Name: large-scale zebrafish embryonic heart dissection for transcriptional analysis
Uploaded: 2015-01-12T13:30:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

Bu protokol Alman ve Berlin eyalet yasasının hayvan bakım kurallarına şöyle; Zebra balığı işleme hayvan koruma (LaGeSo, Berlin-Brandenburg) yerel otorite tarafından izlendi. 1. Kalp Ekstraksiyon için Zebra balığı Embriyolar Alınması <ol><li> Transgenik hat 9 twu34 Kalbe spesifik GFP ile embriyolar elde etmek için: bu tür Tg (EGFP myl7) halinde çapraz kalp raportör zebra balığı. </li><li> İstenilen embriyonik aşamada 10,11 yılına kadar 28.5 ° C&#39;de yumurta suda embriyolar koruyun. Toplanan embriyo nüfus gen ifadesi değişikliklerini sınırlamak için hem genetik ve gelişimsel aşamada homojen olduğundan emin olun. </li><li> Elle embriyolar Dechorionate. </li></ol> 2. Kesme Zebra balığı Embriyonik Kalpler NOT: buz üzerinde tüm çözümleri tutun. Mümkünse, takım çalışması yapın: Bir kişi emb gelen kalpleri masayaryos başka bir kişi floresan stereomikroskop altında kalpleri sıralar ise. Bu, birkaç saat içinde embriyo yüzlerce işlenmesine olanak tanır. <ol><li> 1.5 ml santrifüj tüpüne yaklaşık 100 embriyolar transfer. Tricaine embriyo (E3 ortamı içinde 0.16 mg / ml) anestezisi. Embriyolar açıkça anestezi kez devam edin (onlar yüzmek ve tüpün dibine tortu, ama yürekleri hala yenerek değil). </li><li> Tricaine / E3 çözeltisini çıkarın ve 1 ml L-15/10% FBS ortamı ile bir kez embriyolar yıkama ve buz üzerinde muhafaza. NOT: L-15 /% 10 FBS orta kalpleri RNAlater veya Trizol içine devredilmiştir kadar tüm diseksiyon işlemi sırasında canlı organ ve hücreleri tutmak önemlidir. </li><li> Sarısı tamamen bozulur kadar Yuvarlak Jel Yükleme İpucu yukarı ve aşağı embriyolar ve pipet 5-8 kez 1 ml L-15 /% 10 FBS orta ekleyin. Embriyolar damla damla çözeltinin yüzeyine açılan patlama böylece pipet veEmbriyolar hafifçe bozulur. NOT: yukarı pipetle ve aşağı, yani daha pipet geç evrelerde (örneğin 56 hpf) ihtiyaç olduğu, embriyoların gelişim aşamasında bağlıdır edilecek Nasıl şiddetle ve ne sıklıkta embriyolar var. </li><li> Çok hafifçe pipetle, bazı kalpleri embriyoların takılı kalabilir çünkü disseke embriyoların ilk parti için, embriyoların bütünlüğünü ve bir stereo mikroskop altında disseke kalpleri oranını değerlendirmek. Buna göre diseksiyon sonraki tur için pipetleme şeklini ayarlayın. NOT: plastik yapışmasını kalpleri en aza indirmek için düşük tutma pipet uçları ve mikrosantrifüj tüpleri kullanın. </li><li> 50 ml&#39;lik santrifüj tüpüne yerleştirilen 100 mikron filtre üzerine numuneyi uygulayın; 1 ml L-15/10% FBS ortamı ile 1.5 mi tüp durulama ve sonra, (bir kupa tüpün duvarlarına yapışmasını olabilir) örnek kaybını en aza indirmek amacıyla, filtreye, bu geçerlidir. 1 ml L, iki kez filtre yıkayın-15/10% FBS. Bu adımda, kalpler filtre içinden geçer ve 50 ml&#39;lik bir tüp içinde toplanmıştır. </li><li> 15 ml&#39;lik bir santrifüj tüpüne yerleştirilir, 30 mikron filtre üzerine akış yoluyla uygulanır ve 1 ml L-15/10% FBS ortamı ile bir kez durulayın. Bu ikinci filtrasyon adımında, kalpler filtre muhafaza edilir ve küçük çöp yıkanır. </li><li> Baş aşağı çevirin ve filtreyi 1 ml L-15/10% FBS ile arka arkaya üç yıkar uygulayarak% 1 agaroz kaplı petri içine filtre dışarı kalpleri yıkayın. </li><li> Elle floresan stereomikroskop altında forseps bir çift ile nonfluorescent embriyonik enkaz (örn göz mercekleri) GFP-pozitif kalpleri ayırmak ve yemeğin ortasında onları konsantre. Adım 3&#39;e göre onları toplayın. NOT: embriyolar daha eski 24 hpf iseniz, kalpler, dayak dokular hala fizyolojik normal olduğunu belirten olmalıdır. </li></ol> 3. Dissec mRNA izoleted Kalpler <ol><li> (Buz üzerinde) ve 0.75 ml RNAlater ihtiva eden 1.5 ml&#39;lik bir