This research was supported by grants to M.P. from the Association Retina France, Fondation de France, FMR (Fondation Maladies Rares), BBS (Association du syndrome de Bardet-Biedl), and UNADEV (Union Nationale des Aveugles et D&#233;ficients Visuels) in partnership with ITMO NNP (Institut Th&#233;matique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie) / AVIESAN (Alliance Nationale pour les sciences de la vie et de la sant&#233;).

Generating Retinal Degeneration to Investigate Cellular Repair Strategies in Xenopus

<ol>
	<li>Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cell+reprogramming+and+retina+regeneration.">M&#252;ller glial cell reprogramming and retina regeneration.</a> Nature reviews. Neuroscience. 15 (7), 431-442 (2014).</li><li>Hamon, A., Roger, J. E., Yang, X. -. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+tadpoles+can+regenerate+neural+retina+lost+after+physical+excision+but+cannot+regenerate+photoreceptors+lost+through+targeted+ablation.">Xenopus laevis tadpoles can regenerate neural retina lost after physical excision but cannot regenerate photoreceptors lost through targeted ablation.</a> Investigative Ophthalmology &amp; Visual Science. 54 (3), 1859-1867 (2013).</li><li>Martinez-De Luna, R. I., Kelly, L. E., El-Hodiri, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+homeobox+(Rx)+gene+is+necessary+for+retinal+regeneration.">The retinal homeobox (Rx) gene is necessary for retinal regeneration.</a> Developmental Biology. 353 (1), 10-18 (2011).</li><li>Choi, R. Y., 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=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Investigative Ophthalmology &amp; Visual Science. 52 (1), 364-373 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

Advantages and disadvantages of various retinal injury paradigms in Xenopus tadpoles
Mechanical retinal injury 
Various surgical injuries of the neural retina have been developed in Xenopus tadpoles. The neural retina may either be entirely removed15,16 or only partly excised16,17. The mechanical injury presented here doe...

