, indicating that Vangl2 acts cell-autonomously to recruit Dvl to the plasma membrane. (H–J) Knocking-down endogenous XVangl2 in the DMZ with 50 ng XV-MO injection disrupts Dvl2-EGFP (100 pg RNA injection) enrichment at the plasma membrane (compare H and I). This defect can be rescued by expressing low level (50 pg) of tdT-mVangl2 RNA (compare I and J).

Fly studies on epithelial PCP signaling suggest that the extracellular domain of Vang on the surface of one cell can interact with that of Fz on neighboring cells, thereby allowing Vang to cell-non-autonomously promote Dvl plasma membrane recruitment in opposing cells (16,17). Conversely, biochemical studies indicate that cytoplasmic region of Vangl can also bind to Dvl (24,25,27), and therefore in theory Vangl may directly recruit Dvl to the plasma membrane cell-autonomously. To determine which mode of action accounts for Dvl plasma membrane recruitment by Vangl2 in Xenopus, we performed separate injection of Dvl2-mCh and EGFP-mVangl2 into two adjacent blastomeres at the DMZ region of four-cell stage embryos (Fig. 3E–G). In the resulting DMZ explants, we found that EGFP-mVangl2 remained localized at the plasma membrane, but was unable to recruit Dvl2-mCh to the plasma membrane in adjacent cells. Therefore, Vangl2 acts cell-autonomously to promote Dvl recruitment to the plasma membrane.

To test whether endogenous Vangl2 might also be required for efficient plasma membrane recruitment of Dvl during Xenopus CE, we co-injected 50 ng XV-MO together with 100 pg Dvl2-EGFP into the DMZ at four-cell stage. We found that Dvl plasma membrane localization normally observed in DMZ explant cells during CE was significantly diminished by morpholino-mediated knockdown of XVangl2 (Fig. 3H and I). This defect could be rescued by a small dose of tdT-mVangl2, confirming that the observed reduction of Dvl plasma membrane localization was due to the lack of endogenous Vangl2, but not a non-specific effect of the morpholino. As plasma membrane localization is a pre-requisite for Dvl function in PCP signaling (43,44), this data suggests to us that Vangl2 may synergize with and positively regulate Dvl during CE by promoting efficient plasma membrane recruitment of Dvl.

Vangl2 inhibits Dvl–Daam1 interaction

But how does Vangl2 also antagonize and negatively regulate Dvl function during CE? Since over-expressed Vangl2 can cell-autonomously recruit Dvl to the plasma membrane (Fig. 3E–G), yet functionally inhibits Dvl → Daam1 branch of PCP signaling (Fig. 1; Supplementary Material, Figs S3 and S4), we hypothesize that Vangl2 may exert inhibitory effect by binding to Dvl and locally preventing Dvl from interacting with its other molecular partners, such as Daam1. Using in vitro translated proteins and co-immunoprecipitation (co-IP) assay, we confirmed that Vangl2 could bind directly to Dvl2 (Fig. 4A), as described previously (24,25,27). In addition, Dvl2 could also bind specifically to the C-terminal region of Daam1, C-Daam1 [Fig. 4B and (38)]. When we incubated Dvl2 simultaneously with both Vangl2 and C-Daam1, however, Dvl2 could co-IP with Vangl2 but no longer with C-Daam1, indicating that binding to Vangl2 inhibited Dvl2 from interacting with C-Daam1 (Fig. 4C). In light of the previous studies that have mapped the PDZ domain as the region required for Dvl binding to both Vangl2 (24–27) and Daam1 (38), our data supports the idea that Dvl–Daam1 and Dvl–Vangl2 interactions are mutually exclusive, and that binding to Vangl2 prevents Dvl from interacting with and activating Daam1.

Figure 4.

Figure 4

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Vangl2 inhibits Dvl–Daam1 interaction and a model of PCP signaling during convergent extension. EGFP-Vangl2, GFP-C-Daam1 and flag-Dvl2 were produced by in-vitro transcription/translation and used for immunoprecipitation and western blot assays. (A) Direct binding between Dvl2 and Vangl2: EGFP-mVangl2 can interact with flag-Dvl2. (B) Direct binding between Dvl2 and C-terminal portion of Daam1, C-Daam1: GFP-C-Daam1 can interact with flag-Dvl2. (C) Vangl2 inhibits Dvl interaction with Daam: incubating increasing amount of EGFP-mVangl2 together with flag-Dvl2 and GFP-C-Daam1 disrupts the ability of Dvl2 to co-IP C-Daam1, although Dvl2 can still interact with and co-IP Vangl2. (D) EGFP-Vangl2 and flag-Dvl2 injected into Xenopus embryos can also bind to each other, but their binding is diminished by co-injecting non-canonical Wnt ligand Wnt11. (E) Model: Vangl2 serves as a bi-functional switch for PCP signaling during CE. See text for detail.

