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. 2016 Apr 1;412(1):32-43.
doi: 10.1016/j.ydbio.2016.02.016. Epub 2016 Feb 20.

Lens placode planar cell polarity is dependent on Cdc42-mediated junctional contraction inhibition

Maria Muccioli  1 , Dalya Qaisi  1 , Ken Herman  1 , Timothy F Plageman Jr  1
Affiliations

Lens placode planar cell polarity is dependent on Cdc42-mediated junctional contraction inhibition

Maria Muccioli et al. Dev Biol. .

Abstract

Development of the ocular lens commences with the formation of the lens placode, an epithelial structure that thickens and subsequently bends inward in a process called invagination. Invagination is observed during the development of many embryonic structures, but the spectrum of morphogenetic events driving this process are, in most cases, not fully understood. A characteristic commonly found in embryonic tissues undergoing epithelial reorganization is planar polarity, a property where cells are geometrically and/or molecularly orientated in a specific direction along the plane of an epithelium. Planar polarity is known to drive the morphogenesis of several epithelial structures, however its role during invagination events is less clear. We have found that at the onset of invagination, cells of the lens placode become geometrically planar polarized such that they are orientated toward a central point in the lens placode. Further investigation revealed that this is due to contraction of radially orientated junctions and the elongation of those circumferentially orientated. Radial junctions have an elevated localization of actomyosin and their contraction is dependent on the F-actin and Rho-kinase binding protein, Shroom3. Elongation of circumferential junctions is dependent upon Cdc42, a Rho-GTPase known to regulate polarity via the Par-complex. We determined that Cdc42 and members of the Par-complex inhibit Shroom3-induced contractility and promote anisotropic placode cell geometry through inhibition of junctional contraction. We postulate that invagination of the lens placode requires careful orchestration of these opposing processes which are mediated by the planar polarization of junctional proteins.

