The tumor suppressor merlin is required for cell cycle exit, terminal differentiation, and cell polarity in the developing murine lens

Abstract

PURPOSE. Neurofibromatosis type 2 (NF2) is an autosomal-dominant CNS tumor syndrome that affects 1:25,000 children and young adults. More than 50% of NF2 patients also develop posterior subcapsular cataracts (PSCs). The authors deleted Nf2 from the lens to determine its role in fiber cell differentiation. METHODS. Nf2 was conditionally deleted from murine lenses using the LeCre transgene. Standard histology and immunohistochemical and immunofluorescent methods were used to analyze lens morphology and markers of cell cycle progression, differentiation, and cell junctions in wild-type and knockout lenses from embryonic day 10.5 through postnatal day 3. RESULTS. Fiber cells lacking Nf2 did not fully exit the cell cycle and continued to express epithelial cell markers, such as FoxE3 and E-cadherin, despite expressing the fiber cell marker Prox1. Many fiber cells lost their elongated morphology. Markers of apical-basal polarity, such as ZO-1, were mislocalized along the lateral and basal membranes of fiber cells. The lens vesicle failed to separate from the surface ectoderm, and prospective lens and corneal epithelial cells formed a multilayered mass of cells at the surface of the eye. Herniation of this membrane caused the fiber mass to erupt through the cornea. CONCLUSIONS. Nf2 is required for complete fiber cell terminal differentiation, maintenance of cell polarity, and separation of lens vesicle from corneal epithelium. Defects identified in fiber cell differentiation may explain the formation of PSCs in patients with NF2. The lens provides an assay system to identify pathways critical for fiber cell differentiation and to test therapies for the tumors that occur in patients with NF2.

Figure 1.

Nf2^CKO lenses typically herniated through the area of lens-corneal fusion. (A) By E15.5, Nf2^WT lenses show clear separation between the developing lens and the cornea. (B) Nf2^CKO lenses usually developed lens herniation; fiber cells were expelled through the perforated cornea. (C) At P3, Nf2^WT lenses were normal. (D, D′) Nf2^CKO pups typically are born with abnormally small, disrupted lenses. Higher magnification shows the lens herniation phenotype. Expelled lens material is trapped between the cornea and the closed eyelids (asterisk). Sections in (A) and (B) were labeled with antibody to BrdU. Scale bars: 50 μm (A, B), 200 μm (C, D), 50 μm (D′, area highlighted by the black box in D). c, cornea; Le, lens; El, eyelid; R, retina.

Figure 2.

Nf2^CKO lens fiber cells failed to withdraw from the cell cycle. (A) At E11.5, BrdU labeling was restricted to epithelial cells in Nf2^WT lenses. (B) In Nf2^CKO lenses. BrdU labeling was present in fiber cells throughout the posterior fiber region. (C) Staining for Ki67 was seen only in anterior lens epithelia in Nf2^WT lenses at E13.5. (D) Ki67-positive cells were seen throughout the posterior region in Nf2^CKO lenses. (E) At E11.5, phospho-histone H3 staining was seen only at the apical ends of epithelial cells in Nf2^WT lenses. (F) Phospho-histone H3-positive mitotic figures were present in the fiber mass of Nf2^CKO lenses (arrowheads). (G) At E11.5, Nf2^WT lenses displayed normal levels of apoptosis that accompanied normal development (arrowhead). (H) Nf2^CKO lenses showed increased TUNEL labeling, indicating increased apoptotic cell death, compared with Nf2^WT lenses (arrowheads). Scale bars: 25 μm (A–D, G, H), 50 μm (E, F).

Figure 3.

