This work was supported by Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council (NSERC), Alberta Innovates Technology Futures, and Women and Children&#39;s Health Research Institute (WCHRI).

All methods described here have been approved by the University of Alberta Animal Care and Use Committee.
1. Protocol 1: Visualization of SOS using stereomicroscopy and differential interference contrast (DIC) imaging
<ol>
	<li>Embryo collection
	<ol>
		<li>In a tank of dechlorinated water, prepare crosses of gdf6a+/- zebrafish in the evening by pairing a male zebrafish with a female zebrafish. Be sure to separate the male from the female by using a divider to ensure that...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+for+the+analysis+of+vertebrate+development+in+the+zebrafish.">Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish.</a> Developmental Cell. 21 (1), (2011).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), (2013).</li><li>Bilotta, J., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+visual+system.">The zebrafish as a model visual system.</a> International Journal of Developmental Neuroscience. 19, 621-629 (2001).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book; A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Kimmel, C. 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The authors have no conflicting interests to declare.

Here, we present a standardized series of protocols to observe the SOS in the developing zebrafish embryo. To determine closure delay phenotypes, our protocols have focused on the ability to distinguish the separation of two discrete lobes of the dorsal-nasal and dorsal-temporal sides of the eye, similar to techniques used to visualize choroid fissure closure delay phenotypes in the ventral eye.
These visualization techniques can be used in conjunction with a variety of genetic manipulation te...

The formation of the vertebrate eye is a highly conserved process in which carefully orchestrated intercellular signaling pathways establish tissue types and specify regional identity1. Perturbations to early eye morphogenesis result in profound defects to the architecture of the eye and are frequently blinding2. One such disease results from the failure to close the choroid ocular fissure in the ventral side of the optic cup3. This disorder, known as ocular coloboma, is estimated to occur in 1 out of 4-5000 live births and cause 3-11% of pediatric blindness, commonly manifesting as a keyhole-like structure that protrudes inferiorly from the pupil in the center of the eye4,5,6. The function of the choroid fissure is to provide an entry point for early vasculature growing into the optic cup, after which the sides of the fissure will fuse to enclose the vessels7.
While ocular coloboma has been known since ancient times, we have recently identified a novel subset of coloboma patients with tissue loss affecting the superior/dorsal aspect of the eye. Recent work in our lab has led to the discovery of an ocular structure in the zebrafish dorsal eye, which we refer to as the superior ocular sulcus (SOS) or superior fissure8. It is important to note that the structure has characteristics of both a sulcus and a fissure. Similar to a sulcus, it is a continual tissue layer that spans from the nasal to the temporal retina. In addition, the closure of the structure is not mediated by a fusion of the two opposing basement membrane, and it appears to require a morphogenetic process by which the structure is populated by cells. However, similar to a fissure, it forms a structure that separates the nasal and temporal sides of the dorsal eye with the basement membrane. For consistency, we will refer to it as SOS in this text.
The SOS is evolutionarily conserved across vertebrates, being visible during eye morphogenesis in fish, chick, newt, and mouse8. In contrast to the choroid fissure, which is present from 20-60 hours post-fertilization (hpf) in zebrafish, the SOS is highly transient, being easily visible from 20-23 hpf and absent by 26 hpf8. Recent research in our lab has found that, similar to the choroid fissure, the SOS plays a role in vascular guidance during eye morphogenesis8. Although the factors that control the formation and closure of the SOS are not yet fully understood, our data did highlight roles for dorsal-ventral eye patterning genes8.
Zebrafish is an excellent model organism to study the SOS. As a model system, it provides a number of advantages in studying eye development: it is a vertebrate model; each generation exhibits high fecundity (~200 embryos); its genome has been fully sequenced, which facilitates genetic manipulation; and approximately 70% of human genes have at least one zebrafish orthologue, making it an ideal genetics-based model of human disease9,10. Most importantly, its development takes place externally to the mother, and its larvae are transparent, which allows for the visualization of the developing eye with relative ease11.
In this set of protocols, we describe the techniques through which the SOS can be visualized in zebrafish larvae. The variety of visualization techniques used in this report will allow clear observation of the SOS during normal eye development, as well as the ability to detect SOS closure defects. Our example protocols will feature investigations of Gdf6, a BMP localized to the dorsal eye and known regulator of SOS closure. Further, these techniques can be combined with experimental manipulations to identify genetic factors or pharmacological agents that affect proper SOS formation and closure. In addition, we have included a protocol through which the fluorescent imaging of all cell membranes is possible, allowing the experimenter to observe morphological changes to the cells surrounding the SOS. Our goal is to establish a set of standardized protocols that can be used throughout the scientific community to offer new insights into this novel structure of the developing eye.

<table>
	<tbody>
		<tr>
			<td>1-phenyl 2-thiourea</td>
			<td>Sigma Aldrich</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Corning</td>
			<td>CLS430588</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>BioShop Canada Inc.</td>
			<td>AGA001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC/Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioImager Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope camera</td>
			<td>Qimaging</td>
			<td>MicroPublisher 5.0 RTV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-vacuum grease</td>
			<td>Fisher Scientific</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma Aldrich</td>
			<td>A5040-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Image capture software</td>
			<td>Zeiss</td>
			<td>ZEN</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model 1545</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass (22 mm x 22 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pin</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine Sp6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol pH 6.7 +/- 0.2</td>
			<td>Fisher Scientific</td>
			<td>BP1752-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma Aldrich</td>
			<td>P4850</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-laminin antibody</td>
			<td>Millipore Sigma</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO Dnase (2 U/&#181;L)</td>
			<td>Invitrogen</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure low-melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Sodium Dodecyl Sulfate (SDS)</td>
			<td>Invitrogen</td>
			<td>15525017</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of the superior ocular sulcus during danio rerio embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

The zebrafish SOS appears at 20 hpf in the presumptive dorsal retina8. By 23 hpf the SOS transitions from its initial narrow architecture to a wide indentation and by 26 hpf it is no longer visible8. Therefore, to examine the SOS during normal zebrafish eye development, the embryos must be observed between 20-23 hpf. During this period, the SOS is observable through the dissecting microscope and via DIC imaging as a thin line in the dorsal e...

Watch this Scientific Journal Video about Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis at JoVE.com

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

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.

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Research

JoVE Journal

Developmental Biology

5.3K Görüntüleme.  University of Alberta. Bu standart protokoller kümesi, gelişmekte olan gözün yakın zamanda keşfedilen yapısı olan üstün oküler sulkus veya SOS'a yeni bir bakış açısı sunmak için kullanılabilir. Bu protokoller, dünyanın dört bir yanındaki herhangi bir deneycinin zebra balığı göz gelişimi sırasında SOS'u net bir şekilde görselleştirmesini sağlayacaktır. Heterozigot büyüme farklılaşma faktörü altı Bir erkek ve dişi zebra balıklarını, eşleştirmelerinden önceki akşam klor...