Biz nezaketle betikleri için Dr John Hayes ederim; Dr. Eric Bradley ve konfokal mikroskopi için yardım Christopher Del Negro, Drew Hughes, proje geliştirme ve ön veriler sağlayan çalışmaları için Laura Odorizzi, Alex Garafalo, Rebecca Lowden, ve Liz MacMurray; istatistiksel analize yararlı önerileri için Dr Greg Smith. Bu çalışma MSS bir NIH hibe (NINDS IR15N5067566-01) ve William and Mary College Howard Hughes Tıp Enstitüsü Fen Bilgisi Eğitimi Hibe tarafından desteklenmiştir.

Çıkar çatışması ilan etti.

Tüm omurgalılar arasında muhafaza edilir, iyi karakterize hücre tipleri ile, retinanın hücre tipi özellikleri ve farklılaşma yöneten molekül hücresel süreçleri incelemek için yararlı bir model sağlamaktadır. Birincil hücre kültürü ve gen ekspresyonu, protein dinamiği, ve kalsiyum ve tek bir hücre çözünürlük düzeyde elektrik etkinlik içeren işlemler çok çeşitli araştırma için güçlü bir yöntem tanıyor. Burada varsayımsal Retina gelişimi ve primer hücre kültürü çok erken a?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td></td></tr><tr><td></td><td></td><td></td><td> Diseksiyonlarında ve Kültürleme için </td></tr><tr><td> BD Falcon Kolay Tutuş Doku Kültürü Yemekleri, 35 mm </td><td> Balıkçı </td><td> 08-772A </td><td></td></tr><tr><td> Tek Polistiren Petri Yemekleri, 60 x 15 mm </td><td> Balıkçı </td><td> 0875713A </td><td></td></tr><tr><td> 35 mm Nunclon Yüzey Petri kaplar (Airvent ile) </td><td> Balıkçı </td><td> 12-565-91 </td><td></td></tr><tr><td> Dumont Güzel Forseps (Dumostar # 55) </td><td> Balıkçı </td><td> NC9341917 </td><td></td></tr><tr><td> Cellattice Mikro-yönetilen plastik lamel, 25 mm </td><td> Balıkçı </td><td> 50-313-17 </td><td></td></tr><tr><td> Etil-m-aminobenzoat metansülfonat Tuz (MS-222) </td><td> MP Biomedicals, LLC </td><td> 103106 </td><td></td></tr><tr><td> Gentamisin Sülfat </td><td> Enzo Yaşam Bilimleri </td><td> 380-003-G025 </td><td></td></tr><tr><td> Gibco Tripsin (1:250) Toz </td><td> Yaşam Teknolojileri </td><td> 27250-018 </td><td></td></tr><tr><td> Clostridium histolyticum gelen Kollajenaz B </td><td> Roche </td><td> 11088831001 </td><td></td></tr><tr><td> Penisilin-Streptomisin </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> Nile Blue Sülfat (isteğe bağlı) </td><td> Pfaltz &amp; Bauer </td><td> N05550 </td><td></td></tr><tr><td> 500 ml Vakum Filtre / Depolama Şişe Sistemi, 0.22 mikron gözenek 33.2 cm 2 CN Membran </td><td> Corning </td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ Td&gt; <td></td><td> Kalsiyum For Imaging </td></tr><tr><td> Fluo-4 DMSO içinde 1 mM Çözüm, Daimi Hücre AM </td><td> Yaşam Teknolojileri </td><td> F-14217 </td><td></td></tr><tr><td> Su Pluronic F-127% 10 Çözüm </td><td> Yaşam Teknolojileri </td><td> P-6866 </td><td></td></tr><tr><td> LSM 510 Konfokal Mikroskop Sistemi </td><td> Zeiss </td><td> Model Üretilmeyen </td><td></td></tr><tr><td> Reaktif Engelleme </td><td> Roche </td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> In situ hibridizasyon Floresan (FISH) için </td></tr><tr><td> Anti-digoxigenin-POD, Fab Fragments </td><td> Roche </td><td> 11207733910 </td><td></td></tr><tr><td> Anti-DNP-HRP antikor </td><td> Perkin-Elmer </td><td> NEL747A </td><td></td></tr><tr><td> Cy3NHS ester </td><td> GE Healthcare </td><td> PA13101 </td><td></td></tr><tr><td> NHS-Floresein </td><td> Thermo Scientific </td><td> 46409 </td><td></td></tr><tr><td> Formamid, Deiyonize </td><td> Amresco </td><td> 0606-950ML </td><td></td></tr><tr><td> Torula RNA, tip IX </td><td> Sigma-Aldrich </td><td> R3629 </td><td></td></tr><tr><td> Domuz intestinal mukozadan Heparin Sodyum Tuz, </td><td> Sigma-Aldrich </td><td> H3393-250KU </td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich </td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tablo 2. Özel reaktifleri ve ekipmanlar. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Çözüm </td><td> Referans </td><td> Içindekiler </td></tr><tr><td> Hücre Kültürü Orta </td><td> Chang ve Spitzer, 2009 54 </td><td> 116 mM NaCl 0.67 mM KCl 2 mM CaCl2 1.31 mM MgSO4 4.6 mM Tris % 1 (v: v) Penisilin / Streptomisin HCl ile 7.8 pH ayarlayın. 0.22 mikron CN filtre geçerek sterilize filtreleyin. 4 ° C&#39;de depolayın </td></tr><tr><td> Kalsiyum-Magnezyum Ücretsiz Medium (CMF) </td><td> Gu et al, 1994 42;. Gu ve Spitzer, 1995 55 </td><td> 116 mM NaCl 0.67 mM KCl 4.6 mM Tris 0.4 mM EDTA % 1 (v: v) Penisilin / Streptomisin HCl ile 7.8 pH ayarlayın. 0.22 mikron CN filtre geçerek sterilize filtreleyin. 4 ° C&#39;de depolayın </td></tr><tr><td> Maleik Asit Tampon (MAB) </td><td> Sive ve ark., 2000 53 </td><td> 100 mM maleik asit 150 mM NaCl (pH 7.5). </td></tr><tr><td> 10X Marc Modifiye Ringers (MMR), </td><td> Sive ve ark., 2000 53 </td><td> 100 mM NaCl mM KCl 1 mM MgSO4 2 mM CaCl2 5 mM HEPES pH NaOH ile 7.4 &#39;e ayarlandı 0,1 X MMR da 50 mg / ml gentamisin sülfat ve pH, NaOH ile 7.4 &#39;e ayarlanır içerir. </td></tr><tr><td> MEMFA Çözüm, 10X </td><td> Sive ve ark., 2000 53 </td><td> 0.1 M MOPS (pH 7.4) 2 mM EGTA 1 mM MgSO4 % 3,7 formaldehit 10X bir çözelti, formaldehit olmaksızın, 4 ° C&#39;de saklanabilir Formaldehit (% 37, standart bir stok, 1/10 hacim), taze ilave edilir. </td></tr><tr><td> In situ Hibridizasyon Tamponu In </td><td> SivE ve diğ., 2000 53 </td><td> 50% Formamid 5X SSC 1 mg / ml Torula RNA 100 mg / ml heparin 1X Denhart Çözümü % 0.1 Tween 20 % 0.1 CHAPS 10 mM EDTA. </td></tr></tbody></table> Penisilin ve streptomisin bizim kültür ortamı olarak kullanılabilir yaparken çözümler. * Gentamisin, bir antibiyotik, bizim MMR çözüm olarak kullanılır. </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