Millions of people worldwide are afflicted with various retinal degenerative diseases leading to blindness, such as retinitis pigmentosa, diabetic retinopathy, or age-related macular degeneration (AMD). To date, these conditions remain largely untreatable. Current therapeutic approaches under evaluation include gene therapy, cell or tissue transplantations, neuroprotective treatments, optogenetics, and prosthetic devices. Another emerging strategy is based on self-regeneration through the activation of endogenous cells with stem cell potential. M&#252;ller glial cells, the major glial cell type of the retina, are among cellular sources of interest in this context. Upon injury, they can dedifferentiate, proliferate, and generate neurons1,2,3. Although this process is very effective in zebrafish or Xenopus, it is largely inefficient in mammals.
Nonetheless, it has been shown that appropriate treatments with mitogenic proteins or overexpression of various factors can induce mammalian M&#252;ller glia cell-cycle re-entry and, in some cases, trigger their subsequent&#160;neurogenesis commitment1,2,3,4,5. This remains, however, largely insufficient for treatments. Hence, increasing our knowledge of the molecular mechanisms underlying regeneration is necessary to identify molecules able to efficiently turn M&#252;ller stem-like cell properties into new cellular therapeutic strategies.
With this aim, we developed several injury paradigms in Xenopus that trigger retinal cell degeneration. Here, we present (1) a mechanical retinal injury that is not cell type-specific, (2) a conditional and reversible cell ablation model using the NTR-MTZ system that targets rod cells, (3) a CRISPR/Cas9-mediated rhodopsin knockout, a model of retinitis pigmentosa that triggers progressive rod cell degeneration, and (4) a CoCl2-induced cytotoxic model that according to the dose can specifically target cones or lead to broader retinal cell degeneration. We highlight the particularities, advantages, and disadvantages of each paradigm.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-Propanediol (propyl&#232;ne glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>398039</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol &#8805;99.8%</td>
			<td>VWR chemicals</td>
			<td>20821-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Cleaved Caspase 3 antibody (rabbit)</td>
			<td>Cell signaling</td>
			<td>9661S</td>
			<td>Dilution 1/300</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody (chicken)</td>
			<td>Aveslabs</td>
			<td>GFP-1020</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-M-Opsin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5405</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-mouse secondary antibody, Alexa Fluor 594 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11005</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-Otx2 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>Ab183951</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 488 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11008</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 594 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11012</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-Recoverin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5585</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-Rhodopsin antibody (mouse)</td>
			<td>Sigma-Aldrich</td>
			<td>MABN15</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-S-Opsin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5407</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Apoptotis detection kit (Dead end fluorimetric TUNEL system)</td>
			<td>Promega</td>
			<td>G3250</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>Stock solution 10%</td>
		</tr>
		<tr>
			<td>bisBenzimide H 33258 (Hoechst)</td>
			<td>Sigma-Aldrich</td>
			<td>B2883</td>
			<td>Stock solution 10 mg/mL</td>
		</tr>
		<tr>
			<td>Butanol-1 &#8805;99.5%</td>
			<td>VWR chemicals</td>
			<td>20810.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2, 2H2O)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.02382</td>
			<td>Use at 0.1 M</td>
		</tr>
		<tr>
			<td>Cas9 (EnGen Spy Cas9 NLS)</td>
			<td>New England Biolabs</td>
			<td>M0646T</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark Capillary Glass model GC100TF-10</td>
			<td>Warner Instruments (Harvard Apparatus)</td>
			<td>30-0038</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(II) chloride hexahydrate (CoCl2, 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C8661</td>
			<td>Stock solution 100 mM</td>
		</tr>
		<tr>
			<td>Coverslip 24 x 60 mm</td>
			<td>VWR</td>
			<td>631-1575</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako REAL ab diluent&#160;</td>
			<td>Agilent</td>
			<td>S202230-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Rotary Microtome</td>
			<td>Thermo Scientific</td>
			<td>Microm HM 340E&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin 1% aqueous</td>
			<td>RAL Diagnostics</td>
			<td>312740</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein lysine dextran&#160;&#160;</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>D1822</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin albumin acc. Mallory</td>
			<td>Diapath</td>
			<td>E0012</td>
			<td>Use at 3% in water</td>
		</tr>
		<tr>
			<td>Hematoxylin (Mayer&#39;s Hemalun)</td>
			<td>RAL Diagnostics</td>
			<td>320550</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES potassium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H0527</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin hormone</td>
			<td>MSD Animal Health</td>
			<td>Chorulon 1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid fuming, 37% (HCl)</td>
			<td>Sigma-Aldrich (SAFC)</td>
			<td>1.00314</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7880</td>
			<td>Use at 2% in 0.1x MBS (pH 7.8 - 8.0)</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate (MgSO4, 7H2O)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.05886</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole&#160;</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>M3761</td>
			<td>Use at 10 mM</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller (P-97 Flaming/Brown)</td>
			<td>Sutter Instrument Co.</td>
			<td>Model P-97</td>
			<td>Program : Heat 700 / Pull 100 / Vel 75 / Time 90 / Unlocked p = 500</td>
		</tr>
		<tr>
			<td>Mounting medium to preserve fluorescence, FluorSave Reagent</td>
			<td>Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium, Eukitt</td>
			<td>Chem-Lab</td>
			<td>CL04.0503.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MX35 Ultra Microtome blade</td>
			<td>Epredia</td>
			<td>3053835</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Agani 25 G x 5/8&#39;&#39;</td>
			<td>Terumo</td>
			<td>AN*2516R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filtration tissue (sifting fabric) NITEX, mesh opening 1,000 &#181;m</td>
			<td>Sefar</td>
			<td>06-1000/44</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin histowax without DMSO</td>
			<td>Histolab</td>
			<td>00403</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution (32%)</td>
			<td>Electron Microscopy Sciences</td>
			<td>EM-15714-S</td>
			<td>Use at 4% in 1x PBS pH 7.4</td>
		</tr>
		<tr>
			<td>Peel-A-Way Disposable Embedding Molds</td>
			<td>Epredia</td>
			<td>2219</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>VWR</td>
			<td>431-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 100 mm</td>
			<td>Corning Gosselin</td>
			<td>SB93-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 55 mm</td>
			<td>Corning Gosselin</td>
			<td>BP53-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline Solution (PBS) 10x</td>
			<td>Euromedex</td>
			<td>ET330-A</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoSpritzer Microinjection system</td>
			<td>Parker Instrumentation Products</td>
			<td>PicoSpritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Pins&#160;</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysucrose (Ficoll PM 400 )</td>
			<td>Sigma-Aldrich</td>
			<td>F4375</td>
			<td>Use at 3% in 0.1x MBS</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Powdered fry food : sera Micron Nature</td>
			<td>sera</td>
			<td>45475 (00720)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors dissection</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Superfrost&#160;</td>
			<td>&#160;KNITTEL Glass</td>
			<td>VS11171076FKA&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide warmer</td>
			<td>Kunz instruments</td>
			<td>HP-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate trisodium salt dihydrate (C6H5Na3O7, 2H2O)</td>
			<td>VWR chemicals</td>
			<td>27833.294</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.06329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide 30% aqueous solution (NaOH)</td>
			<td>VWR chemicals</td>
			<td>28217-292</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes Omnifix-F Solo Single-use Syringes 1 mL</td>
			<td>B-BRAUN</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-activating crRNA (tracrRNA)</td>
			<td>Integrated DNA Technologies</td>
			<td>1072533</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
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generating retinal injury models in xenopus tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Mechanical retinal injury 
Retinal sections of tadpoles subjected to the mechanical injury described in protocol section 1 show that the retinal lesion encompasses all layers of the tissue while remaining limited to the puncture site (Figure 2A,B).
Conditional rod cell ablation using the NTR-MTZ system 
The eyes of anesthetized Tg(rho:GFP-NTR) transgenic tadpoles treated with MTZ treatment, as d...

Watch this Scientific Journal Video about Exploring Retinal Regeneration Mechanisms in the Xenopus Frog at JoVE.com

Author Spotlight: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

generating-retinal-injury-models-in-xenopus-tadpoles

Research

JoVE Journal

Developmental Biology

1.3K Views.  Université Paris-Saclay. We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Video: Author Spotlight: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog 

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...