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Figures

Fig. 1.
Fig. 1.
(A) Drawings of cross-sections of the corresponding lens invagination stages. The red boxes indicate the region magnified in (B′–E″) (B–E) β-catenin immunofluorescent labeling of whole-mount wild-type mouse embryos at four distinct lens invagination stages. The yellow squared region are magnified in the panels below. Note that the central region of Stage II/V placodes are out of the focal plane. Scale bars=50 μm (B–E); 10 μm (B′–E″). (F, G). The average apical length of cells measured in their circumferential (F) or radial dimensions (G) at five distances from the center of the placode at Stage 0 and Stage I. For the peripheral images, the placode center is to the right of the image. Note that the peripheral cell geometries become anisotropic at Stage I. Error bars show the standard error and the asterisks indicate p<0.05.
Fig. 2.
Fig. 2.
(A, B) Still images from movies of lens placode cells from Ecad-mCFP transgenic embryos changing their apical geometries. Cells are depicted that contract isotropically where both radially and circumferentially orientated junctions shorten (A), or cells contract their radial junctions but elongate their circumferential junctions (B). The top panels show two time-points of cells changing their apical shape (radial=up/down; circumferential=left/right), while the bottom two panels are an aggregate of images of specific junctions changing over the course of several frames (10 min intervals). White brackets indicate the junctions magnified in the bottom panels, and the scale bar=10 μm. (C, D). A graph of the measurements of the length of four apical or circumferential junctions contracting (C), or four circumferential junctions elongating (D) over the imaging time period.
Fig. 3.
Fig. 3.
(A–H) Apical view of whole-mounted lens placodes immunofluorescently labeled for Myosin IIb (A–B), Phalloidin (C–D), Shroom3 (E–F), or p120-catenin (G–H). The images are taken of either the center of the placode (A,C,E,G) or 150–200 μm away from the center (peripheral, B,D,F,H). Closed and open arrowheads indicate radially or circumferentially orientated junctions, respectively. The yellow brackets indicate the regions magnified in B′–H′. (I) A depiction of how lens placode junction angles were measured for the following intensity measurements. (J) The graphs indicate the average intensity of immunofluorescent labeling simultaneously measured with the junctional angle with respect to the placodal radius. Note that as the angle increases, and is more radially orientated, the greater the junctional intensity. (K,L) Representative regions of the peripheral lens placode 150–200 μm away from the center of wild-type (K) or Shroom3 deficient (L) embryos immunofluorescently labeled with β-catenin. The placode center is orientated to the right of the images. (M, N) The graphs depict the average dimension ratios (M) and circumferential and radial length (N) of placodal cells from Shroom3Gt/Gt mutant embryos at five different distances from the placode center. Error bars represent the standard error and scale bars=10 μm.
Fig. 4.
Fig. 4.
(A, B) Representative regions of the peripheral lens placode 150–200 μm away from the center of wild-type (a) or Cdc42 deficient (B) embryos immunofluorescently labeled for β-catenin. (C) The graphs depict the average dimension ratios of cells from control or Cdc42 mutant placodes at Stage 0 and Stage I located at the indicated distance from the center. Note that differences are not observed until Stage I. (D) The graphs depict the average circumferential and radial length of placodal cells from Cdc42 mutant embryos located at the indicated distances from the placode center at Stage I. (E) The average cell dimensions from control and mutant placodes at the indicated distance from center were used to shape hexagons to model the average shape of cells in distinct placodal regions from different genotypes. (F–K) Representative regions from the placodes of control or Cdc42 mutant whole mounted embryos immunofluorescently labeled for Shroom3 (F, G) or Myosin IIb (I, J). Arrowheads indicate circumferentially orientated junctions, and the white bracketed region is magnified in the lower panels. The graphs (H, K) indicate the average intensity of immunofluorescent labeling simultaneously measured with the junctional angle with respect to the placodal radius. Note that the more radial measurements show significant differences in junctional intensity of both Shroom3 and Myosin IIb. Placode centers are orientated to the right of all images. Error bars represent the standard error, the scale bars=10 μm, and the asterisks indicate p<0.05.
Fig. 5.
Fig. 5.
(A, B) Representative regions of the central lens placode of wild-type (A) or Cdc42 deficient (B) embryos immunofluorescently labeled for Myosin IIb. (C) The graphs depict the average apical area of cells within a 50 μm of the placode center or 150–200 μm away from the placode center at Stage 0 and Stage I. (D) The average cell dimensions from control and mutant Stage 0 placodes at the indicated distance from center were used to shape hexagons to model the average shape of cells in distinct placodal regions from different genotypes. Note that the apical areas are smaller in the absence of Cdc42. (E, F) Representative regions of the central lens placode (E) or surface head ectoderm (F) from whole-mounted wild-type embryos immunofluorently labeled for Cdc42. The bracketed area of E indicates a representative region of central placodal cells with lower junctional Cdc42 localization. (G) A magnified view of a portion of the region depicted in panel E showing co-labeling of Cdc42 and β-catenin. The yellow circles and asterisks mark cells with high or low levels of junctional Cdc42, respectively. (H) The graph depicts the average junctional intensity of central placodal cells with apical areas less or greater than 25 μm2. Note, that the junctional intensity is significantly reduced in cells with cells with smaller apical areas. Error bars represent the standard error, the scale bars=10 μ, and the black asterisks indicate p<0.05.
Fig. 6.
Fig. 6.
(A–H) Apical view of representative transgenic MDCK cells (asterisks) expressing the indicated plasmids immunofluorescently labeled for β-catenin (white) and the indicated transgene (green, inset). (I) The graph depicts comparisons between the average apical area of cells transfected with distinct combinations of indicated plasmids. The number of cells quantified for each group are listed below. The asterisk and pound-sign indicate averages that are significantly distinct from Shroom3 transfected cells or Shroom3 and Cdc42 co-transfected cells, respectively. Error bars represent the standard error, the scale bars=10 μm, and the black asterisks indicate p<0.05.
Fig. 7.
Fig. 7.
(A, B) Representative regions of the peripheral lens placode 150–200 μm away from the center of Stage I wild-type (A) or Cdc42 deficient (B) embryos immunofluorescently labeled for aPKC. The white bracketed region is magnified to the right and arrowheads point out radial junctions. The scale bar=10 μm and the placode centers are orientated to the right of the images. (C) The graph depicts the average intensity of immunofluorescent labeling of junctional aPKC simultaneously measured with the junctional angle with respect to the placodal radius. Note that the more radial measurements show significant increases in junctional intensity for aPKC. Error bars represent the standard error, and the asterisks indicate p<0.05.
Fig. 8.
Fig. 8.
The average apical cell shape of Stage I wild-type and Cdc42 deficient peripheral cells are modeled here along with an exaggerated depiction of the relative localization of the color-matched junctional proteins, where the line thickness correlates with greater abundance. Junctional contraction is thought to occur when the sum of junctional contraction protein (Shroom3, Rock, Myosin IIb) activity conceptually outweighs that of the inhibitory proteins (Cdc42 and aPKC). Junctional elongation is thought to be permitted when the activity of the inhibitory proteins conceptually outweigh that of the contractility function. See the discussion for a full description.
Movie 1.
Movie 1.
Apical view of a placode cell from a Ecad-mCFP transgenic embryo over the course of 230 min (each frame is 10 min). Note that in this cell, junctional contraction occurs in both circumferentially (aligned left to right) and radially (aligned up and down) orientated junctions with respect to the placode center. A video clip is available online.Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.ydbio.2016.02.016.
Movie 2.
Movie 2.
Apical view of a group of placode cells from a Ecad-mCFP transgenic embryo over the course of 230 min (each frame is 10 min). Note that in these cells, junctional contraction occurs in radially orientated junctions (aligned top to bottom), while elongation occurs in those orientated circumferentially (aligned left to right).Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.ydbio.2016.02.016.
See this image and copyright information in PMC

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