Most Nf2^CKO fiber cells express fiber-specific differentiation markers, but many coexpress epithelium-specific proteins. (A) At E13.5, high levels of p57^KIP2 were present in nuclei at the lens equator and throughout the posterior fiber region of Nf2^WT lenses. (B) In Nf2^CKO lenses, many cells had weak expression or lacked detectable p57^KIP2 (arrow and arrowheads). (C) In E12.5 Nf2^WT lenses, antibodies to Prox1 labeled the nuclei of cells near the equator and throughout the posterior fiber region. (D) As in WT lenses, Prox1 staining was first detected in nuclei at the equator of Nf2^CKO lenses. Careful examination revealed that all cells in the fiber region of the knockout lenses expressed Prox1, though levels were low in some cells. (C′) FoxE3 staining was present in the nuclei of all Nf2^WT epithelial cells. Notably, several nuclei near the lens equator were stained with both antibodies. (D′) Antibodies to FoxE3 stained many cells in the posterior fiber compartment of these lenses. Therefore, all fiber cells in Nf2^CKO lenses that expressed FoxE3 were also Prox1-positive. (E) In Nf2^WT lenses, c-Maf is strongly expressed in the nuclei of all elongating fiber cells. (F) In Nf2^CKO lenses, c-Maf is also localized to the nuclei of many fiber cells. However, there are many fiber cells that display either weak labeling or that lack positive labeling for c-Maf (arrowheads). This is especially evident in the clusters of cells near the equatorial region (arrow). Even when the images were contrast enhanced to give the impression of overstaining, the fiber cells in Nf2^CKO lenses appeared unstained (arrow and arrowheads). (G, H) At E12.5, antibody to MIP stained fiber cells in Nf2^WT and Nf2^CKO lenses. However, not all Nf2^CKO cells in the fiber compartment were stained. Cells in the epithelioid clusters near the equator had little or no detectable MIP staining. Scale bars: 25 μm (A, B, E, F), 50 μm (C, C′, D, D′), 20 μm (G, H).

Figure 4.

Nf2^CKO lens fiber cells retained expression of the epithelial cell marker E-cadherin, and some lost their elongated morphology. (A) Staining WT lenses with fluorescent-conjugated phalloidin to label F-actin showed prominent actin staining throughout the lens. Higher levels of F-actin were especially visible at the apical ends of both epithelial and fiber cells. E-cadherin expression labeled cell–cell junctions in the epithelial layer but was absent from differentiated fiber cells (arrow). F-actin colocalized with E-cadherin at the apical ends of epithelial cells. (B) F-actin was also more prominent at the apical ends of elongating fibers cells in Nf2^CKO lenses. F-actin was typically seen along the lateral membranes and at the basal ends of Nf2^CKO fiber cells. The image shown here is an extreme case of apical F-actin in Nf2^CKO lenses, but it also shows double labeling of F-actin and E-cadherin. In Nf2^CKO lenses, E-cadherin expression was retained in many fiber cells. Although not completely colocalized, there were areas in the fiber region of CKO lenses in which F-actin colocalized with E-cadherin (arrows). (C) In Nf2^WT lenses, β-catenin labeled cellular junctions throughout the lens and specifically colocalized with E-cadherin in the epithelium. (D) In Nf2^CKO lenses, E-cadherin that was retained in the posterior fiber zone was completely colocalized with β-catenin and in the epithelium, as would be expected (arrows). (E, F) There were no notable differences in the amount and organization of F-actin, β-catenin, or N-cadherin between Nf2^WT and Nf2^CKO lenses. Scale bars: 50 μm (A, B, E, F); 20 μm (C, D).

Figure 5.

Nf2^CKO lens cells lost apical-basal polarity. (A) Staining for ZO-1 was restricted to the apical ends of WT cells in the invaginating lens vesicles. (B) In Nf2^CKO lens vesicle cells, ZO-1 was also present along the lateral (arrow) and basal (arrowheads) ends. (C) At E12.5, ZO-1 was expressed at the apical ends of epithelial and fiber cells of Nf2^WT lenses, especially near the lens equator (arrowheads). (D) In Nf2^CKO lenses, ZO-1 was expressed throughout the fiber region, especially along fiber cell lateral membranes (arrowheads). Scale bars: 20 μm (A, B); 50 μm (C, D).

Figure 6.

Diagram of lens vesicle formation. (A) At E10, the lens placode invaginates, forming the lens pit. The letters A and B represent cells located on either side of the lens pit. (B) At E10.5, the sides of the lens pit fuse. (C) By E11, the lens vesicle separates from the surface ectoderm that will become the corneal epithelium. This process involves the fusion of epithelia, the exchange of cell neighbors (some A cells now lie next to B cells) with concomitant destruction and reformation of cell–cell junctions, the destruction of part of the original basal lamina, and the production of a new basal lamina where the epithelia fuse and separate.