A model for Vangl2 action during CE

Based on our findings, we propose a new model for Vangl2 action during CE (Fig. 4E). First, Vangl2 binds to Dvl to recruit it to the plasma membrane and keeps Dvl in an inactive state by, at least in part, sequestering Dvl from its downstream effector Daam1. At the same time, Vangl2-mediated plasma membrane recruitment also helps to spatially enrich and poise Dvl for efficient activation, likely by Fz upon the presence of non-canonical Wnt ligands. To test this model, we performed co-injection using Wnt11, a non-canonical Wnt ligand that can activate PCP signaling during CE (47,48). We found that Wnt11 co-injection could indeed diminish Vangl2–Dvl interaction in vivo in gastrulating Xenopus embryos (Fig. 4D). Given that Dvl controls both the stability and orientation of lamellipodia during CE (41), the dual positive- and negative-regulation exerted by Vangl2 may provide the dynamic and spatial modulation of Dvl required to achieve polarized cell intercalation during CE.

Although Vangl2–Dvl interaction has been reported previously, the functional significance of their interaction was not known (24–27). By integrating functional, imaging and biochemical data in this study, the model we proposed above argues that direct binding between Vangl2 and Dvl provides a central bi-functional switch for PCP signaling during CE (Fig. 4E). Fly studies of epithelial PCP demonstrated clearly that Fz is required to recruit Dsh/Dvl to the plasma membrane (8,19,49). In contrast, our findings here indicate that during CE efficient plasma membrane recruitment of Dvl also relies on Vangl2 (Fig. 3). We interpret this result to imply that during CE, endogenous Fz may not be sufficient to bring Dvl to the plasma membrane directly, and that a relay mechanism through Vangl2 is necessary. Given that Daam1 is localized at the plasma membrane (Supplementary Material, Fig. S6) and that Vangl2 prevents Dvl–Daam1 interaction, Vangl2 mediated membrane recruitment may have the advantage of bringing Dvl close to both Fz and Daam1, yet avoiding accidental PCP activation in the absence of non-canonical Wnt.

Whether our findings may have any relevance to epithelial PCP remains to be tested. Early studies proposed that Fz recruits Dsh/Dvl to the plasma membrane during PCP establishment in fly wing epithelium. It is interesting to note, however, that in Fz mutant flies a weak peri-membranous enrichment of Dsh was maintained during early wing development (49). Other studies further found that at this early stage of wing development, Dsh/Dvl co-localize with Vang in a symmetric pattern (24). Therefore, it is possible that during epithelial PCP, Dsh/Dvl may also be recruited initially to the plasma membrane by Vang, and relayed subsequently to Fz. Additional studies on the potential role of Vang in Dsh/Dvl localization during early fly wing development will help address this question.

Our model is able to reconcile the seemingly contradictory results that both reducing and increasing Dvl dosage in Vangl2Lp/+ heterozygous mice can abolish CE [(21); Supplementary Material, Fig. S1). Previous cell culture and mouse genetic studies show that the point mutation in Vangl2Lp not only blocks the endoplasmic reticulum to Golgi transport of mutant Vangl2Lp protein per se, but also affects plasma membrane trafficking of wild-type Vangl2 and Vangl1 protein owing to oligomerization (32,33,50). We confirmed this finding in Xenopus: when expressed in DMZ cells, mCh-mVangl2Lp failed to reach to the cell surface, and caused co-expressed wild-type EGFP-mVangl2 to be retained in the cytoplasm and form ectopic puncta together (Supplementary Material, Fig. S7A–D). We postulate that in Vangl2Lp/+ heterozygous mutants, Dvl membrane recruitment is significantly decreased because the total level of Vangl1/2 on the plasma membrane is diminished. Consequently, further reduction of Dvl gene dosage can readily abolish PCP activation in Vangl2Lp/+ embryos.

On the other hand, our biochemical studies show that Vangl2Lp has significant reduced ability to interact with Dvl2 in Xenopus (Supplementary Material, Fig. S7E), consistent with the previous yeast-two hybrid results (27). Functionally, whereas wild-type Vangl2 suppresses Dvl-overexpression induced CE defects, Vangl2Lp fails to do so but instead slightly enhances the CE defects (Supplementary Material, Fig. S7F–J). We reason that in Vangl2Lp/+ heterozygous mutants, the ability of Vangl to inhibit Dvl is also reduced, and therefore a further increase in Dvl dosage can readily disrupt CE because of unrestrained PCP activity.

