Name: visualization of the superior ocular sulcus during danio rerio embryogenesis
Uploaded: 2019-03-27T14:30:00
Description: Watch this Scientific Journal Video about Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis at JoVE.com

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>
	<li>Chow, R. L., Lang, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+development+in+vertebrates.">Early eye development in vertebrates.</a> Annual Review of Cell and Developmental Biology. 17, (2001).</li><li>Slavotinek, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+development+genes+and+known+syndromes.">Eye development genes and known syndromes.</a> Molecular Genetics and Metabolism. 104 (448-456), (2011).</li><li>Gregory-Evans, C. Y., Williams, M. J., Halford, S., Gregory-Evans, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+coloboma:+a+reassessment+in+the+age+of+molecular+neuroscience.">Ocular coloboma: a reassessment in the age of molecular neuroscience.</a> Journal of Medical Genetics. 41 (12), (2004).</li><li>Onwochei, B. C., Simon, J. W., Bateman, J. B., Couture, K. C., Mir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+colobomata.">Ocular colobomata.</a> Survey of Ophthalmolgy. 45, 175-194 (2000).</li><li>Williamson, K. A., FitzPatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+architecture+of+microphthalmia,+anophthalmia+and+coloboma.">The genetic architecture of microphthalmia, anophthalmia and coloboma.</a> European Journal of Medical Genetics. 57, 369-380 (2014).</li><li>Chang, L., Blain, D., Bertuzzi, S., Brooks, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uveal+coloboma:+clinical+and+basic+science+update.">Uveal coloboma: clinical and basic science update.</a> Current Opinion in Ophthalmology. 17, 447-470 (2006).</li><li>Kaufman, R., 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=Development+and+origins+of+Zebrafish+ocular+vasculature.">Development and origins of Zebrafish ocular vasculature.</a> BMC Developmental Biology. 15 (18), (2015).</li><li>Hocking, J. C., 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=Morphogenetic+defects+underlie+Superior+Coloboma,+a+newly+identified+closure+disorder+of+the+dorsal+eye.">Morphogenetic defects underlie Superior Coloboma, a newly identified closure disorder of the dorsal eye.</a> PLOS Genetics. 14 (3), (2018).</li><li>Lawson, N. D., Wolfe, S. 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. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Distel, M., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+time-lapse+imaging+of+zebrafish+embryonic+development.">In vivo time-lapse imaging of zebrafish embryonic development.</a> Cold Spring Harbor Protocols. , (2007).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature Protocols. 3, 59-69 (2008).</li><li>Kwan, K. M., Otsuna, H., Kidokoro, H., Carney, K. R., Saijoh, Y., Chien, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+choreography+of+cell+movements+shapes+the+vertebrate+eye.">A complex choreography of cell movements shapes the vertebrate eye.</a> Development. 139, 359-372 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullman, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Embryonic+Development+of+the+Zebrafish.">Stages of Embryonic Development of the Zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Gfrerer, L., Dougherty, M., Liao, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Craniofacial+Development+in+the+sox10:+kaede+Transgenic+Zebrafish+Line+Using+Time-lapse+Confocal+Microscopy.">Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy.</a> J. Vis. Exp. (79), e50525 (2013).</li><li>Percival, S. M., Parant, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+Mitotic+Division+and+Dynamics+in+a+Live+Zebrafish+Embryo.">Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo.</a> J. Vis. Exp. (113), e54218 (2016).</li></ol>

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 ...

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