Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse

Abstract

The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

Keywords: Cell adhesion; Cell polarity; Epithelial to mesenchymal transition; Lens development; PDZ proteins; Scrib.

Figure 1. Generation of Scrib conditional knockout mice

A: Shown are exons 1–10 of the wildtype (WT) Scrib allele. The targeting allele (TA) shown illustrates loxP sites that flank exons 2–8 and frt sites that flank a positive selection neomycin (NEO) marker. Mice containing the targeting allele were mated to mice expressing Flpase, which removed the neo marker and generated the floxed (F) allele. Arrows indicate locations of PCR primers a (ScribF), b (ScribwtR), and c (ScribckoR) used to identify if exons 2–8 had been deleted and d (Scrib1-X2-R) used in combination with a to routinely genotype the floxed and wt alleles. The mutant allele was generated through mating with Lens-Cre. B: Immunofluoresence demonstrating localization of Scrib (red) in the E10.5 lens vesicle (lv). Arrow indicates concentrated apical localization in the lens vesicle. oc, optic cup. C: GFP fluorescence indicating cre activity specifically in the E10.5 ScribLecre vesicle. D: Lens specific cre-mediated deletion of exons 2–8. PCR primer a, b, and c were used to amplify DNA fragments from lens, eye and tail samples. The wt band=437bp, the null band=193bp and the floxed band=325bp. ScribLeCre lenses had the 193bp null band. E: Whole cell lysates from lenses of P2 and corneas of P10 control and ScribLeCre mice were subjected to immunoblot analysis for Scrib and reprobed for Gapdh as a loading control. Scrib was reduced in lenses from ScribLeCre mice as compared controls and absent in the cornea lysate from ScribLeCre mice. Protein lysates from Scrib wt and null brain were included to demonstrate the specificity of the antibody.

Figure 2. Morphological defects in the eyes of postnatal day 10 (P10) ScribLeCre mice

A–B: Eyes (A) and lenses (B) were isolated from control and ScribLeCre mice and viewed under a dissecting microscope. A: P10 ScribLeCre eyes were noticeably smaller (right) compared to controls (left). B: ScribLeCre lenses were smaller than controls and also had an opaque center indicating a cataract (arrow). C–J: Longitudinally oriented paraffin embedded sections of eyes from P10 control (C, E, G) and ScribLeCre (D, F, H) mice were stained with hematoxylin and eosin. C–D: Sections of controls (C) and ScribLeCre eyes (D) showing that mutant lenses were vacuolated (arrow) and nuclei were scattered throughout the lens (arrowhead). Also, the iris was hyperplastic (asterisk). E–F: Higher magnification of the lens epithelium in control (E) and ScribLeCre (F) lenses highlighting the flattened and elongated cells in the epithelium of the mutant as compared to controls (arrowheads). G–H: Higher magnification image of control (G) and ScribLeCre (H) corneas showing that the epithelium of mutant corneas lacked an organized stratified epithelium with cuboidal shaped cells in the basal layer (arrowheads). Insets show higher magnification images of the regions indicated by black boxes. ac, anterior chamber; c, cornea; e, epithelium; en, endothelium; f, fiber cells; i, iris; l, lens; s, stroma. Bar = 50µm for C, D and 25µm for E–H.

Figure 3. Epithelial proteins are downregulated and mesenchymal proteins are upregulated in the P10 ScribLeCre lens epithelium