In conclusion, our model argues that regulating Vangl–Dvl binding may underlie the central logic of PCP signaling during CE. One important question to be addressed in the future is how Dvl can be transferred from Vangl2 to Fz. As we demonstrated in Fig. 4D, PCP ligand Wnt11 can release Dvl from Vangl2 in vivo, therefore may promote transfer of Dvl from Vangl2 to Fz. Interestingly, previous studies showed that another PCP ligand, Wnt5a, can signal through co-receptor Ror2 to induce Vangl2 phosphorylation on two clusters of serine and threonine residues (14). Whether or how this phosphorylation event may affect Vangl2–Dvl binding will need to be investigated in the future. Furthermore, several proteins, including Pk, Diego and Dapper/Dact, can bind to both Dvl and Vangl, and positively or negatively regulate PCP signaling (19,24,26,51,52). Understanding how they modulate spatial and/or temporal dynamics of Dvl–Vangl interaction and the potential transfer of Dvl from Vangl to Fz will also help us decipher the mechanism of PCP signaling during morphogenesis, and how their mutations (9,11,53,54) may lead to congenital birth defects in humans.

Materials and Methods

Mouse strains and crosses

Mice carrying Vangl2Lp mutation was obtained from the Jackson Laboratory originally and genotyped as described (21). Vangl2Lp/+ mice were mated to Dvl2-EGFP BAC transgenic mice to generate Dvl2-EGFP; Vangl2Lp/+ mice. Dvl2-EGFP; Vangl2Lp/+ males were then crossed to Dvl2-EGFP/Dvl2-EGFP homozygous females to generate Dvl2-EGFP; Dvl2-EGFP; Vangl2Lp/+ mutants and control littermates. Genotyping of Dvl2-EGFP was carried out as previously described (21) and illustrated in Supplementary Material, Fig. S1B.

Xenopus embryo manipulation and imaging

Embryos were obtained, maintained and microinjected with invitro synthesized RNAs or a morpholino as previously described (55). Briefly, RNAs or morpholino were injected into either animal region of both blastomeres of two-cell-stage embryos (perspective ectoderm), or DMZ of four-cell-stage embryos (perspective dorsal mesoderm). For morphological assessment, the DMZ-injected embryos were fixed at tail bud stages, and embryo images were captured using Leica DFC 490 camera. For animal cap assay, ectodermal explants were isolated at stages 8 or 9 and incubated in 0.5× MMR solution containing 10 ng/ml of Activin B (R&D cat# 659-AB-005). The CE phenotype was quantified by measuring the length of the resulting explants.

For imaging of protein localization, ectoderm explants (animal cap) of the injected embryos were separated at stages 8 and 9, and dorsal mesoderm explants were isolated at stage at 10–10.5. The isolated tissues were subjected to confocal imaging analysis as described (41). The inner layer of the animal caps and the pre-involuting mesodermal tissues were imaged in these experiments.

RNAs, DNAs and Xenopus Vangl2–morpholino

EGFP-Vangl2, tdTomato-Vangl2, Dvl2-EGFP, Dvl2-mCherry, flag-Dvl2, Xdd1, Dsh-MA, GFP-Daam1, GFP-N-Daam1 and Wnt11 were transcribed invitro using mMESSAGE mMACHINE® SP6 Transcription Kit (Ambion cat#1340). For GFP-C-Daam1, plasmid DNA was used for injection. Xenopus Vangl2–morpholino (XV-MO) was the same as previously described (37). The dosage of each RNA or XV-MO is described in each figure.

In vitro and in vivo co-IP assay and western blot

Flag-Dvl2, EGFP-Vangl2 and GFP-C-Daam1 were transcribed and translated in vitro in separate reactions using Promega TnT® SP6 Quick Coupled Transcription/Translation System (Cat# L2080). The protein products were combined and incubated overnight at 4 °C. Sigma Anti-FLAG® M2 Magnetic Beads (Cat# 8823) was used to pull down Flag-Dvl2. For western blot, rabbit anti-GFP antibody (Santa Cruz, Cat# sc-8334) and mouse anti-flag M2 antibody (Sigma, Cat# M8823) were used to detect proteins using LI-COR Odyssey® CLx imaging system.

For in vivo co-IP assay, Xenopus embryos, injected with Flag-Dvl2, EGFP-Vangl2 and Wnt11, were lysed as described (56). Protein lysate were used for pull down with anti-Flag antibody and western blot analysis as described above.

Supplementary Material

Supplementary Material is available at HMG online.

Supplementary Material

Supplementary Data
Click here for additional data file. (5.2MB, zip)

Acknowledgements

We thank Drs Danelle Devenport and John Wallingford for Vangl2 and Dsh-MA constructs.

Conflict of Interest statement. None declared.

Funding

This work was supported by the National Institute of Health (R01 HL109130 to J.W., R01 GM098566 to C.C. and 2R01-GM078172 to R.H.); and the American Heart Association (14GRNT20380467 to J.W. and C.C.).

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