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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01181

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The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development

Christoph Rahner^*, Masayuki Fukuhara^*, Shaomin Peng, Shota Kojima and Lawrence J. Rizzolo

Department of Surgery, Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06520-8062, USA

View larger version (145K): [in a new window]   Fig. 1. Freeze-fracture replicas show the assembly of tight junctional strands during normal development. Sheets of RPE and choroid were isolated from embryos of the indicated age. Microvilli (m) at the top of each panel indicate the apical end of the lateral membrane. (A-C) For E7, no tight junctional strands are evident in (A), but gap junctions (g) are evident. Single strands (short arrows), associated with gap junctions at their tips, are evident in (B) and a patch of strands with open-ended strands (long arrows) is evident in (C). (D,E) For E10, two images show a loose network of strands. A group of strands is separated from the rest of the junction by discontinuities (arrowheads), and the overlying junction consists of only two strands (D). A discontinuity is embedded in a more continuous network (E). (F) For E14, all replicas exhibit a continuous network of anastomosing strands. Gap junctions are bounded by a strand on at least one side. EF, E-face; PF, P-face; Bar, 0.25 µm.

View larger version (18K): [in a new window]   Fig. 2. The number of strands parallel to the plane of the monolayer and the depth of the junctions increased between E10 and E14. The patches of strands evident on E7 were compared with the junctional networks of E10 and E14. The number of strands and the junctional depth (distance between the apical- and basal-most strands) increased during the intermediate phase of development to form a homogenous structure.

View larger version (69K): [in a new window]   Fig. 3. Expression of claudins in freshly isolated RPE, retina and choroid. (A,B) The RT-PCR reaction was used to amplify claudin mRNAs from the indicated tissues. The positions of standards derived from a Hae III digest of X174 (603, 281 and 118 base pairs) are indicated on the right. A single reaction product of the expected length was obtained in each case. No reaction product was detected when reverse transcriptase was omitted from the reaction (data not shown). (A) Ocular tissues. (B) Claudins undetected in ocular tissues could be amplified from other tissues. (C) Real-time RT-PCR was used to quantify the amount of claudin mRNA. Data points, with the standard error, represent the average of 6-9 experiments. (D) RPE stained for the indicated claudin were viewed en face by epifluorescence microscopy. Double staining with ZO-1 (not shown) was used to adjust the focal plane to the junctional region of the lateral membranes. Bar, 10 µm.

View larger version (125K): [in a new window]   Fig. 4. E14 retinal conditioned medium promotes the formation and assembly of tight junctional strands in RPE that is cultured in minimal medium. RPE was isolated from E7 (A,B) and E14 (C,D) embryos and cultured with SF3 medium in the basal chamber and either SF3 (A,C) or rcSF3 (B,D) in the apical chamber. (A) A loose, shallow network of strands was commonly observed in E7 cultures in SF3. (B) When rcSF3 was added to the apical medium chamber, the depth, complexity and number of strands parallel to the apical membrane increased. (C) A rare image from E14 cultures in SF3 shows a loosely organized cluster of junctional strands. (D) When rcSF3 was added to the apical medium chamber, continuous, well-organized networks of tight junctional strands were always observed. Arrowheads, discontinuity; EF, E-face; PF, P-face. Bar, 0.25 µm.

View larger version (23K): [in a new window]   Fig. 5. Quantification of the effects of rcSF3 on the morphology of tight junctions. Strand number (A,C) and depth (B,D) were determined for the E7 (A,B) and E14 (C,D) cultures depicted in Fig. 4. The apical medium chamber contained either SF3 () or rcSF3 (). In E7 cultures, the junctions were heterogeneous with respect to strand number and depth. Although the junctional networks remained heterogeneous, retinal conditioned medium increased the number of strands and the depth. In E14 cultures, the rare junctions that were observed in the absence of rcSF3 had a structure that was similar to those found in the presence of rcSF3. In E14 cultures, the principal effect was to convert the rare patches into the continuous, circumferential network that typifies tight junction morphology.

