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Diffusible, retinal factors stimulate the barrier properties of junctional complexes in the retinal pigment
1Department of Anatomy and Cell Biology, Emory University, Atlanta, GA 30322.
Insights
Neural retinas release factors that promote the development of the blood-retinal barrier in the retinal pigment epithelium. These factors enhance tight junction formation and Na+/K+-ATPase polarization in cultured cells.
Area of Science:
- Developmental Biology
- Ophthalmology
- Cell Biology
Background:
- The blood-retinal barrier (BRB) is crucial for retinal function, formed by the retinal pigment epithelium (RPE).
- BRB integrity relies on polarized RPE cells with tight junctions and specific membrane protein distribution.
- These features develop gradually during chicken embryogenesis.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the gradual development of RPE polarity and barrier function.
- To identify diffusible factors from the neural retina that stimulate RPE barrier maturation.
Main Methods:
- Primary RPE cell culture on filters to assess barrier properties.
- Treatment of cultured RPE with conditioned medium from neural retinas of different embryonic ages.
- Measurement of transepithelial electrical resistance (TEER) and [3H]inulin diffusion to evaluate barrier function.
- Immunofluorescence to determine Na+/K+-ATPase localization.
Main Results:
- Early embryonic RPE cultures exhibited non-polarized Na+/K+-ATPase and leaky tight junctions (low TEER).
- Conditioned medium from embryonic neural retinas significantly increased TEER and improved Na+/K+-ATPase polarization in RPE cultures.
- The effectiveness of the conditioned medium increased with the age of the neural retina, suggesting age-dependent production of inductive factors.
Conclusions:
- Diffusible factors produced by the developing neural retina are essential for inducing RPE barrier maturation.
- These factors stimulate both tight junction closure and Na+/K+-ATPase polarization.
- The study highlights a paracrine signaling mechanism crucial for establishing the blood-retinal barrier during development.
Abstract:
The retinal pigment epithelium lies at the interface between the neural retina and the choriocapillaris where it forms a blood-retinal barrier. Barrier function requires a polarized distribution of plasma membrane proteins and 'tight' tight junctions. During chicken embryogenesis, these features develop gradually. Although terminal junctional complexes are established by embryonic day 4, the distribution of the Na+/K(+)-APTase is not polarized in all cells of the epithelium until embryonic day 11. Similarly, the tight junctions of early embryos are leaky, but become tight by hatching (embryonic day 21). We used primary cell culture to examine the molecular basis of this gradual induction of polarized function. Pigment epithelium harvested from embryonic day 7, and cultured on filters, formed monolayers coupled by junctional complexes. The distribution of the Na+/K(+)-ATPase was non-polarized and the tight junctions were leaky with a transepithelial electrical resistance of 20-30 omega cm2. To isolate diffusible factors that stimulate the transepithelial electrical resistance, neural retinas from embryonic day 7, 14 or 16 embryos were incubated at 37 degrees C in base medium for 6 hours. The conditioned medium was added to the apical chamber of freshly cultured pigment epithelium. The distribution of the Na+/K(+)-ATPase became basolateral, and the electrical resistance gradually increased two to three times over 6 days. The increase in electrical resistance corresponded to a decrease in the rate of [3H]inulin diffusion across the monolayer. The effectiveness of the conditioned medium increased steadily with increasing age of the neural retina. Rather than increased production of an active factor, apparently different active factors were produced at different ages.(ABSTRACT TRUNCATED AT 250 WORDS)