Development of a macromolecular diffusion pathway in the lens

Abstract

The mammalian lens consists of an aged core of quiescent cells enveloped by a layer of synthetically active cells. Abundant gap junctions within and between these cell populations ensure that the lens functions as an electrical syncytium and facilitates the exchange of small molecules between surface and core cells. In the present study, we utilized an in vivo mouse model to characterize the properties of an additional pathway, permeable to macromolecules, which co-exists with gap-junction-mediated communication in the lens core. The TgN(GFPU)5Nagy strain of mice carries a green fluorescent protein (GFP) transgene. In the lenses of hemizyous animals, GFP was expressed in a variegated fashion, allowing diffusion of GFP to be visualized directly. Early in development, GFP expression in scattered fiber cells resulted in a checkerboard fluorescence pattern in the lens. However, at E15 and later, the centrally located fiber cells became uniformly fluorescent. In the adult lens, a superficial layer of cells, approximately 100 μm thick, retained the original mosaic fluorescence pattern, but the remainder, and majority, of the tissue was uniformly fluorescent. We reasoned that at the border between the two distinct labeling patterns, a macromolecule-permeable intercellular pathway was established. To test this hypothesis, we microinjected 10 kDa fluorescent dextran into individual fiber cells and followed its diffusion by time-lapse microscopy. Injections at depths of >100 μm resulted in intercellular diffusion of dextran from injected cells. By contrast, when injections were made into superficial fiber cells, the injected cell invariably retained the dextran. Together, these data suggest that, in addition to being coupled by gap junctions, cells in the lens core are interconnected by a macromolecule-permeable pathway. At all ages examined, a significant proportion of the nucleated fiber cell population of the lens was located within this region of the lens.

Connexin 46 (Cx46) Gap Junctions Provide a Pathway for the Delivery of Glutathione to the Lens Nucleus

“…Although it is possible that gap junctions may allow diffusion of the divalent ion to the periphery from the center because the coupling conductance of fiber cells is typically very high, other pathways for GSSG efflux/ breakdown might operate in the lens. Cell-cell diffusion of molecules in the lens may also be mediated by membrane fusions between adjacent fiber cells (39,40). Unlike gap junction channels, which exclude molecules above 1 kDa, membrane fusions can allow the diffusion of very large molecules such as green fluorescent protein (GFP) and Alexa 488 conjugated to dextran between adjacent cells (39,40).…”

“…Cell-cell diffusion of molecules in the lens may also be mediated by membrane fusions between adjacent fiber cells (39,40). Unlike gap junction channels, which exclude molecules above 1 kDa, membrane fusions can allow the diffusion of very large molecules such as green fluorescent protein (GFP) and Alexa 488 conjugated to dextran between adjacent cells (39,40). These fusions have been identified in adult lenses from several species, including humans and rodents, and require the presence of Lim2 (also known as MP20) (41).…”

Signaling through FGF receptor‐2 is required for lens cell survival and for withdrawal from the cell cycle during lens fiber cell differentiation

“…As fiber cells differentiate, they rapidly increase in length and volume and accumulate high levels of crystallins, the proteins that account for the transparency and high refractive index of the lens. After they complete the process of elongation, fiber cells partially fuse with their neighbors and degrade all membrane bound organelles (Bassnett, 2002;Shestopalov and Bassnett, 2003).…”

Inducible gene expression in the lens using tamoxifen and a GFP reporter

“…Thus, it appears that the mosaicism of the Z/EG transgene places an upper limit on the number of cells that express GFP following tamoxifen treatment. Mosaic expression of transgenes in the lens is not uncommon (Shestopalov and Bassnett, 2003) and limits the utility of the Cre-ER ™ ;Z/EG system in this tissue. However, for our purposes, which involve labeling individual cells within the intact lens at specific stages of development, the relatively low induction efficiency was advantageous.…”

“…Fiber cell GFP expression was also evident at later stages, but the fluorescence was diffuse in fibers located deep within the lens. This may reflect the assimilation of those cells into the lens syncytium, a region of the lens core in which proteins are thought to diffuse between cells, perhaps as a result of the partial fusion of neighboring fibers (Shestopalov and Bassnett, 2003).…”