We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their excellent technical assistance.

All experiments adhered to the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology (ARVO).
1. Animals
<ol>
	<li>Maintain the wild-type zebrafish (Danio rerio) of the AB (Oregon) strain aged between 6-12 months under standard conditions in water with a temperature of 26.5 &#176;C and a 14/10 hr light/dark cycle 15.</li>
	<li>Follow the animal care guidelines of the involved institutions for the animal experiments after approval by the Cantonal Veterinary Office.</li>
</ol>
2. MNU Treatment
<ol>
	<li>Prepare water containing 150 mg/L dry substance of N-methyl-N-nitrosourea (MNU). CAUTION! MNU is toxic; may cause cancer, heritable genetic damage or harm to the unborn child.</li>
	<li>Incubate zebrafish in water containing the MNU for 60 min at room temperature.</li>
	<li>Rinse zebrafish quickly with fresh water and put the fish to a new fish tank without MNU. Maintain fish under standard conditions as long as desired for the experiments.</li>
</ol>
3. Visual Acuity Measurement
<ol>
	<li>Start the optomotor system, choose &#39;testing&#39; from the menu and set the options to &#8220;Simple Staircase&#8221;, &#8220;Acuity (Freq)&#8221; and &#8220;Randomized/Separate&#8221; 16.</li>
	<li>Install an infusion bottle filled with 500 ml water about 1 m above the optomotor system.</li>
	<li>Place one zebrafish in the examination chamber and connect it to the infusion bottle. Place the examination chamber in the optomotor system.</li>
	<li>Start the measurement and observe the eye movement in real time on the computer screen. Thereby, a positive response (&#8220;yes&#8221;) is defined as consecutive saccades in the correct direction, whereas a negative response (&#8220;no&#8221;) represents random eye movements similar to the ones observed with stationary gratings. If the eyes of the zebrafish exhibit three or more subsequent optokinetic responses (OKR), press &#39;yes&#39;; if not, press &#39;no&#39;.</li>
	<li>Extract the visual acuity, which has been calculated by the software by determining the threshold of the spatial frequency of the optokinetic stimulus, under &#8220;Results&#8221; in the menu.</li>
</ol>
4. Histology
<ol>
	<li>Euthanize the zebrafish by an overdose of ethyl 3-aminobenzoate methanesulfonate (200-300 mg/L) and enucleate the eyes.</li>
	<li>Fix the whole eyes in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C for 12 hr and then dehydrate the samples in a graded alcohol series.</li>
	<li>Embed the samples in paraffin, cut 5 &#181;m sections through the optic nerve head (ONH) and mount them on a glass slides.</li>
	<li>Stain the deparaffized paraffin sections with hemalum for 4 min and dip the slides two times in distilled water followed by 0.2% hydrochloric acid and 0.8% ammonia. Stain the sections with eosin for 3 min after development of the hemalum staining in tap water for at least 10 min (H&#38;E).</li>
	<li>Embed the dehydrated slides in mounting medium and observe the slides under the light microscope.</li>
</ol>
5. Immunohistochemistry
<ol>
	<li>Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
	<ol>
		<li>Incubate the deparaffinized sections with 20 &#181;g/ml proteinase K at room temperature for 15 min. Wash the sections three times with Tris buffered saline (TBS) for 5 min each.</li>
		<li>Incubate the sections with 50 &#181;l TUNEL reaction mixture (10% labeling solution and 90% enzyme solution) in a humidified chamber at 37 &#176;C for 60 min. Wash three times with TBS for 5 min each.</li>
		<li>Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.</li>
	</ol>
	</li>
	<li>Proliferating Cell Nuclear Antigen (PCNA) staining
	<ol>
		<li>Boil the deparaffinized sections in antigen retrieval buffer (Tris-EDTA + 0.05% Tween 20, pH 9.0) for 3 min and wash three times with TBS for 5 min each. Block with 100 &#181;l blocking solution (TBS + 10% goat normal serum + 1% bovine serum albumin, pH7.6) at room temperature for 1 hr.</li>
		<li>Stain with anti-PCNA primary antibodies in a 1:200 dilution in a humidified chamber at 4 &#176;C overnight. Wash three times with TBS + Tween 20 for 5 min each.</li>
		<li>Finish the detection with the appropriate secondary antibodies in a 1:500 dilution at room temperature for 1 hr.</li>
		<li>Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa.">Retinitis pigmentosa.</a> Lancet. 368, 1795-1809 (2006).</li><li>Tsubura, A., Yoshizawa, K., Kuwata, M., Uehara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+retinitis+pigmentosa+induced+by+MNU;+disease+progression,+mechanisms+and+therapeutic+trials.">Animal models for retinitis pigmentosa induced by MNU; disease progression, mechanisms and therapeutic trials.</a> Histol. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+adult+and+developing+mouse+spatial+vision+using+a+virtual+optomotor+system.">Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.</a> Invest. Ophthalmol Vis. Sci. 45 (12), 4611-4616 (2004).</li><li>Beck, J. C., Gilland, E., Tank, D. W., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+ontogeny+of+optokinetic+and+vestibulo-ocular+behaviors+in+zebrafish,+medaka,+and+goldfish.">Quantifying the ontogeny of optokinetic and vestibulo-ocular behaviors in zebrafish, medaka, and goldfish.</a> J. Neurophysiol. 92 (6), 3546-3561 (2004).</li><li>Fimbel, S. M., Montgomery, J. E., Burket, C. T., Hyde, D. 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The authors have nothing to disclose.