View larger version (185K): [in a new window]   Fig. 6. Effect of bovine pituitary extract and E14 retinal conditioned media on E7, E10 and E14 RPE in culture. RPE was isolated from E7 (A,B), E10 (C,D) and E14 (E,F) embryos and cultured with SF2 medium in the basal chamber and either SF3 (A,C,E) or rcSF3 (B,D,F) in the apical chamber. (A,C,E) Bovine pituitary extract (SF2) in the basal chamber supported an epithelioid morphology and tight junctions in each embryonic stage studied. (B,D,F) Retinal conditioned media (rcSF3) in the apical chamber induced higher complexity, and increased the number of strands and junctional depth in each culture. Discontinuities were not evident. Notably, E14 cells cultured in rcSF3/SF2 show the most in vivo-like tight junction structure with frequent gap junctions, and reduced tight junctional depth. Arrowheads, discontinuity; Arrow, free-floating strand. Bar, 0.25 µm.

View larger version (28K): [in a new window]   Fig. 7. The interaction of bovine pituitary extract and E14 retinal conditioned medium depended on the developmental stage of the RPE. Strand number (A,C,E) and depth (B,D,F) were determined for RPE that was isolated from E7 (A, B), E10 (C,D) and E14 (E,F) embryos and cultured with SF2 medium in the basal chamber and either SF3 () or rcSF3 () in the apical chamber. In E7 cultures, rcSF3 had minimal effects on strand number, but increased depth in some regions to create a bimodal distribution. In the E10 and E14 cultures, the effect of rcSF3 was to create a more homogeneous junction that was similar to those of E14 RPE in vivo. Notably, the E14 cultures in rcSF3 had the highest TER, but as in vivo (Fig. 2), had fewer strands and reduced depth.

View larger version (166K): [in a new window]   Fig. 8. Medium conditioned by embryonic fibroblasts (CEF) induced the formation of heterogeneous tight junctions. RPE was isolated from E14 embryos and cultured with CEF medium in the basal chamber and either SF3 (A) or rcSF3 (B) in the apical chamber. (A) CEF medium alone induced the formation of loosely organized junctions that could be very deep with many strands parallel to the plane of the monolayer. Discontinuities (arrowheads) were evident. (B) When rcSF3 was added to the apical medium chamber, strand number increased as the junctions became deeper and more complex. Discontinuities were evident in the basal portions of the junctions. A similar distribution and density of gap junctions and microvilli were observed in E14 RPE in vivo (Fig. 1F). g, gap junctions; m, microvilli. Bar, 0.5 µm.

View larger version (20K): [in a new window]   Fig. 9. Quantification of the effects of CEF and rcSF3 media on cultures of E14 RPE. Strand number (A) and depth (B) were determined for cultures depicted in Fig. 8. The basal medium chamber contained CEF medium, but the apical chamber contained either SF3 () or rcSF3 (). A continuous network of junctional strands induced by CEF, with some regions exhibiting very high number of parallel strands with great depth. The effect of rcSF3 was to increase strand number and depth. Curiously, there was a bimodal distribution of junctional strands in each growth condition. Nonetheless, the more prominent mode in rcSF3 resembled E14 RPE in vivo.

View larger version (72K): [in a new window]   Fig. 10. Expression of claudins in cultured RPE. RPE was isolated from E7 or E14 embryos and cultured for 9 days with the various media in the apical and basal medium chambers. Total RNA was isolated and analyzed using real-time RT-PCR (A-C). (A) Basal SF3 and apical rcSF3. (B) Basal SF2 and apical SF3 or rcSF3, as indicated. (C) Basal CEF conditioned medium and apical SF3 or rcSF3, as indicated. Data, with standard error, represent the average of 3-5 determinations. *Significant effect of rcSF3 (P<0.05); Significant difference between E14 and E7 RPE that were maintained in the same culture conditions (P<0.05). (D) Cultures stained for the indicated claudin were viewed en face by epifluorescence microscopy. Double staining with ZO-1 (not shown) was used to adjust the focal plane to the junctional region of the lateral membranes. Bar, 10 µm.

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