Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis

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.

Figure 1

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).

Figure 2

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.

Figure 3

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).

Figure 4

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.