Previously, our group has transferred the MNU model of photoreceptor degeneration from rodents into the zebrafish system 9. The ensuing events were described and followed for up to 30 days. In this time period complete retinal morphological degeneration and regeneration occurred after the initial treatment. First, histology reveals a reduced rod cell count from day 3 on with a minimum at day 8. Correspondingly, TUNEL staining identifies apoptosis of rod photoreceptors 3 days after MNU treatment. Regeneration s...

Vision is the most essential sense for the human being and its impairment has a high socio-economic impact. In the developed world, retinal degenerative diseases are the leading cause of vision loss and blindness among older adults 1. The cause of most degenerative retinal diseases is only partly understood and therapeutical solutions to regain lost vision are very limited. Retinitis pigmentosa is a typical example of a retinal degenerative disease with primary photoreceptor loss 2-3. N-methyl-N-nitrosourea (MNU) induces retinal degeneration and is therefore widely used in rodents to model diseases with primary photoreceptor cell death 4. It is an alkylating agent and leads to benign and malignant tumors, which usually appear several months after exposure 5-7. In addition, it causes specific photoreceptor cell death within a short term observation period. Thereby, the loss of the retinal layer structure and significant retinal thinning were observed in a concentration-dependent manner. Retinal glia cells were activated, but no changes in the retinal pigment epithelium (RPE) were found. Endoplasmic reticulum (ER) stress-related apoptosis appears to be the main pathway of MNU action in the retina 8.
We have recently introduced MNU as a chemical model to induce photoreceptor degeneration in zebrafish 9. Amongst other reasons, the zebrafish (Danio rerio) has become important in visual research because of the similarities of its visual system to that of other vertebrates 10. The outer retina contains the photoreceptors, which can be grouped into four different cone types with peak sensitivities in the ultraviolet, short, middle, and long wavelength of the visible spectra and one rod photoreceptor type. In the inner nuclear layer (INL), the cell bodies of bipolar, horizontal, and amacrine interneurons are found, as well as the cell soma of Muller glia cells. In the outer plexiform layer (OPL) the synaptic contacts between photoreceptors and the inner retina are formed, whereas the cell layer closest to the lens is the ganglion cell layer (GC), which components form long axons comprising the optic nerve and the optic tract. Synaptic contacts between ganglion cells and the cells in the inner nuclear layer are formed in the inner plexiform layer (IPL) 11. The RPE lies outside the neurosensory retina and surrounds the photoreceptor outer segments with long apical microvilli 12. Furthermore, the zebrafish is highly regenerative and able to regrow lesioned brain, retina, spinal cord, heart, and other tissues 13. When retinal damage occurs, cells in the INL, which are thought to be M&#252;ller cells, are activated and have the potential to differentiate into various retinal cell types. In addition, they also generate rod progenitors, which are located in the ONL. Another source that supplies the retina of adult zebrafish with new cells is the ciliary marginal zone. This source is needed to achieve a constant density of rod photoreceptors in the continuously growing zebrafish eye 14.
The MNU model can be used as a simple and reproducible degeneration/regeneration approach for retinal tissue. Due to certain similarities of biological processes in zebrafish and in humans this could open the doors to identify involved cell death pathways and to screen potential neuroprotective drugs. Based on a previous study from our group, we now illustrate the methods of this MNU-induced zebrafish model of retinal de- and consequent regeneration including functional changes with according laboratory videos 9.