tüp içine mümkün olan en küçük hacimde kalpleri (örneğin, 10 | il) ve pipet toplayın. Hiçbir kalpler bir floresan stereomikroskop altında görüntüleyerek pipet kalır emin olun. </li><li> Alternatif olarak, bir kimyasal başlık floresan mikroskop hemen yakınında varsa, kimyasal başlık altında Trizol (DİKKAT) toplanır kalpleri aktarmak. Bu pipet ve aynı zamanda kalplerin santrifüj sırasında yapışmasını kalplerin olası kaybını circumvents (3.4, 3.5 ve 3.7 adımları). Trizol ile aynı tüpe izolasyon birkaç tur kalpleri havuzu ve 3.8 adıma doğrudan gitmek; Nihai hacim orijinal Trizol hacminin% 10 geçmemelidir. NOT: Kullanmadan önce Trizol Malzeme Güvenlik Bilgi Formu (MSDS) okuyun. Bir başlık altında Trizol reaktif Kulp ve Kişisel Koruyucu Ekipman tavsiye aşınma. Cilt, göz ve clothi ile temastan kaçınınng. Tüm tutuşma kaynaklarını uzaklaştırın. </li><li> RNA izolasyonu etkinliği toplanan doku miktarı ile iyileştikçe, (buz üzerinde) RNAlater aynı tüp içine tecrit birkaç tur kalpleri havuzda toplayın. Toplam örnek hacmi, orijinal RNAlater hacminin% 10&#39;unu aşması halinde, (buz üzerinde) depolama için 0.75 ml RNAlater ile ek bir tüp kullanın. </li><li> Kalpleri çökeltmek için 4 ° C&#39;de 20 dakika boyunca 15.700 x g&#39;de örnekleri santrifüjleyin. </li><li> Flöresanlı bir stereomikroskop altında, 3 ml L-15/10% FBS ortamı içeren bir% 1 agaroz kaplı petri içine mümkün olduğu kadar çok dikkatli bir şekilde süpernatan toplamak ama santrifüj tüpünün tabanına tabakalandığı kalpleri rahatsız yoktur. Kalpleri% 15 e kadar bağlı RNAlater yüksek bir viskoziteye sahip süpernatan içinde kalabildiği için aşama 3.7&#39;de tarif edildiği gibi, bunları almak. </li><li> Bir kimyasal Kaputun altında, sedimante kalpleri içeren tüp 0.5 ml Trizol ekleyin ve buz üzerinde tutmak. </li><li> Floresan microsco altındaPE, sulandırmak için 3 ml L-15/10% FBS ile bir% 1 agaroz-kaplanmış çanak içine RNAlater / l-15/10% FBS çözeltisi ihtiva eden,% 1 agaroz-kaplanmış çanak (adım 3.5 den) kalpleri aktarmak RNAlater dışarı. Petri kasesinin ortasına kalpleri toplamak ve kimyasal bir başlık altında Trizol tabakalandığı kalplerin tüp içine, küçük bir hacim içinde aktarın. </li><li> Girdap Trizol kalpleri ihtiva eden 1.5 ml&#39;lik bir tüp kalpleri bozabilir ve oda sıcaklığında 5 dakika boyunca inkübasyona. </li><li> Bir kimyasal Kaputun altında, 100 ul kloroform (DİKKAT) ekleyin, iyice karıştırın ve (kullanmadan önce maksimum hızda santrifüj 30 sn) 1.5 ml önceden bükülmüş PLG tüp Trizol / kloroform çözüm aktarın. Oda sıcaklığında 3 dakika boyunca inkübe edin. NOT: Kullanmadan önce kloroform MSDS okuyunuz. Bir başlık altında kloroform reaktif Kulp ve Kişisel Koruyucu Ekipman tavsiye aşınma. Deri, göz ve giysilere dokunmayınız. Alkaliler, metal gibi uzak Uygunsuz maddelerden tutunuz. </li><li> Santrifüjge 4 ° C&#39;de 15 dakika boyunca 15.700 x g&#39;de numune ve yeni bir 1.5 ml&#39;lik bir tüp içine, sulu faz aktarılır. </li><li> 5-10 mikrogram glikojen ve izopropanol (-20 ° C&#39;de) soğutulmuş 250 ul ekleyin; iyice karıştırılır ve -20 ° C &#39;de gece boyunca inkübe edilir. </li><li> 4 ° C&#39;de 30 dakika boyunca 15,700 x g&#39;de santrifüjleyin ve süpernatant atılır. </li><li> 4 ° C&#39;de 15 dakika boyunca 15.700 x g&#39;de 1 mi% 75 etanol, santrifüj ile pelet yıkayın ve süpernatant atılır. </li><li> Bu (10-15 dakika, örneğin), beyaz hale gelinceye kadar oda sıcaklığında pelet kurutulmasıyla etanol buharlaşmasını olsun. </li><li> Pelet 20 ul RNase içermeyen GKD 2 O ekleyin ve RNA çözmek üzere 55 ° C de 10-15 dakika inkübe edilir; Daha sonra buz üzerinde tutmak. </li><li> Absorbans ölçümü RNA konsantrasyonu ve saflığı değerlendirin. Bir% 1 agaroz jeli üzerinde örneği çalışmadan RNA bütünlüğü değerlendirin. </li></ol>

