Name: assessment of zebrafish lens nucleus localization and sutural integrity
Uploaded: 2019-05-06T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity at JoVE.com

We would like to acknowledge our funding source: NIH R01 EY05661 to J.E.H, Ines Gehring for assisting with generating the aqp0a/b double mutants and zebrafish husbandry, Dr Daniel Dranow for discussions leading to generation of the transgenics, Dr Bruce Blumberg and Dr Ken Cho&#8217;s labs for use of their dissecting microscopes, and Dr Megan Smith for help with statistical analysis.

The animal protocols used in this study adhere to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and have been approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, Irvine.

1. Zebrafish Husbandry and Euthanasia
<ol>
	<li>Raise and maintain zebrafish (AB strain) under standard laboratory conditions12. Raise embryos in embryo medium (EM)12. Add 0.003% PTU to EM from 2...

<ol>
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Analysis of zebrafish lens morphology is the initial step in understanding phenotypes in mutants, or the effects of pharmacological interventions aimed at studying biology of the ocular lens. We outline methods to analyze lens sutures, cortical fiber cell morphology and aspects of the lens nucleus. These approaches are a combination of in vitro and in vivo (compared in Table 1). The in vitro methods allow for greater detail of the outer cortical cell morphology, as well as acce...

The zebrafish is an excellent model for studying lens development and physiology due to the anatomical similarities to mammalian lenses, relative ease of genetic and pharmacological manipulation, speed of embryonic eye development, small size and transparency at early stages allowing for in vivo imaging. The methods described here were developed to analyze zebrafish lens morphology at embryonic and adult stages with a focus on sutural integrity, cortical membrane morphology in vitro and in vivo, and location of lens nuclear position along the anterior-posterior axis ex vivo. These methods serve as a starting point for functional studies of lens development and physiology, as well as reverse genetic screens for lens phenotypes in zebrafish.
Imaging zebrafish lens morphology
Aquaporin 0 (AQP0) is the most abundant lens membrane protein and is critical for both, lens development and clarity, due to multiple essential functions in mammals. Zebrafish have two Aqp0s (Aqp0a and Aqp0b) and we have developed methods to analyze loss of their functions in both embryonic and adult lenses. Our studies reveal that aqp0a-/- mutants develop anterior polar opacity due to instability of the anterior suture, and aqp0a/b double mutants develop nuclear opacity1. AQP0 has been shown to play roles in water transport2, adhesion3,4, cytoskeletal anchoring5 and generation of the refractive index gradient6, but these studies have largely been performed in vitro. The zebrafish provides a unique opportunity to study how loss of function, or perturbed function of Aqp0a or Aqp0b would affect morphology and function in a living lens. To assess lens cell morphology and sutural integrity during development, we modified existing in vitro immunohistochemical methods7 for use in embryonic and adult lenses, and generated transgenics to monitor this process in vivo.
Immunohistochemical analysis of plasma membrane structure and sutural integrity was performed in whole fixed embryos and adult lenses. Zebrafish lenses are extremely small (lens diameter is ~100 &#181;m in embryos and up to 1 mm in adults) compared with their mammalian counterparts and have point sutures8, which are infrequently captured in cryosections. Thus, whole lenses are essential for analyzing sutural integrity. For in vivo analysis of anterior suture formation, and imaging of precise lens cell architecture, we generated transgenics expressing mApple specifically labeling lens membranes.
Advantages of live imaging of lens membrane transgenics include: 1) avoiding fixation artifacts, 2) studying dynamic morphological changes in time-lapse movies, and 3) enabling longitudinal studies in which earlier events can be correlated with later phenotypes. Pigmentation of the iris normally prevents clear imaging of the lens periphery. Addition of 1-phenyl 2-thiourea (PTU) before the primordia-5 (prim-5) stage9 prevents melanogenesis and eye pigmentation up to around 4 days postfertilization (dpf). However, after 4 dpf, the lens periphery is obscured in vivo, particularly at older stages. Furthermore, the density of the lens itself obscures imaging of its posterior pole. Therefore, to study morphology of the lens periphery, or the posterior suture, after 4 dpf, lenses need to be excised and fixed.
Transgenic zebrafish lines have been used to analyze embryonic lens membrane structure in vivo10. The Q01 transgenic expresses a cyan fluorescent protein fused to a membrane targeting sequence, Gap43, driven by the EF1&#945; promoter and a hexamer of the DF4 pax6 enhancer element ubiquitously in lens fiber cells11. Q01 does have extra-lenticular expression, including amacrine cells in the retina, which adds background signal if the primary interest is the lens. We developed a novel transgenic line that expresses a membrane-tethered mApple specifically in the lens, with the aim of avoiding any extra-lenticular signal.
Lens nucleus localization
We discovered that the lens nucleus moves from an initial anterior location in larval zebrafish to a central location in the anterior-posterior axis in adult lenses. This shift in the position of the high refractive index lens nucleus is thought to be a requirement for normal development of zebrafish lens optics1. Our methods allow quantification of lens nuclear centralization in relation to the lens radius. Using this method, we showed that Aqp0a is required for lens nuclear centralization1, and this simple method can be applied to other studies of the development and physiology of the lens and its optical properties in the zebrafish model.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">P7629</td>
			<td style="width:258px;">CAUTION &#8211; very toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:310px;">4% Paraformaldehyde aqueous solution</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:118px;">RT 157-4</td>
