Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area

Abstract

Purpose: The corneal endothelium is widely believed to consist of geometrically regular cells interconnected by junctional complexes. However, while en face visualization of the endothelial apical surface reveals characteristic polygonal borders, the overall form of the component cells has rarely been observed.

Methods: To visualize the shape of individual endothelial cells within the native monolayer, two independent Cre/LoxP-based cell labeling approaches were used. In the first, a P0-Cre mouse driver strain was bred to an R26-tdTomato reporter line to map neural crest-derived endothelial cells with cytosolic red fluorescent protein. In the second, HPRT-Cre induction of small numbers of green and red fluorescent protein-filled cells within a background of unlabeled cells was achieved using a dual-color reporter system, mosaic analysis with double markers (MADM). Selective imaging of the endothelial lateral membranes at different apicobasal levels was accomplished after staining with antibodies to ZO-1 and the neural cell adhesion molecule (NCAM).

Results: When viewed in their entirety in whole-mount preparations, fluorescent protein-filled cells appear star-shaped, extending multiple dendritic processes that radiate outward in the plane of the monolayer. Examination of rare cases where cells expressing different fluorescent proteins lie directly adjacent to one another reveals that these long processes undergo extensive interdigitation. The resulting overlap allows individual cells to extend over a greater area than if the cell boundaries were mutually exclusive. Anti-NCAM staining of these interlocking peripheral cell extensions reveals an elaborate system of lateral membrane folds that, when viewed in optical sections, increase in complexity from the apical to the basal pole. This not only produces a substantial increase in the basolateral, relative to the apical, membrane but also greatly extends the paracellular pathway as a highly convoluted space.

Conclusions: Our analysis indicates that, far from being simple polygonal prisms, endothelial cells possess an elaborate multipolar shape. Their unusual geometry may be essential for the endothelium to carry out its role as the principal regulator of corneal extracellular fluid flux, and thus ultimately of tissue clarity.

Figure 1

Reporter gene expression in mouse corneal endothelial cells. A–C: P0-Cre-driven expression of tdTomato. A: In this low-power confocal projection image, the labeled cells exhibit irregular projections that extend in the plane of the monolayer. Asterisks mark areas of non-expressing cells, one of which (indicated by the box) is viewed at higher power in (B) and (C). B: Optical section at 0.3 µm from the apical pole reveals the linear borders of cells where they are connected by junctions (arrows). C: At 2.1 µm relative to the apical pole, fluorescent protein expression fills the pseudopod-like processes (arrowheads) that extend into the territory of a non-expressing cell. D-H: HPRT-Cre-mediated mosaic analysis with double markers (MADM) labeling. D: Cells marked due to interchromosomal recombination events (see Methods) are scattered among mainly unlabeled endothelial cells in this cornea flatmount viewed at low magnification. This overlay image shows green fluorescent protein (GFP)-expressing (green) cells, c-Myc-red fluorescent protein (RFP)-expressing (red), and cells expressing both fluorescent proteins (yellow). E–G: A field of view similar to that in D is seen at higher power. Where a green cell and a yellow cell occur closely adjacent to one another, their processes can be seen to overlap and interdigitate (arrow in E). Images collected in the RFP and GFP channels (F and G, respectively) compose the overlay image in (E). H: In this high-magnification image of nearby but non-adjacent MADM-labeled cells, the distinctive star-shaped morphology of corneal endothelial cells, with their branched dendritic processes, can be clearly appreciated. Images D–H represent projections of all optical sections in the data set.

Figure 2

Gallery of endothelial cell surface representations obtained following 3D reconstruction from confocal image stacks. A–F: Viewed from their anterior surface at 3/4 perspective, mosaic analysis with double markers (MADM)-labeled corneal endothelial cells present flat apical membranes in the form of a plateau region (indicated by asterisks in B, D, and F) that overlaps and partially obscures the dendritic lateral processes. In some cases, the processes can be seen to taper as they extend peripherally and ultimately give rise to smaller branches (arrows in A and C).

Figure 3

Immunolocalization of corneal endothelial cell lateral membrane markers by widefield fluorescence microscopy. A: Reaction of intact tissues with ZO-1 antibody highlights the regular polygonal outlines of cells seen at the level of tight junctions. B: Staining for the cell adhesion protein neural cell adhesion molecule (NCAM), however, reveals that much of the interacting surface is in the form of a complex arrangement of membrane folds. When viewing whole cells, this gives the impression of extensive membrane ruffling.

Figure 4

Variation in intercellular boundaries viewed at different apicobasal levels. A: ZO-1 antibody delineates the borders of individual cells near the apical corneal endothelial cell (CEC) surface (0.3 µm from the apical pole). B–D: Imaging of anti-neural cell adhesion molecule (NCAM)-labeled membranes at (B) 0.3 µm, (C) 1.2 µm, and (D) 2.1 µm from the apical pole. In (B), the cell boundaries outlined by the NCAM antibody deviate somewhat from those visualized with anti-ZO-1 labeling. A progressive expansion of the CEC lateral membranes is seen as the plane of the optical section passes nearer to the endothelium basal pole (C, D). E: Boxed area in panel (A) overlaid with the corresponding region from panel (D), shown at higher magnification. Note that the subapical NCAM-containing membranes form expansions that extend centrally and peripherally to the apical (anti-ZO-1-labeled) borders. Using the overlay in (E), outlines of a single cell have been drawn at its apical and basal poles (F). All images represent single confocal optical sections.