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First published online 17 October 2006
doi: 10.1242/jcs.03202

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Connexin43 is required for production of the aqueous humor in the murine eye

¹ Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
² Department of Neurobiology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
³ Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA

View larger version (104K): [in a new window]   Fig. 1. Localization of the ß-galactosidase reaction product in 5.5-week-old nestin-cre/ROSA26 mice. (A) At low power, the blue reaction product is evident in the retina, lens and ciliary body. Owing to the heavily pigmented PE and iridal epithelial cells, the reaction product is difficult to see in these locations. (Inset A) Control specimen showing no reaction product. (B) At higher magnification, ß-galactosidase activity is clearly visible in the NPE cells (black arrows) and, with favorable planes of section, in the PE cells (white arrows). The zonular fibers joining the ciliary body to the lens are indicated.

View larger version (106K): [in a new window]   Fig. 2. Nestin-cre reduces Cx43 expression in the ciliary epithelium and iris by embryonic day 16-18. Eyes from nestin-cre/Cx43+/flox (A,B) and nestin-cre/Cx43flox/flox (C,D) mice were harvested, treated and sectioned as described in Materials and Methods section. Photomicrographs of immunostaining using anti-Cx43 antibody (A,C) and H and E stained (B,D) sections from the same specimens were taken. (A) In the heterozygote, Cx43-positive staining is seen in the ciliary epithelium (CE) and presumptive iris (PI) and in the corneal epithelium (ep) and endothelium (en) and the retinal pigment epithelium (RPE). The lens staining is non-specific (see Fig. 3). (C) In the homozygote, a reduction of staining is evident in the ciliary epithelium and presumptive iris. Bars, 20 µm.

View larger version (117K): [in a new window]   Fig. 3. Immunohistochemistry on paraffin sections of Cx43+/– (A) and Cx43–/– (B) eyes from E15 mouse embryos using the Sigma anti-Cx43 reagent. (A) In the heterozygote, strong signal is seen in the lens, ciliary body (CB), RPE (arrows, a), corneal epithelium (arrows, b). (B) In the homozygous Cx43 knockout, the lens staining persists, indicating a non-specific signal. The staining is completely removed, however, from the ciliary body, cornea and RPE.

View larger version (91K): [in a new window]   Fig. 4. Fluorescence images of stripped capsules with attached lens epithelial cells from 6-week-old nestin-cre positive Cx43+/flox (A,B) and Cx43flox/flox (C,D) lenses. Cx43 is stained with the Chemicon anti-Cx43 reagent (A,C) and nuclei are visualized with Hoechst staining (B,D). There is a loss of the punctate gap junctional staining in the homozygote (C) demonstrating loss of Cx43 from the lens epithelium.

View larger version (108K): [in a new window]   Fig. 5. Cx43 expression is abolished by nestin-cre in the pigmented epithelium of the ciliary body and retinal pigment epithelium of Cx43flox/flox mice. At P0 (B) and at P14 (D) a loss of detectable Cx43 staining is observed in the pigmented epithelium (PE) but not in the non-pigmented epithelium (NPE) of the ciliary processes in the nestin-cre+/Cx43flox/flox (B,D) as compared with the nestin-cre+/Cx43+/flox mice (A,C). Cx43 staining in the homozygote is also lost in the retinal pigment epithelium (arrows, A,B). There is no apparent change in Cx43 immunoreactivity in the lens due to the non-specific staining illustrated in Fig. 3. Bars, 50 µm (A,B); 20 µm (C,D).

View larger version (90K): [in a new window]   Fig. 6. Eyes from Cx43-deficient mice display structural changes. Eye sections from nestin-cre/Cx43+/flox (A,C) and nestin-cre/Cx43flox/flox (B,D) mice at P7 were used for H and E staining (A,B) and anti-Cx43 immunostaining (C,D). (A,B) Clear changes are evident in the anterior eye in the homozygote at 1 week of age. The posterior pigmented epithelium of the iris detached from the myoepithelium, leaving a precipitate-filled space (B, asterisk). A precipitate is also visible in the anterior chamber (B, double asterisk) and in the posterior chamber adjacent to the ciliary body and ora serrata (B, triple asterisk). The insets A and B compare the ciliary body, lens bow (lb) and iris close to the corneal angle (CA). In the homozygote, there is a variable separation of the PE from the NPE leaving clear vacuoles (arrows, inset B). (C,D) Immunostaining reveals a loss of Cx43 from the pigmented epithelium (PE) and from the iris (which has split, leaving the posterior pigmented epithelium on the surface of the lens. Bars, 100 µm (insets at the same magnification).