Nöral plaka ön bölgede gözdeki doğurur hangi süreci klinik ve temel hem de araştırma önemli bir odak noktası olmaya devam ediyor. Retina hastalığı anlaşılması ve tedavi edilmesi için, bariz bir tıbbi uygunluk ilave olarak, omurgalı retina gelişimi nöronal hücre tipi anlaşılmasını kararlılık ve farklılaşması için 1-16 ve önemli bir şık model sistemi olarak hizmet etmektedir. Nöral retinada basmakalıp katmanlar halinde düzenlenmiş altı ayrı hücre tipleri (gangliyon, yatay, amacrine fotoreseptör, bipolar hücreleri ve Müller glial hücreler), büyük ölçüde tüm omurgalıların 12,14-18 arasında korunduğunu bir deseni oluşur. Sağlam gelişen embriyo retina okuyan açıkça bu kompleks organ katmanlı bir yapıya önbeyin bir çıkıntı gelişir nasıl anlaşılması için gerekli iken, fro yararlanacak pek çok soru varm varsayımsal retina hücrelerinin primer hücre kültürü 7,19-23 kullanarak yaklaşımlarının uygulanması. Örneğin, farklı aşamalarında kaldırılır ve ayrışmış dokulardan hücrelerin analiz biri farklı gelişim aşamalarında tek tek hücrelerin belirlenmesi ve karakterize edilmesi durumu ayırt etmek için izin verir, bu ise, komşu dokulara 8,19-22 ile etkileşimi yokluğunda hücrelerin kaderini ,24-33. Primer hücre kültürü de araştırmacının belirli reaktifleri ile kültür tedavi ve tek hücre düzeyinde 5,8,21,24,27-30,33-39 üzerindeki sonuçlarını analiz etmeyi sağlar. Xenopus laevis, çalışma için klasik bir model sistem erken nöral gelişim 19,27,29,31-32,40-42, retina birincil hücre kültürü 10,38,43-45 için özellikle uygun bir sistem olarak hizmet vermektedir. Presumptive retina dokusunun hemen nöral indüksiyon 25,38,43 takiben, gelişiminin erken aşamalarında erişilebilir. Buna ek olarak, inci verilmişembriyo her hücre sarısı bir kaynağı da içerir, retina hücreleri tamponlanmış bir tuz çözeltisi, çok basit bir tanımlandığı ortamda kültüre edilebilir, böylece kuluçka veya diğer serum bazlı ürünlerin 10,24,44-45 şaşırtıcı etkileri kaldırma . Bununla birlikte, çevre dokular ve daha sonraki işleme retina dokusunun izolasyon zordur. Burada da, tek bir hücre çözünürlükte kalsiyum etkinlik ve gen ekspresyonu için analiz edilecek birincil hücre kültürleri hazırlamak için kullanılır Xenopus laevis retina hücreleri disseksiyon ve ayrılma için bir yöntem sunmaktır. Bu yazıda sunulan konuyu kendiliğinden kalsiyum Transientlerin analiz ederken, teknik araştırma soruları ve yaklaşımlar (Şekil 1), geniş bir dizi geniş uygulanabilir.