Longitudinally oriented, paraffin embedded (A–H, M–P) or cryogenic (I–L) sections from P10 control and ScribLeCre eyes were immunostained with antibodies against epithelial proteins (Pax6; A–D and E-Cadherin; E–H), the mesenchymal protein, αSMA (I–L), and Smad4 (M–P). Sections were counterstained with either propidium iodide (red, A, C), To-Pro3 (blue, I, K, M, O) or anti-α-catenin antibodies (E, G). A–D: Pax6 (green) was found consistently in the nuclei of epithelial cells from control lenses (A–B) whereas nuclear Pax6 staining was absent from (arrows; C, D) or reduced in nuclei of ScribLeCre epithelial cells. E–F: E-cadherin (green) colocalized (yellow) with α-catenin (red) along all membranes of control epithelium. G–H: In the flattened ScribLeCre epithelium there were areas where E-cadherin and α-catenin were absent from basal and apical membranes (arrows). I–L: αSMA was observed only in the iris (i) of control lenses (I, L), but was found in both the iris and the epithelium (arrow) of ScribLeCre lenses (K–L). The red box inset (L) shows a higher magnification image of the region indicated by the white box. M–P: Smad4 (red) was cytoplasmic in control epithelial cells (N) but was concentrated in the nuclei of ScribLeCre epithelial cells (O, P, arrows). e, epithelium; f, fiber cells; i, iris. Bar = 50µm.

Figure 4. E-cadherin and Pax6 levels are reduced in ScribLeCre lenses

Protein lysates from P2 control and ScribLeCre mice were subjected to western blot analysis using anti-E-cadherin (A) and anti-Pax6 (B) antibodies. Blots were reprobed for Gapdh as a loading control. For E-cadherin, three independent pools of protein were analyzed on three different blots. For Pax6 two independent pools were analyzed on two different blots. Bands were quantified by phosphorimager analysis and for E-cadherin statistical analysis was conducted using the two-sided Wilcoxon Rank Sum test. The levels of E-Cadherin were reduced 35% (p=0.04) in ScribLeCre lenses as compared to controls. The levels of Pax6 were reduced 55% in ScribLeCre lenses as compared to controls.

Figure 5. E-cadherin is progressively lost from ScribLeCre lenses

Longitudinally oriented, paraffin embedded sections from E11.5 (A–D), E13.5 (E–H) and E17.5 (I–L) control (A, B, E, F, I, J) and ScribLeCre (C, D, G, H, K, L) embryos (A–H) and eyes (I–L) were immunostained with anti-E-cadherin (green) and anti-α-catenin (red) antibodies. A–B: Colocalization (yellow) was prominent at the apical surface of cells in the anterior lens vesicle (A, arrow). E-cadherin was punctate in this region (B). C–D: E-cadherin staining was reduced in the anterior region of the ScribLeCre lens vesicles (arrows). E–F: Colocalization was observed along the basal and lateral membranes of controls at E13.5 (E). E-cadherin was punctate along the apical surface (F, arrow). G–H: In E13.5 ScribLeCre lenses, E-cadherin was specifically reduced along the basal and lateral membranes surface (arrows). I–J: In E17.5 control lenses, colocalization was found along all surfaces of the epithelium (I). E-cadherin was punctate at the apical surface (J). K–L: In E17.5 ScribLeCre lenses, E-cadherin was absent or reduced along some basal, lateral membranes (arrows). Red boxes show higher magnification images of the regions indicated by white boxes. ce, corneal epithelium; le, lens epithelium; lf, fiber cells; lv, lens vesicle; s, corneal stroma. Bar= 50 µm.

Figure 6. The tight junction protein, ZO-1, is gradually lost from the apical surface of ScribLeCre lens epithelial cells

Longitudinally oriented, paraffin embedded sections of E13.5 embryos (A, B), E15.5 heads (C, D), and E17.5 eyes (E, F) from control (A, C, E) and ScribLeCre (B, D, F) mice were immunostained with anti-ZO-1 antibodies (green) and the nuclei counterstained with propidium iodide (red). A–B: At E13.5, ZO-1 staining was observed at the apical surfaces of epithelial cells in the control lenses (arrows) but was reduced or absent (B, arrow) on the apical surface of the ScribLeCre lenses. C–D: At E15.5, ZO-1 staining was observed at the epithelial-fiber interface in control lenses (C, arrows) but was reduced or absent (D, arrows) on the apical surface of the epithelial cells of the ScribLeCre lenses. E–F: At E17.5, punctate ZO-1 staining was observed on the apical membrane of the epithelial cells at the epithelial-fiber interface (E, arrows) but was further reduced or absent (F, arrows) on the apical membrane of the epithelial cells of the ScribLeCre lenses. The staining pattern in the lens fiber cells was also disrupted at E17.5. e, epithelium; f, fiber cells. Bar= 50 µm.

