Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning

Abstract

Defining the three body axes is a central event of vertebrate morphogenesis. Establishment of left-right (L-R) asymmetry in development follows the determination of dorsal-ventral and anterior-posterior (A-P) body axes, although the molecular mechanism underlying precise L-R symmetry breaking in reference to the other two axes is still poorly understood. Here, by removing both Vangl1 and Vangl2, the two mouse homologues of a Drosophila core planar cell polarity (PCP) gene Van Gogh (Vang), we reveal a previously unrecognized function of PCP in the initial breaking of lateral symmetry. The leftward nodal flow across the posterior notochord (PNC) has been identified as the earliest event in the de novo formation of L-R asymmetry. We show that PCP is essential in interpreting the A-P patterning information and linking it to L-R asymmetry. In the absence of Vangl1 and Vangl2, cilia are positioned randomly around the centre of the PNC cells and nodal flow is turbulent, which results in disrupted L-R asymmetry. PCP in mouse, unlike what has been implicated in other vertebrate species, is not required for ciliogenesis, cilium motility, Sonic hedgehog (Shh) signalling or apical docking of basal bodies in ciliated tracheal epithelial cells. Our data suggest that PCP acts earlier than the unidirectional nodal flow during bilateral symmetry breaking in vertebrates and provide insight into the functional mechanism of PCP in organizing the vertebrate tissues in development.

Figure 1. The Vangl2^Δ/Δ; Vangl1^gt/gt embryos exhibit the most severe polarity defects

a, Asymmetrical localization of Vangl1 protein (green) in the sensory hair cells of the Vangl2^Δ/Δ cochlea at E18.5. b, The sensory hair cell polarity at E18.5 is shown by the orientation of actin-based stereocilium bundles (red) and the microtubule-based kinocilia (green). c, Quantification of misoriented hair cells in embryos with different genotypes. Data are means ±SD from 3 samples. The orientation defects in the cochlea ranged from few misoriented cells (Vangl2^Δ/+;Vangl1^gt/gt) to several affected cells (Vangl2^Δ/Δ, Vangl2^Lp/+;Vangl1^gt/gt and Vangl2 ^Lp̃Lp) to almost complete loss of polarity (Vangl2^Δ/Δ; Vangl1^gt/gt).

Figure 2. Laterality defects in the Vangl2^Δ/Δ; Vangl1^gt/gt embryos

a, Failure of embryonic turning and heart (marked by Nkx2.5 expression) looping in the Vangl2^Δ/Δ; Vangl1^gt/gt embryos. The Vangl2^Δ/Δ; Vangl1^gt/gt lung has three lobes (outlined) instead of four on the right side. R: right; L: left. b, Perinodal Nodal expression is enhanced on the right side of the Vangl2^Δ/Δ; Vangl1^gt/gt embryo (arrow). c, Ventral views of Nodal expression in LPM (upper panel) and around the node (lower panel, arrows). Weak perinodal Nodal expression was outlined. d, Lefty1 expression (orange arrow) on the left side of prospective floor plate and Lefty2 expression (white arrow) in the LPM were shown in ventral views (upper panel) and transverse sections (lower panel). e, Ventral views of Pitx2 expression.

Figure 3. Randomized ciliary positioning in the Vangl2^Δ/Δ; Vangl1^gt/gt embryos

a, Scanning electron microscopy of the PNC showing normal cilia at E8.0. b, Normal cilia (acetylated tubulin, red) in the PNC of the Vangl2^Δ/Δ; Vangl1^gt/gt embryo at E8.0. c, Basal 16 bodies (γ–tubulin, green) and cell boundaries (F-actin, red) of PNC cells at somite 0 stage. White lines indicate the middle position along the A-P axis of each PNC cell. A: anterior; P: posterior. d, Relative positions of basal bodies to the midline (along the A-P axis) of the PNC cells were plotted. Posteriorly biased basal body positioning was lost in the Vangl2^Δ/Δ; Vangl1^gt/gt embryos.

Figure 4. Vangl1 and Vangl2 are required for normal leftward nodal flow

a, Nodal flow shown by tracing latex bead movement across the PNC with lines in different colors (Supplementary movie 1, 2). A: anterior; P: posterior; L: left; R: right. b, Ciliary beating was recorded by live imaging (Supplementary movie 3, 4) and the mean beating frequency was calculated from at least 10 nodal cilia of 3 embryos with indicated genotypes. Data are means ±SD. c, Intracellular calcium release around the node, intensity scales are shown in the lower panels. d, Distribution of embryos (percentage and number) according to calcium patterns. e, Schematic diagram showing the role of Vangl1 and Vangl2 in breaking the L-R symmetry. I. A mouse embryo at E8.0. (II) Enlarged distal view of (I). (III) Normal nodal flow generated by posteriorly positioned cilia. A side view of ciliated cells are shown in the lower panel. (IV) In the Vangl2^Δ/Δ; Vangl1^gt/gt embryo, randomly positioned cilia around the center of the PNC cells resulted in turbulent nodal flow.