Başarılı bir şekilde parçalanmış, optik vezikülleri (aşama 25) ve retinae (aşama 35) örnekleri Şekiller 2E ve 2J gösterilmiştir. Bu protokol geliştirme çeşitli aşamalarında kullanılabilir olsa da, daha fazla deneyler için doğruluğunu sağlamak için sadece retina dokusunun elde etmek önemlidir. Dikkatle tüm aşamalarında epidermisin kaldırmak ve forseps retina dokusunun ponksiyon olmamasını sağlamak. Aşama 35 veya daha büyük ise, mercek retina üzerinde bir katman açık...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis hücre kaderinin şartname ve primer hücre kültüründe bireyin retina hücrelerinin fizyolojik fonksiyonu eğitim için ideal bir model sistem sağlar. Burada retina dokuları diseksiyon ve kalsiyum etkinliği için görüntülü ve in situ hibridizasyon ile analiz edilir primer hücre kültürlerinde üretmek için bir yöntem mevcut.

Diseksiyon, Kültür ve Analizi<em&gt; Xenopus laevis</em&gt; Embriyonik Retina Doku

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

Research

JoVE Journal

Neuroscience

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

13.8K Görüntüleme.  College of William and Mary. Xenopus laevis hücre kaderinin şartname ve primer hücre kültüründe bireyin retina hücrelerinin fizyolojik fonksiyonu eğitim için ideal bir model sistem sağlar. Burada retina dokuları diseksiyon ve kalsiyum etkinliği için görüntülü ve in situ hibridizasyon ile analiz edilir primer hücre kültürlerinde üretmek için bir yöntem mevcut.

Video: Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

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.

Kök hücre benzeri<em&gt; Xenopus</em&gt; Embriyonik Eksplantlarındaki Erken Nöral Gelişim Özellikleri Okuyacak<em&gt; İn Vitro</em&gt; Ve<em&gt; İn Vivo</em

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

Gelişim Biyolojisi

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Larva, Pupa ve Erişkin diseksiyonu ve İmmünohistokimya<em&gt; Drosophila</em&gt; Retinaları

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Nörobilim

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Drosophila pupa Retina diseksiyon immünhistokimya, Batı analiz ve RNA izolasyon için

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Xenopus laevis Embriyolarında Göz tomurcuklarına DNA'nın mikroenjeksiyonu ve GFP'nin Sağlam Optik Aksonal Arbors'u İfade Etme Görüntülemesi, Yaşayan Xenopus Kurbağaları

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Test Görsel Fonksiyonu için Basit Bir Davranış Testi<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biyoloji

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebra balığı Embriyonik Göz Dokular, Mikrodisseksiyon

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Xenopus'ta Hücresel Onarım Stratejilerini Araştırmak için Retina Dejenerasyonu Oluşturma

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.

Yazar Spotlight: Xenopus Kurbağasında Retinal Yenilenme Mekanizmalarını Keşfetmek 

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.

Xenopus Kurbağa Yavrularında Retina Yaralanma Modellerinin Oluşturulması

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

Diseksiyon, Kültür ve Analizi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Kök hücre benzeri

Larva, Pupa ve Erişkin diseksiyonu ve İmmünohistokimya

Drosophila pupa Retina diseksiyon immünhistokimya, Batı analiz ve RNA izolasyon için

Xenopus laevis Embriyolarında Göz tomurcuklarına DNA'nın mikroenjeksiyonu ve GFP'nin Sağlam Optik Aksonal Arbors'u İfade Etme Görüntülemesi, Yaşayan Xenopus Kurbağaları

Test Görsel Fonksiyonu için Basit Bir Davranış Testi

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