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The authors have nothing to disclose.

Bu protokol gen ifadesi için Zebra balığı Embriyonik kalp dokusunun hızlı zenginleşme analizleri verir. RNA arıtma sadece yeterli çalışır çünkü, kalplerin kaybı protokolü her aşamasında önlenir, ilk numune miktarı büyük ölçüde artırıldı: kardiyak spesifik RNA örnek miktarı ve kalitesi büyük ölçüde bir kaç önemli adımlar bağlıdır başlangıç ​​malzemesi. İkinci olarak, deneyi ile tamamen bağlıdır numune saflığı, sıralama ve sıkı bir şekilde diğer dokuları hariç...

Balığı (Danio rerio), yaygın küçük olması nedeniyle ve floresan proteinleri dokuya-özel ifadesi transjenik raportör hatları durumu ile birleştirilmiş olan, hızlı, şeffaf ve rahim dışı embriyonik gelişme, in vivo olarak organogenez çalışma gelişim biyolojisi kullanılır. Bu küçük omurgalı, özellikle de erken Zebra balığı embriyonun oksijenlenme, kalp atışı ve kan akımı dayanmaz çünkü kalp gelişimini incelemek için uygundur; Bu özellikler kardiyovasküler mutantların 1,2 sayıda karakterizasyonu izin ve zebra balığı artık kalp hastalıkları 3 incelemek için yaygın olarak tanınan bir model organizmadır. Embriyo gelişimi esnasında gen ekspresyonunu incelemek için, transkriptler genellikle tüm montaj in situ melezleşme (VVISH) 4 ile analiz edilmiştir, RT-qPCR 5, mikrodiziler 6, ya da bir sonraki nesil sıralama (RNA Dizi) 7. SüreWISH analiz tüm embriyo olan gen ifadesinin uzamsal ve zamansal analizi, transkript seviyeleri, genellikle, RT-qPCR mikrodizilerdeki ya da RNA-Seq yaklaşımlarla değerlendirilir sağlar. Bununla birlikte, bu yöntemler, belirli bir gen ekspresyonu profillemesi doku zenginleştirme gerektirir. Zebra balığı Embriyonik kalp tüm embriyo küçük bir kısmını temsil ettiğinden dolayı, kalp gelişimi sırasında transcriptome çalışmaları diseksiyon ve kalpleri zenginleştirilmesi için bir protokol gerektirir. Buna ek olarak, fizyolojik olarak ilgili veri elde etmek için, bu RNA ekstraksiyonu kadar tam olarak işlevsel kalp dokusunu muhafaza etmek önemlidir. Burada, biz hızla fizyolojik, normal izole ve verimli bir şekilde ileri analizler için yüksek kaliteli RNA örnekleri elde etmek için Zebra balığı embriyolarının yüzlerce kalpleri yenerek bir protokol açıklar. Bu yöntem Burns ve MACRAE 2006 8 tarafından bildirilen protokol dayanmaktadır. Kalp dokusunun zenginleşmesi için, her iki yöntem bir transgenik miyokard muhabiri hattını kullanmakve diğer dokulara karşı Zebra balığı embriyonik kalpleri diferansiyel yapışma özelliklerine yararlanmak. Kısaca, dar bir pipet ucu ile yukarı ve aşağı doğru çok sayıda embriyolar pipetleme, kalpler aynı embriyonik gövdelerinden serbest bırakılır ve daha sonra, iki hızlı bir filtreleme adımlarından embriyonik enkaz ayrılır; floresan etiketli kalpler sonra el kalan enkaz sıralanır ve daha fazla işlem için toplanır.