View larger version (91K): [in a new window]   Fig. 7. Ablation of Cx43 by nestin-cre causes a complete loss of the vitreal space and alterations in the ciliary body, retina and lens. (A) At 5 weeks of age, the nestin-cre+/Cx43flox/flox eye shows a precipitated material in the anterior chamber beneath the cornea. The vitreal space is almost completely absent, with the lens in close apposition to the inner limiting membrane of the retina. The iris remains separated into two layers, the posterior pigmented epithelium adherent to the anterior surface of the lens and to the vitreal side of the ciliary body. The retinal layers are wavy and non-uniform in thickness. (B) At higher power, clear lakes have appeared in the lens where lens fibers have fused. The ciliary body appears disorganized, with a loss of recognizable ciliary processes. The posterior pigmented epithelium of the iris can be seen more clearly adherent to the anterior surface of the lens. Both A and B are sectioned para-saggitally to the pupil. Bars, 100 µm (A); 50 µm (B).

View larger version (58K): [in a new window]   Fig. 8. Silver-stained SDS-PAGE of aqueous humor from Cx43+/flox (lane A) and Cx43flox/flox (lane B) reveals increased protein levels in the homozygote. Numbers on the right indicate the locations of the molecular weight standards. Arrows NC 1-7 indicate locations were the gel was sampled for mass spectroscopy. The MS results are shown in the supplementary material Table S1, corresponding to each of the NC samples.

View larger version (110K): [in a new window]   Fig. 9. Cx43 inactivation results in back-diffusion of plasma proteins from the veins into the eye chambers. (A) HRP reaction product stains the subendothelial connective tissue and vasculature in the stroma of the ciliary body (asterisk arrows) of a nestin-cre+/Cx43+/flox mouse as visualized by light microscopy. The HRP is also visible in an iridal artery. (B) In a nestin-cre+/Cx43flox/flox mouse, the HRP has entered the anterior chamber where H and E staining revealed a precipitate (see Fig. 6B and Fig. 7). HRP was contained in iridal blood vessels, notably in an indicated artery. Inset: electron microscopy demonstrates that the blood-ocular barrier at the level of the iridal (inset a) and retinal (inset b) blood vessels was not disrupted by Cx43 removal. These images show capillaries exhibiting containment of the HRP reaction product within the lumen of the vessel. The iridal vessel contains a peroxidase-positive erythrocyte. Bars, 20 µm (A); 10 µm (B); 500 nm (insets).

View larger version (168K): [in a new window]   Fig. 10. Tight junctions are functional at 2.5 weeks in the Cx43-deficient mice. The figure and inset B are from a nestin-cre+/Cx43+/flox animal, and inset A is from a nestin-cre+/Cx43flox/flox mouse. Five minutes following intravenous HRP injection, the reaction product can be seen by electron microscopy in stromal blood vessels of the ciliary body (BV), in the intercellular spaces between pigmented epithelial (PE) cells (white arrow) and at the interface between the (PE) and non-pigmented epithelium (NPE) (black arrows). In the insets, HRP diffusion can be seen blocked by tight junctions (TJ) so that no reaction product can be seen in the intercellular spaces either between the NPE cells (asterisks) or in the posterior chamber (PC). Bars, 500 nm.

View larger version (118K): [in a new window]   Fig. 11. A representative example of the PE/NPE interface in nestin-cre+/Cx43+/flox (A) and nestin-cre+/Cx43flox/flox (B) ciliary bodies of 2.5-week-old animals. In the heterozygote, gap junctions between PE and NPE are very frequent, as indicated by the black arrows. Less frequently, gap junctions can also be observed between NPE cells (white arrows). In the homozygote, gap junctions are not observed at the PE/NPE interface. As described in Fig. 10, tight junctions (TJ) can also be seen limiting the diffusion of HRP between the NPE cells (asterisks).

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