Related Experiment Videos
A culture model of development reveals multiple properties of RPE tight junctions
1Department of Surgery, Yale University, New Haven, CT 06520-8062, USA.
Insights
Tight junctions in retinal pigment epithelium (RPE) develop independent charge and size discrimination during development. RPE barrier function changes are not predicted by ZO-1 protein levels.
Area of Science:
- Ophthalmology
- Cell Biology
- Physiology
Background:
- The retinal pigment epithelium (RPE) forms a critical barrier in the eye.
- Tight junctions (TJs) are key structures regulating RPE barrier function.
- Understanding TJ development is crucial for ocular health.
Purpose of the Study:
- To investigate the developmental regulation of tight junction barrier properties in the retinal pigment epithelium (RPE).
- To examine how RPE tight junctions discriminate between solutes based on size and charge during development.
- To determine the relationship between TJ protein expression and barrier function.
Main Methods:
- Chick RPE cells were cultured at different embryonic ages (E7, E10, E14).
- Barrier properties were assessed using permeability assays (horseradish peroxidase, inulin, mannitol) and transepithelial electrical resistance (TER).
- Expression of TJ proteins ZO-1 and ZO-1LP was analyzed by immunoblotting.
Main Results:
- RPE cultures exhibited age-dependent decreases in permeability, with distinct regulation of ion and non-ionic solute flux.
- Developing RPE tight junctions demonstrated independent discrimination of solutes by size and charge.
- Retinal conditioned medium reduced permeability but did not alter ZO-1 or ZO-1LP expression.
Conclusions:
- Tight junction charge and size discrimination in RPE develop independently.
- RPE barrier function development is not solely dependent on ZO-1 expression levels.
Purpose:
A culture model was used to examine the development of tight junctions in the retinal pigment epithelium (RPE).
Methods:
Chick RPE was isolated on embryonic day 7 (E7), E10 or E14 and cultured on laminin-coated filters. Barrier properties were stimulated with E14 retinal conditioned medium. Morphology was characterized by confocal microscopy. Permeability was determined by measuring the flux of horseradish peroxidase (HRP), radiolabeled inulin and mannitol, and the transepithelial electrical resistance (TER). Changes in the expression of ZO-1 and a related protein, ZO-1LP, were determined by immunoblotting.
Results:
RPE from each age formed epithelial monolayers of similar height, but the density of the cultures varied in parallel with density changes in vivo. The cultures appeared to regulate the permeability to ions and nonionic solutes independently. With embryonic age, there was a progressive decrease in permeability that first affected larger and then smaller tracers. Despite a small decrease in the permeability to mannitol, there was a large decrease in the permeability to ions. This suggests that in E14 cultures tight junctions discriminated by charge, as well as size. Although E14 retinal conditioned medium reduced the permeability to all solutes, it appeared to regulate size discrimination more than charge discrimination. Despite large effects on permeability, conditioned medium had no effect on the expression of ZO-1 or ZO-1LP.
Conclusions:
The ability of tight junctions to discriminate on the basis of charge and size is regulated independently during development. The permeability of tight junctions cannot be predicted by the level of ZO-1 expression.