Our protocols provide a starting point for functional studies of lens development and physiology and for reverse genetic screening of lens phenotypes in zebrafish. Our protocols bridge in vitro and in vivo analyses of zebrafish lens membrane morphology and provide a new and straightforward method for quantifying the development of zebrafish lens optics. Analyzing zebrafish lens morphology and optics leads to insights into mechanisms controlling lens development, normal physiology, and pathophysiology.
These insights can in turn lead to an ability to delay or prevent cataract. There have been no reports in species other than zebrafish of asymmetrically localized lens nuclei. Identifying lens sutures is critical for correctly orienting the lens to measure nucleus localization.
After confirmation of sufficient sedation with Tricaine, use microdissection scissors to immediately excise both eyes from either an adult or a larval zebrafish and place both eyes into a 35-millimeter custom-made Petri dish with a silicone dissection mold filled with PBS. To harvest the lens from an adult zebrafish, place the eye posterior side up and insert forceps at a less than 45-degree angle through the optic disc. Use dissection scissors to make two or three radial incisions through the retina and sclera from the optic disc to the ciliary zone in the immobilized eye and peel back the retina and sclera like flower petals.
Invert the eye, cornea-side up, and use the flat side of the scissors to immobilize the lens indirectly via manipulation of the sclera and cornea by using forceps to pull away the retina and attached tissues, then carefully trim away any excess tissue from the lens. For larval zebrafish lens harvest, place a larval eye posterior side up onto the flat part of a silicone dish of PBS and use a sharpened tungsten needle to make radial cuts through the retina and sclera while immobilizing the eye with another needle or forceps, then gently scoop the lens from the dissociated eye with the blunt side of a custom-made tungsten-wire needle and carefully pull away the attached tissue. To measure the anterior-posterior axis lens nucleus localization, orient the freshly-excised lenses axially in PBS in a 35-millimeter dish with a cover glass bottom with the poles and sutures oriented parallel to the plane of focus.
To identify the lens nucleus, look for a difference in the refractive index, which usually occurs at the interface of the lens cortex and lens nucleus. If lens sutures are not apparent, a slight lick with forceps to the lens capsule reveals the sutures and the lens nucleus, helping orient the lens for the measurement. Place the dish on the stage of a dissection microscope equipped with a camera and image the lenses with the lens nucleus in focus under brightfield illumination or with differential interference contrast optics.
Image a micrometer under the same magnification for calibration and click Live View. Click Snapshot to obtain an image and save the image as a TIFF file. To calibrate the acquired lens images, in an appropriate image-processing software program, select the straight line tool and draw a line of known length on the micrometer image.
Click Analyze and Set Scale and enter the known distance and units, then select Global calibration and click OK.To measure the distance from the center of the lens nucleus to the anterior pole, identify the location of the lens suture and use the straight line tool to draw a line across the imaged center of the lens nucleus in an axial orientation. The center of this line is the center of the lens nucleus. Draw another line from this point to the anterior pole of the lens and select Measure in the Analyze menu to measure the distance.
Draw another line from the anterior pole to the posterior pole and measure this distance as the lens diameter, then copy these lengths from the Results window to a spreadsheet and calculate the normalized localization of the lens nucleus with respect to the lens radius. By three days post-fertilization, anterior sutures form at the anterior pole and the striking convergence of cells is clearly visualized by phalloidin staining of the narrow fiber cell membranes in fixed embryos. Stronger broad-fiber cell labeling in lens membrane localized mApple transgenic allows live visualization of membrane subdomains but lacks the sutural convergence.
Posterior sutures can be visualized both in vitro and in vivo at three days post-fertilization. In an equatorial plane, phalloidin strongly labels the outer cortical fiber cells, revealing a flattened hexagonal shape. Phalloidin is excluded from the compacted lens nucleus and wild type and mutant lens look indistinguishable.
The strong labeling of broad fiber cells in lens membrane-localized mApple mosaics transgenics reveal disruptions in cell volume within the mutant lens. As transgene expression is not limited by permeability, some mosaics reveal lens nucleus labeling. In wild type animals, the lens nucleus starts closer to the anterior lens pole during the early stages of development before centralizing during adulthood.
Expression of green-tagged proteins in a transgenic host in which the cell membranes fluoresce red allows simultaneous protein localization as well as assessment of fiber cell morphology in vivo. Zebrafish are especially well suited for in vivo studies. Using the tools described here, we can probe lens mechanisms, largely studied in vitro, in an in vivo system.