<table><tbody><tr><td>Equipment for raising fish and collecting eggs&amp;nbsp;</td><td></td><td></td><td>see the Zebrafish Book&lt;sup&gt;11&lt;/sup&gt; for details</td></tr><tr><td>Fluorescence stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Refrigerated Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>UV-Spectrophotometer</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Thermo Scientific Nanodrop 2000</td></tr><tr><td>Nucleic acid electrophoresis chamber</td><td></td><td></td><td></td></tr><tr><td>Petri dishes 4 cm &amp;Oslash;, coated with 1% agarose in E3 medium</td><td></td><td></td><td></td></tr><tr><td>Micropipettes and tips (P20, P100, P1000)</td><td></td><td></td><td></td></tr><tr><td>1.5 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>Pair of Dumont #5 forceps</td><td></td><td></td><td></td></tr><tr><td>ExactaCruz&amp;trade; Round Gel Loading Tips in Sterile Rack, 1-200&amp;mu;l&amp;nbsp;</td><td>Santa Cruz</td><td>sc-201732</td><td></td></tr><tr><td>100 &amp;mu;m filter (BD Falcon 100 mm Cell Strainer)</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>30 &amp;mu;m filter (Pre-Separation Filters-30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Phase lock gel, heavy, 1.5 ml tubes&amp;nbsp;</td><td>Prime</td><td>2302810</td><td></td></tr><tr><td>Egg water medium</td><td></td><td></td><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td></tr><tr><td>E3 medium&amp;nbsp;</td><td></td><td></td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td>A-5040</td><td>4 mg/ml Tricaine stock solution, pH 7</td></tr><tr><td>1% agarose (in E3 medium)</td><td></td><td></td><td></td></tr><tr><td>1% agarose gel (in TBE buffer)</td><td></td><td></td><td></td></tr><tr><td>Leibovitz&amp;acute;s L-15 medium&amp;nbsp;</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7020</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>1559606</td><td></td></tr><tr><td>Glycogen&amp;nbsp;</td><td>Invitrogen</td><td>10814-010</td><td>20 &amp;micro;g/&amp;micro;l in RNase-free water</td></tr><tr><td>chloroform</td><td></td><td></td><td></td></tr><tr><td>isopropanol</td><td></td><td></td><td></td></tr><tr><td>75% ethanol (in DEPC-ddH2O)</td><td></td><td></td><td></td></tr><tr><td>Nuclease-free water or sterilized DEPC treated ddH2O</td><td></td><td></td><td></td></tr><tr><td>Nucleic acid loading buffer</td><td></td><td></td><td></td></tr><tr><td>TBE (Tris/Borate/EDTA) buffer for electrophoresis</td><td></td><td></td><td></td></tr></tbody></table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

Zebra balığı Embriyonik kalp tüm embriyonun sadece küçük bir kısmını temsil eden, sadece birkaç yüz hücrelerden oluşur. Bu nedenle, genel embriyonik transcriptome tarafından maskelenen kardiyak transcriptome önlemek için, ayrıca analiz için kalpleri yeterli sayıda toplamak için gereklidir. Zebra balığı kalp geliştirme hızla devam Dahası, kalp toplanması ve RNA ekstraksiyonu yöntemleri numunelerin homojen olmasını sağlamak için hızlı olması gerekir. Burada, biz Zebra balığı embriyolarının fonksiyonel / yenerek kalpleri toplamak için hızlı manuel diseksiyon protokolü sunuyoruz. Bu transcriptome analizleri ile bu kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (RT-qPCR) halinde, kalp-özgü gen ekspresyon seviyelerini belirlemek için daha sonra, kalp-özel RNA ekstraksiyonu için bir temel oluşturur. yöntem, diğer dokulara göre zebra balığı embriyonik kalp diferansiyel yapışma özellikleri dayanmaktadır; Bu hızlı laşılmakla sağlarakışkan kesme kuvveti bozulması, aşamalı filtrasyon ve transgenik floresan etiketli kalplerin manuel toplama bir kombinasyonu ile dışı dokusundan kalp ceği ayrılması.

Sadece miyokardın (. Şekil 1) içinde twu34 transgenik hattı 9, yeşil floresan proteininin (GFP) ifade eder: Burada, biz zebrabalıkları Tg (GFP myl7) kullanarak bir temsilci kalp diseksiyon deneyi tarif. Biz disseke kalpleri ve kalp numunenin saflığını değerlendirmek için elde edilmiştir hangi embriyolar hem topladı. Kısaca, homozigot Tg (myl7 GFP) twu34 zebra balığı 9, embriyonik kalpleri GFP etiketli ki, vahşi tip ile ç...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Zebra balığı kalp gelişimi sırasında kalp gen ekspresyon profillerini analiz etmek, toplam RNA izole kalpleri elde edilecek olan. Burada, biz kalp-özel mRNA elde etmek Zebra balığı embriyolarının gelen hızlı manuel diseksiyon ile işlevsel / yenerek kalpleri toplamak için bir protokol mevcut.

Transkripsiyonal Analiz Büyük ölçekli Zebra balığı Embriyonik Kalp Diseksiyon

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

14.4K Görüntüleme.  Max Delbrück Center for Molecular Medicine.  Zebra balığı kalp gelişimi sırasında kalp gen ekspresyon profillerini analiz etmek, toplam RNA izole kalpleri elde edilecek olan. Burada, biz kalp-özel mRNA elde etmek Zebra balığı embriyolarının gelen hızlı manuel diseksiyon ile işlevsel / yenerek kalpleri toplamak için bir protokol mevcut. 

Video: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

İzolasyon ve Zebra balığı Embriyolar gelen Tek Hücre Karakterizasyonu

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Gelişim Biyolojisi

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Larva Zebra balığı Gonadal Doku Diseksiyon

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Miyokardiyal Mekanik Deformasyonun Yer Değiştirme Analizi (DIAMOND) Embriyonik Zebrabalığında Kardiyak Fonksiyonun Segmental Heterojenliğini Ortaya Çıkarır

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Yamultma stres gelişmekte olan zebra balığı kalp oransal etkileri 4 boyutlu görüntülerini yakalamak için ışık sayfalık floresans mikroskobu

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Biyomühendislik

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Larvaların Kalp Diseksiyon, Çocuk ve Yetişkin Zebra balığı, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Biyoloji

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebra balığı Embriyonik Kalp Dissected, immün

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Zebra Balığı Embriyolarında Hızlı Kardiyak Dinamiklerin Işık Tabakası Mikroskopisi

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Zebra Balığı Kalplerinde CRISPR-Cas9 Aracılı Hassas Knock-In Düzenlemeleri

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...