This set of standardizes protocols can be used to offer new insight into the superior ocular sulcus, or SOS, a recently discovered structure of the developing eye. These protocols will allow any experimenter around the world to clearly visualize the SOS during zebrafish eye development. Begin by placing heterozygous growth differentiation factor six A male and female zebrafish on separate sides of a divider in a tank of dechlorinated water the evening before their pairing.
The next morning, remove the divider and allow the fish to breed for no more than 30 minutes. At the end of the breeding period, collect the embryos into Petri dishes containing E3 medium for their incubation in a 28.5 degree incubator. At 20 hours post-fertilization, replace the supernatant with E3 medium supplemented 0.004%1-Phenyl-2-thiourea to prevent pigment production and examine the embryos by light microscopy to confirm they are all at the appropriate developmental stage.
Place the samples under a dissecting microscope. Discard any developmentally immature embryos and use fine forceps to gently pull apart the chorions. Visualize the SOS, which may appear as an indentation or line at the dorsal margin of the eye.
Then, sort the embryos that show SOS closure delay from those that do not. To photograph these embryos using a dissecting microscope, prepare a Petri dish containing 1%agarose in E3 medium and lightly prick the center of the agarose to create a shallow hole into which the yolk of the embryo can be placed. Then, place an anesthetized embryo laterally onto the agarose.
To image the embryos using a compound or confocal microscope, transfer an embryo into a 35 millimeter Petri dish containing a small bolus of non-gelled 1%low melting point agarose into E3 medium and use fine fishing line to quickly position the embryo in the lateral position. When the agarose has cooled, pour enough E3 medium into the dish to cover the agarose and use a 20x water immersion objective lens to visualize the SOS. After imaging, gently pull the agarose from the embryo and fix the tissue in 4%paraformaldehyde or allow the zebrafish to continue its development.
To visualize the SOS by fluorescent mRNA expression, use a microinjection apparatus to inject 300 picograms of enhanced GFP-CAX mRNA into embryos at the one-cell stage. Then, use a fluorescent stereoscope to screen for embryos with bright enhanced GFP expression and obtain images of the embryos by one of the imaging methods as demonstrated. For whole-mount immunofluorescent staining of embryonic zebrafish laminin, dechorionate the embryos as demonstrated and fix the embryos in a microcentrifuge tube in freshly prepared 4%paraformaldehyde for two hours on a room temperature shaker.
At the end of the fixation, wash the embryos four times in PBS plus Tween, or PBST, for five minutes per wash followed by permeabilization in 10 micrograms per milliliter or Proteinase K at room temperature for five minutes. Wash the permeabilized embryos four times in PBST as demonstrated and block the embryos in 5%goat serum and two milligrams per milliliter of bovine serum albumin in PBST for one to two hours on a room temperature shaker. At the end of the blocking incubation, incubate the embryos in the primary anti-laminin antibody of interest at four degrees Celsius overnight on a shaker.
The next morning, wash the embryos five times in PBST for 15 minutes per wash and label the tissues with an appropriate secondary antibody overnight at four degrees Celsius on the shaker protected from light. The next morning, wash the embryos four times in PBST for 15 minutes per wash and place the embryos in a small Petri dish containing fresh PBST. Using fine forceps, gently disrupt the yolks and remove the yolk cells through mild scraping of the yolk sacs.
Transfer the deyolked embryos into freshly prepared 30%glycerol in PBS, making sure to place the embryos on top of the solution and transferring as little of the previous solution as possible and wait for the embryos to sink to the bottom of the tube. When the embryos have reached the bottom of the container, transfer them to sequential 50%and 70%glycerol in PBS solutions, as just demonstrated. After the embryos have sunk to the bottom of the 70%glycerol container, transfer the samples to a small plastic dish for dissections and sever the embryos posterior to the hindbrains.
Using fine dissection tools, move one head to a glass slide with as little glycerol as possible and gently insert a fine minutien pin into the forebrain ventricle from the interior, pushing downward to separate the right and left halves of the head from each other. When the head has been completely filleted down the midline, mount each side of the head midline down, eye-side up. The SOS is observable through the dissecting microscope at 20 to 23 hours post-fertilization and via differential interference contrast imaging as a thin line in the dorsal eye that separates the nasal and temporal halves of the developing retina.
In addition, a subtle indentation may be visible in the dorsal boundary of the eye. Following the immunofluorescent staining of laminin, the thin line can be confirmed as the basement membrane. When SOS closure is delayed, its prolonged presence can be observed as a pronounced cleft in the dorsal side of the eye under a dissecting microscope.
When observed under a compound microscope using differential interference contrast imaging, this feature is even more prominent with the nasal and temporal sides of the eye separated by the SOS, which is clearly visible as a line in the dorsal eye. By 28 hours post-fertilization in wildtype zebrafish, the laminin staining demonstrates clearly that the SOS is completely closed, making this the ideal stage for monitoring delays in fissure closure. The injection of enhanced GFP-CAX mRNA allows the visualization of the cell membranes of alive or fixed embryo for tracking changes in the morphology of the cells that lead to SOS closure.
Visualizing the SOS can be difficult, as it is narrow and transient. However, it is imperative that you maintain a consistent scoring method when evaluating closure delays at later timepoints. These techniques can be combined with experimental manipulations for identifying genetic factors or pharmacological agents that affect proper SOS formation and closure.
