Constant lens fiber cell thickness in fish suggests crystallin transport to denucleated cells.

The crystalline lens of the vertebrate eye grows throughout life. This growth may be enormous in fish, while the lens must be functional from larva to adult. During growth, the fiber cells of the lens must increase the concentration of specific proteins (crystallins) in the cytoplasm to increase refractive index. However, the bulk of the fiber cells in a vertebrate lens are denucleated and have no organelles to synthesize proteins. To study how this problem is solved, we first measured lens fiber cell thickness in the Nile tilapia, a teleost fish. In the lenses from 25 fish, in two size groups, fibers were considerably thinner than in other vertebrates. Fiber thickness was about constant along the radius of the lens and the same between the size groups. Since our results provided no evidence for shrinkage of lens fiber cells with growth (expected if protein concentration is increased by expelling water) we included eight additional teleost species to elucidate the mechanism by which the cells increase crystallin concentration. In all species, fiber cell thickness was about constant throughout the lens, with species-specific values. The changes in fiber cell thickness expected from an increase in crystallin concentration by removal of water were modeled. Shrinkage in cell thickness by up to 66% would have been necessary to reach the required crystallin concentration. We conclude that crystallin concentration in denucleated lens fiber cells is increased by transport of proteins from synthetically competent cells in the periphery of the lens.

Visualization of adult fish lens fiber cells.

The crystalline lens of a vertebrate eye is a gradient-index lens and grows throughout life by addition of new lens fiber cells in the periphery. In fish, the growing ball-shaped lens maintains sophisticated optical properties throughout life by maintaining the distribution of refractive index relative to the increasing radius of the lens. During this process, the central fibers must increase refractive index by increasing the cytosolic concentration of crystallin proteins. However, only the youngest, most peripheral lens fiber cells have the ability to synthesize proteins. Unfortunately, the hardness of fish lenses makes investigation of the cellular anatomy impossible with traditional histological methods. We have developed a method for visualizing lens fiber cells across the diameter of the lens in adult fish. The method relies on sectioning embedded lenses with a high-speed power saw and observing the cut surface with a scanning electron microscope (SEM). The combination of SEM and image analysis allowed for precise tracking of the positions of individual cell fiber cells. As an application of the method, we present a cell thickness profile, i.e. the distribution of cells thicknesses and their relative positions along the lens's radius. Combined with detailed optical studies, which by mathematical reasons only are possible on ball-shaped lenses, our method can lead to new insights into the mechanism governing the functional and cellular development of vertebrate lenses.

Osmotic Concentration of Zebrafish (Danio rerio) Body Fluids Is Lower in Larvae than in Adults.

We intended to perform optical and structural measurements on larval zebrafish eyes at 5 days post fertilization, that is, the earliest age at which zebrafish show visually guided behavior. However, excised larval crystalline lenses deteriorated quickly if immersed in a medium that gives good results with adult lenses from a variety of fish species. We suspected that the larvae have body fluids of lower osmolality and tested a medium with 240 mOsm, which is 75% of the established adult value of 320 mOsm. The optical quality of freshly excised and immersed lenses was used to judge the osmotic matches. In addition, we tested how well the shape of the eye is preserved in fixatives of different osmolalities. In both cases, 240 mOsm produced the best results. Immersed lenses performed better and the fixed eyes had a more natural shape. Our findings indicate that zebrafish body fluids have lower osmolality in larvae than in adults. This is probably due to an unfavorable body surface-to-volume ratio and incompletely developed regulatory mechanisms. Body fluid osmolality deviating from the adult value has to be taken into account in optical and histological work.