Figure 7. Downregulation of Pax6 and upregulation of αSMA and nuclear Smad4 are observed in the lens epithelium of E17.5 ScribLeCre embryos

Longitudinally oriented, paraffin embedded (A–D, I–L) or cryogenic (E–H) sections from eyes of E17.5 control (A, B, E, F, I, J) and ScribLeCre (C, D, G, H, K, L) embryos were immunostained for Pax6 (A–D, green), αSMA (E–H, red) or Smad4 (I–L, red) and the nuclei counterstained with propidium idodide (A, C, red) or To-Pro3 (E, G, I, K, blue). A–D: Pax6 staining was observed uniformly in the nuclei of lens epithelium from control mice (A, B) whereas the intensity of Pax6 staining was variable in the nuclei of the epithelium from ScribLeCre mice with some nuclei exhibiting markedly reduced staining (C, D) and some of these nuclei appeared flattened rather than rounded (C, D, arrowhead). E–H: Staining for αSMA was observed in the iris but not the lens of control eyes (E, F) whereas staining was observed in both the lens epithelium (arrows) and iris of the ScribLeCre eyes (G, H). Note that the iris does not extend across the entire ScribLeCre eye at E17.5 as it does in P10 ScribLeCre eyes (see Figure 2D). The hyperplastic iris was only observed in eyes of postnatal ScribLeCre mice. Red boxes show higher magnification images of regions indicated by white boxes. I–L: Smad4 staining (red) was cytoplasmic in control epithelial cells (I, J) whereas it was concentrated in the nuclei of some of the ScribLeCre epithelial cells (arrows) and the nuclei appeared flattened rather than rounded (K, L). e, epithelium f, fibers i, iris. Bar = 50µm.

Figure 8. Nuclear Smad3 and Snail are detected in lens epithelium of ScribLeCre mice

Longitudinally oriented, paraffin embedded eye sections from control (A, C, E, G) and ScribLeCre (B, D, F, H) E17.5 embryos (A, B, E, F) or P10 mice (C, D, G, H) were immunostained for Smad3 (A–D) or Snail (E–H) and counterstained with hematoxylin. A–D: Nuclear immunoreactivity for Smad3 was not observed in the lens epithelium of E17.5 (A) or P10 (C) control eyes whereas nuclear Smad3 staining was observed in some nuclei of the lens epithelium of E17.5 (B, arrows) and P10 (D, arrows) ScribLeCre mice. E–H: Nuclear immunoreactivity for Snail was not observed in nuclei of E17.5 (E) or P10 (G) control lens epithelium whereas nuclear staining was observed in ScribLeCre lenses at both E17.5 (F, arrows) and P10 (H, arrows). ce, corneal epithelium, le, lens epithelium; lf, lens fiber cells; s, corneal stroma. Bar = 25µm.

Figure 9. The corneal epithelium in the ScribLeCre mice acquires EMT characteristics

Longitudinally oriented, cryogenic sections from P10 control and ScribLeCre mice were immunostained for E-cadherin (A–D, green), cytokeratin 12 (K12, E–H, red) and αSMA (I–L, red). Nuclei were counterstained with propidium iodide (red, A, C) or To-Pro3 (blue, E, G, I–L). A–D: E-cadherin (green) is strongly expressed along all cell surfaces of the corneal epithelial cells of controls (A–B), but is weakly expressed or absent in the corneal epithelial cells from the ScribLeCre mice (D arrows). E–H: K12 (red) is expressed throughout the corneal epithelium of control mice but is reduced and discontinuous in corneal epithelium of ScribLeCre mice. (G, H, arrows). I–L: αSMA (red) was expressed only in ScribLeCre corneal epithelium and endothelium (K–L, arrows). Red boxes show higher magnification images of epithelium indicated by the white boxes. e, epithelium; s, stroma; en, endothelium Bars=50µm.