			<td>CAUTION &#8211; health hazard, combustible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Confocal microscope</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;">Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Cryostat</td>
			<td style="width:196px;">Leica</td>
			<td style="width:118px;">CM3050S</td>
			<td style="width:258px;">Objective and chamber temperature set to -21&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">DAPI</td>
			<td style="width:196px;">Invitrogen</td>
			<td style="width:118px;">D1306</td>
			<td>CAUTION &#8211; irritating to eyes and skin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">D128</td>
			<td style="width:258px;">CAUTION &#8211; combustible, penetrates skin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Disposable base mold</td>
			<td style="width:196px;">VWR Scientific</td>
			<td style="width:118px;">15154-631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Disposable Pasteur glass pipets</td>
			<td style="width:196px;">Fisherbrand</td>
			<td style="width:118px;">13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dumont # 5 forceps</td>
			<td style="width:196px;">Dumont &#38; Fils</td>
			<td style="width:118px;"></td>
			<td>Keep forceps sharpened</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:118px;">A5040</td>
			<td>CAUTION - toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Glass bottom microwell dish (35mm petri dish, 14mm microwell, #1.5 coverglass)</td>
			<td style="width:196px;">MatTek Corporation</td>
			<td style="width:118px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Glycerol</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hu&#946;B1cry:mAppleCAAX DNA construct</td>
			<td style="width:196px;">Addgene</td>
			<td style="width:118px;">ID:122451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">ImageJ</td>
			<td style="width:196px;">Wayne Rasband, NIH</td>
			<td style="width:118px;">v1.51n</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Low Melt Agarose (LMA)</td>
			<td style="width:196px;">Apex</td>
			<td style="width:118px;">902-36-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">NIS-Elements</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;">V 4.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">NIS-Elements AR software</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus&#160; with a model 2.1.1.183 controller</td>
			<td style="width:196px;">Olympus Corp</td>
			<td style="width:118px;">DP70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus microscope&#160;</td>
			<td style="width:196px;">Olympus Corp</td>
			<td style="width:118px;">SZX12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phalloidin-Alexa Flour 546</td>
			<td style="width:196px;">Thermo Fisher</td>
			<td style="width:118px;">A22283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phenol Red indicator (1% w/v)</td>
			<td style="width:196px;">Ricca Chemical Company</td>
			<td style="width:118px;">5725-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phosphate buffered saline (PBS)</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">BP399</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Photoshop</td>
			<td style="width:196px;">Adobe</td>
			<td style="width:118px;">CS5 v12.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Photoshop software</td>
			<td style="width:196px;">Adobe</td>
			<td style="width:118px;">CS5 v12.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plan Apo 60x/1.2 WD objective</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Power source</td>
			<td style="width:196px;">Wild Heerbrugg</td>
			<td style="width:118px;">MTr 22</td>
			<td>Or equivalent power source&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Slide warmer model No. 26020FS</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">12-594</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:310px;">Sodium Hydroxide beads</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">S612-3</td>
			<td style="width:258px;">CAUTION - corrosive/irritating to eyes and skin, target organ - respiratory system, corrosive to metals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Superfrost/Plus microscope slide</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Sylgard 184 silicone Dow Corning</td>
			<td style="width:196px;">World Prevision Instruments</td>
			<td style="width:118px;">SYLG184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:196px;">Sakura Finetek</td>
			<td style="width:118px;">4583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Triton X-100 BioXtra</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">T9284</td>
			<td style="width:258px;">CAUTION &#8211; Toxic, hazardous to aquatic environment, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Vannas micro-dissection scissors</td>
			<td style="width:196px;">Ted Pella Inc</td>
			<td style="width:118px;">1346</td>
			<td>Sharp/sharp straight tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Vectashield antifade mouting medium</td>
			<td style="width:196px;">Vector laboratories</td>
			<td style="width:118px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Wheat Germ Agglutinin (WGA)-Alexa Flour-594</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:118px;">W11262</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of zebrafish lens nucleus localization and sutural integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

Adult zebrafish eye anatomy closely resembles that of mammals (Figure 1A). Despite some differences between zebrafish and mammalian eyes, such as having a ciliary zone instead of a ciliary body17, differences in optical properties18, and differences in morphogenesis during embryonic development19, the zebrafish eye is an excellent model for studying eye development and understanding ophth...

Watch this Scientific Journal Video about Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity at JoVE.com

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

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.

Research

JoVE Journal

Biology

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Studying Protein Expression During Chicken Lens Development

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens (Video) | JoVE

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...