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. 2010 Feb 15;338(2):193-201.
doi: 10.1016/j.ydbio.2009.11.033. Epub 2009 Dec 5.

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

Yuki Sugiyama  1 , Richard J W Stump, Anke Nguyen, Li Wen, Yongjuan Chen, Yanshu Wang, Jennifer N Murdoch, Frank J Lovicu, John W McAvoy
Affiliations

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

Yuki Sugiyama et al. Dev Biol. .

Abstract

Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles.

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Figures

Fig 1
Fig 1. Diagrammatic representations of the lens
(A) Sagittal section of eye. (B,C) Horizontal sections from regions indicated by the horizontal dotted lines in (A). Abbreviations: CE, central epithelium; GZ, germinative zone; TZ, transitional zone; OCF, outer cortical fibers; ICF, inner cortical fibers; NF, nuclear fibers.
Fig 2
Fig 2. Centrosome/cilium polarisation in lens cells
(A–E) Pericentrin (green) immuno-reactivity localises the centrosome/cilium and β-catenin (purple) localisation demarcates cell margins in lens whole mounts. In OCF (A, B) and CE (E), the centrosome/cilium is clearly associated with the cell margin proximal to the anterior pole. Arrow in (A) points to anterior pole. Scale bar, 20µm. (F) Histogram showing results from a quantitative analysis of centrosome/cilium polarisation in the different cellular compartments. A superimposed grid was used to determine the position of the centrosome/cilium in the apical region of each cell in relation to four quadrants (colour-coded) and a central region (c). Cells from at least 3 independent adult rat lenses were assessed and the total number of cells counted per compartment were as follows: OCF 247, ICF 331, GZ 639, CE 158. In all compartments, except for GZ, more centrosomes/cilia were in quadrant 1 than any other quadrant. (G) Higher magnification of a region in B. Scale bar, 10µm. (H) TEM shows basal bodies, cilium axoneme (small arrow) and striated rootlet (large arrow) at the apical end of a cortical fiber cell. Central microtubules are absent (inset). (I) TEM shows a pair of basal bodies with associated striated rootlets. Scale bar, 500 nm. (J) Diagrammatic summary with arrows representing regions of lens exhibiting PCP.
Fig 3
Fig 3. PCP proteins (green) are partitioned to cellular domains
(A) At apical tips of OCF, Fz6, Vangl2 and Abi2 are predominantly associated with the short side of each cell proximal to anterior pole. In contrast, Pk1 is predominantly localised to the long sides. Dvl2/Dvl3 tend to be localised to all sides of each cell with some cells showing a little more intense reactivity associated with the short side proximal to anterior pole. β-catenin (purple) demarcates cell margins and co-localises with Pk1, Dvl2 and Dvl3. Arrow in (A) points to anterior pole. Scale bar, 20 µm. (B) Representation of the apical tips of fibers (left) and a single fiber (right). Abbreviations: Abi2, Abl-interactor-2; Dvl2/3, Dishevelled 2/3; Fz6, Frizzled 6; Inv, Inversin; OCF, outer cortical fibers; Pk1, Prickle 1; Vangl2, Van Gogh-like 2.
Fig 4
Fig 4. Lp/Lp and Crsh/Crsh mouse lenses lack the alignment/orientation required to form normal sutures
(A,B) Sagittal histological sections of P0 lenses stained with haematoxylin and eosin. The Lp/Lp lens clearly has abnormal dimensions being flatter and wider than the lens of the wild-type (WT) littermate (+/+). Scale bar, 200 µm. (C–F) With phalloidin staining confocal microscopy from the anterior polar aspect of P0 lenses shows that fibers in the WT exhibit regular parallel alignment and orientiation so that they form a distinctive Y-shaped suture (C, E). In contrast, fibers in the Lp/Lp lens are not regularly aligned and lack the orientation required to form a normal Y-shaped suture (D, F). Note that images C and D are from horizontal slices closer to the anterior polar surface than images in E and F. (G,H) Phalloidin staining confocal microscopy of E18.5 Crsh/Crsh lens shows fibers are not regularly aligned and lack the orientation required to form a normal Y-shaped suture. Scale bars C-H, 50 µm.
Fig 5
Fig 5. Mice overexpressing Sfrp2 (Sfrp2+) in lens exhibit multiple and disordered suture-like arrangements
(A,B) With lectin staining confocal microscopy shows that the distinctive Y-shaped suture present in the P3 WT lens is absent from the Sfrp2+ littermate. Scale bar, 100 µm. (C,D) Histological cross sections of P3 lenses stained for β-catenin show that compared with WT lens, fiber orientation in the Sfrp2+ lens appears to be random. However, groups of fibers exhibit some local order and when groups with differing orientations meet, suture-like arrangements (arrows in C) or boundaries (arrows in D) are evident. Scale bar, 100 µm. (E) Pericentrin (green) immuno-reactivity localises the centrosome/cilium and β-catenin (purple) localisation demarcates cell margins in lens whole mounts from WT and Sfrp2+ littermates. Note that in virtually all cells in both WT and sfrp2+ lenses the centrosome/cilium is clearly polarised to one side of the cell. Scale bar, 10 µm.
Fig 6
Fig 6. Lens epithelial packing is disrupted in Sfrp2+ and Lp/Lp lenses
(A,B) The epithelial layer of lectin-stained whole lenses from WT (A) and Sfrp2+ (B) mice (P4) viewed from the anterior pole. Scale bar, 100 µm. (C,D) A higher magnification view of the epithelial packing arrangement of WT (C) and Sfrp2+ (D) lenses similar to that used for quantitative analysis. Scale bar, 25 µm. Inset in (D) shows a typical rosette-like arrangement frequently observed in the Sfrp2+ lens epithelium. (E) Distribution (%) of cells with 3 or 4 (4>), 5 and 6 or more (6<) cell-cell contacts. This shows a significant decrease in abundance of cells with 6 or more cell-cell contacts (hexagonal-like packing) and a significant increase in cells with 3–4 cell-cell contacts (rosette-like packing) in Sfrp2+ mice compared with WT mice. Cell-cell contacts were counted in pairs of WT and Sfrp2+ lenses from mice of 3 independent litters (P3/P4) and subjected for chi square analysis (Χ2=71.4; n=2,039; P<0.01). (F) A similar analysis conducted with 4 Lp/+ and 3 Lp/Lp lenses (E18.5) also showed a significant reduction in numbers of cell-cell contacts in Lp/Lp lenses (Χ2=14.89; n=1,594; P<0.01). (G) Analysis of cell density in Sfrp2+ and Lp/Lp lenses. Cell numbers in a 100 µm square are shown as means ± S.D (P values by two-tailed Student's t test). Cells counted and numbers of lenses assessed were as follows: WT, 427 (n=3); Sfrp2+ 523 (n=3); Lp/+, 608 (n=4); Lp/Lp, 930 (n=5). This shows a slight, but significant increase in cell density in Sfrp2+ and Lp/Lp lenses compared with their WT and Lp/+ counterparts.
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