<table><tbody><tr><td>Acetic acid</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>A6283</td><td></td></tr><tr><td>Ammonia</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>294993</td><td>0.80%</td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>05470</td><td></td></tr><tr><td>Dako Pen</td><td>Dako, Glostrup, Danmark</td><td>S2002</td><td></td></tr><tr><td>DAPI mounting medium</td><td>Vector Labs, Burlingame, CA, USA</td><td>H-1200</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>45260</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>2860</td><td>100%, 96%, 70%</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>ED</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>E10521</td><td>Tricaine</td></tr><tr><td>Eukitt</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>3989</td><td></td></tr><tr><td>Goat anti-rabbit Alexa 594</td><td>Life Technologies, Zug, Switzerland&amp;nbsp;</td><td>A11012</td><td></td></tr><tr><td>Goat normal serum</td><td>Dako, Glostrup, Danmark</td><td>X0907</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>320331</td><td>0.20%</td></tr><tr><td>In situ Cell Death Detection Kit</td><td>Roche Applied Sciences, Rotkreuz, Switzerland</td><td>11684795910</td><td>TUNEL Kit</td></tr><tr><td>Mayer&#039;s hemalum solution</td><td>Merck, Darmstadt, Germany</td><td>109249</td><td></td></tr><tr><td>Methylnitrosourea (MNU)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>N4766</td><td>Toxic</td></tr><tr><td>OptoMotry</td><td>CerebralMechanics, Lethbridge, AB, Canada</td><td>n.a.</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P6148</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5368</td><td></td></tr><tr><td>Proteinase K</td><td>Dako, Glostrup, Danmark</td><td>S3004</td><td></td></tr><tr><td>Rabbit anti-PCNA&amp;nbsp;</td><td>Santa Cruz Biotechnology, Santa Cruz, USA</td><td>sc-33756</td><td></td></tr><tr><td>Superfrost Plus glass slides</td><td>Gehard Menzel GmbH, Braunschweig, Germany</td><td>10149870</td><td></td></tr><tr><td>Tris buffered saline (TBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5912</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>T1503</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P1379</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>534056</td><td></td></tr><tr><td>Zebrafish (Danio rerio) AB (Oregon) strain</td><td>University of Fribourg, Dept. of Biology</td><td>n.a.</td><td>Own fish facility</td></tr></tbody></table>

methylnitrosourea (mnu)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Visual acuity:
The experimental set-up [spatial frequency: 0.042 circles/degree (c/d); contrast: 100%; drift speed: 20 degrees/second (d/src); back light luminance: 152 cd/m2] of this study enabled OKR assessment of adult zebrafish. The mean duration of VA measurement was about 5 &#8211; 10 minutes for each zebrafish, which tolerated the procedure well. Visual acuity before MNU exposure was 0.577 &#177; 0.014 cycles/degree (c/d). Figure 1 shows the visual acuity cou...

Watch this Scientific Journal Video about Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics at JoVE.com

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

methylnitrosourea-mnu-induced-retinal-degeneration-regeneration

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.4K Views.  University of Bern. Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Video: Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Developmental Biology

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

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 can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

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 ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Neuroscience

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Immunology and Infection

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

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 neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

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

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Summary

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Chapters in this video

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Microbead Implantation in the Zebrafish Embryo

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

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

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium