الكتاب أشكر كريستا هاس، دوريس ويبر وبريجيت كورتي للحصول على المساعدة الفنية. ويدعم العمل في المختبر Weidinger من المنح المقدمة من جمعية الألمانية للبحوث WE 4223 / 3-1، WE 4223 / 4-1 ودويتشه FÜR غزلشافت Kardiologie عن طريق أوسكار-لاب-Stipendium وكلاوس جورج اوند-Sigrid- Hengstberger-Forschungsstipendium.

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li><li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish.">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, V., 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=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. 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الكتاب ليس لديهم ما يكشف.

الزرد الكبار لديه قدرة مذهلة على تجديد العديد من الأعضاء الداخلية والزوائد بنجاح. فهم شامل للالآليات الجزيئية والخلوية تشارك يتطلب تحليل الأنسجة محددة من وظائف الجينات ومسارات الإشارات. لتحقيق هذا، ويوفر النظام تيتون أداة فعالة للتعبير عن الجينات التي تسيطر عليها ...

الزرد هو كائن نموذج الفقاريات راسخة لدراسة العديد من جوانب التنمية، علم وظائف الأعضاء، والمرض، وتجديد. مع اعتماد متزايد من الزرد كنموذج للعمليات البيولوجية بعد الجنينية، والأدوات التجريبية للمحرض، والتلاعب الأنسجة محددة التعبير الجيني أو مسارات الإشارات أصبحت ذات أهمية متزايدة. بشكل خاص، عانت الدراسات إلى الجهاز وتجديد أطرافهم في الزرد الكبار من عدم وجود أدوات للتشريح من المتطلبات المكانية والزمانية لمسارات الإشارات خلال هذه العمليات التجدد. حاليا، وقد استخدمت ثلاثة أنظمة مختلفة لتحقيق المشروط، الأنسجة محددة التعبير الجيني في تجديد أجهزة الزرد الكبار: نظام جنة المساواة العرقية، السلمون المدخن، والتعبير فسيفساء من حرارة صدمة الجينات المحورة محرض باستخدام بوساطة ينقول transgenesis الجسدية، ونظام تيتون 1 -3. تيتون يشير إلى متغير من التتراسيكلين-تابعتوالت نظام تفعيل النسخي، حيث يتم تنشيط التعبير في وجود المضادات الحيوية التتراسيكلين أو مشتق، على سبيل المثال الدوكسيسيكلين. نظام جنة المساواة العرقية، السلمون المدخن، حيث تم حتى الآن استخدامها في الأسماك البالغة، يعتمد على لجنة المساواة العرقية recombinase تسيطر تاموكسيفين (CreERT2)، الذي يقتصر مكانيا من قبل عناصر تنظيمية الأنسجة محددة التعبير. إزالة لجنة المساواة العرقية يحركها من شريط إيقاف تسهل التعبير عن الجينات في المصالح يقودها المروج التي يجب أن تكون نشطة في جميع أنواع الخلايا 1. إنشاء بوساطة ينقول من الجينات المحورة جسدية أعرب mosaically يوفر نظام لمحرض التعبير التحوير في الأنساب الخلية الفردية. حقن الأجنة الزرد مع ينقول Tol2 يحمل الجين من الفائدة تحت السيطرة النسخي من الصدمة الحرارية النتائج المروج في الأفراد خيالية تحمل عادة التحوير فقط في الأنساب خلية منفصلة من جهاز تجديد 2. في حين أن كلا المنظومتين تتيح للالمشروط الأنسجة جمعية مهندسي البترولcific التعبير الجيني، ونظام لجنة المساواة العرقية، السلمون المدخن لا يمكن عكسها، واستراتيجية باستخدام القائم على ينقول وضع العلامات نسيلي يعاني من طبيعتها العشوائية. وبالتالي، نحن وغيرنا لقد تأقلم مؤخرا أنظمة تيتون المعدلة وراثيا لاستخدامها في الزرد، والتي تسهل زمانيا ومكانيا التعبير الجيني للرقابة التي بالإضافة الانضباطي وعكسها 3-5. يتألف نظام تيتون المستخدمة هنا خط سائق المعدلة وراثيا (TetActivator) فيه دوكسي (DOX) المنشط النسخي -inducible (تحسين transactivator التتراسيكلين العكسي، irtTA، باختصار تيتا) هو تحت سيطرة تسلسل الجينوم التنظيمية الأنسجة محددة. ثانيا، فإنه يتطلب خط الرد المعدلة وراثيا (TetResponder) التي تؤوي الجين من الفائدة تحت السيطرة النسخي المشغل التتراسيكلين (عنصر استجابة تيت؛ TetRE) (الشكل 1A). وهكذا، واستخدام مجموعات محددة من TetActivator وTetResponder خطوط يسمح للالمشروط الأنسجة SPECIالتلاعب المرورية في التعبير الجيني. لقد استخدمت مؤخرا نظام تيتون للتحقيق لوظائف الأنسجة محددة من WNT / بيتا catenin الإشارات في الكبار تجديد الذيل الزعنفة الزرد 3. في البروتوكول المذكورة هنا نحن تصف تدفق العمل لانشاء واستخدام نظام تيتون في الزرد، ولا سيما بالنسبة للدراسات تجديد الزعانف. هذا يتضمن تعليمات مفصلة حول كيفية توليد TetActivator وTetResponder خطوط المعدلة وراثيا مستقرة وبروتوكول لتحريض التحوير في الأجنة والزرد الكبار. وعلاوة على ذلك، وصفنا تقنيات للتحقق من الأنسجة محددة التعبير الجيني في تجديد الزعانف، بما في ذلك بروتوكول لإعداد cryosections زعانف الزرد الكبار. بالإضافة إلى ذلك، نحن نناقش اعتبارات لتصميم التحوير TetActivator، واختيار طريقة transgenesis، والكشف عن TetResponder التعبير. وبالتالي، فإن الهدف العام من هذا البروتوكول هو بمثابة مخططلوضع المتابعة نظام تيتون وظيفية في الزرد لتحقيق المشروط الأنسجة محددة التعبير الجيني، والتي يمكن تطبيقها على أي نوع من الأنسجة في المصالح. أنشأنا ناقلات TetActivator السماح لI-SCEI أو الجيل Tol2 بوساطة خطوط TetActivator مستقرة باستخدام قصيرة تسلسل الجينوم التنظيمية (شظايا محسن؛ Weidinger مختبر قاعدة البيانات البلازميد أي 1247؛ الشكل 1B). يحتوي هذا البناء الكاسيت TetActivator تتكون من البديل متحولة M2 من العكسي تيت مجال كاظمة تنصهر مع فيروس الهربس البسيط VP16 transactivation مجال مشتقة 3F [irtTAM2 (3F)]. التعبير عن TetActivator (تيتا) يمكن رصدها بسهولة لأنها مع AmCyan fluorophore من نفس إطار القراءة المفتوح أعرب المشارك؛ الببتيد P2A يتوسط الريباسي الطفر، والتي ينبغي أن يؤدي إلى إنتاج تيتا وAmCyan كما البروتينات منفصلة في نسبة 1: 1 5،6. يحتوي بناء أيضا poylinker 5 &#39;من TetActivator كاسيت لتسهيل إدراج تسلسل الجينوم التنظيمية ذات الاهتمام باستخدام أساليب الاستنساخ التقليدية. بالإضافة إلى ذلك، أنشأنا بناء تتكون من الكاسيت أعلاه وصف TetActivator بالإضافة إلى مجموعة كاسيت كانامايسين (Weidinger قاعدة البيانات البلازميد مختبر أي 1180؛ الشكل 1C)، والتي يمكن معاد في الكروموسوم الاصطناعي البكتيري (BAC) تحتوي على منطقة الجينومية كبيرة ( عادة في كودون بداية الجين الذي التعبير النمط أن يحاكي قبل التحوير). تتوفر كل من يبني من المختبر Weidinger بناء على طلبها.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breeding boxes</td>
			<td>Aqua Schwarz</td>
			<td>AquaBox 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>e.g. Leica, Thermo-Scientific</td>
			<td>varies with the manufacturer</td>
			<td>for cryosectioning</td>
		</tr>
		<tr>
			<td>4&#8217;, 6- diamidino-2-phenylinodole (Dapi)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use 1/5000 in PBS 
			for visualization of nuclei</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM) 
			for TetResponder induction</td>
		</tr>
		<tr>
			<td>E3 embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2&#903; 2 H2O, 0.33 mM MgSO4&#903;7 H2O, 0.2 &#8240; (w/v) methylene blue, pH 6.5 
			for embryo/larvae husbandry</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>4 % (w/v) paraformaldehyde in PBS, pH 7.5 
			for fixation</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline (PBS)</td>
			<td></td>
			<td></td>
			<td>1.7 mM KH2PO4, 5.2 mM Na2HPO4, 150 mM NaCl, pH 7.5</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline + Tween 20 (PBT)</td>
			<td></td>
			<td></td>
			<td>1x PBS with 0.1 % Tween 20</td>
		</tr>
		<tr>
			<td>Superfrost Ultra Plus adhesion microscope slides</td>
			<td>Thermo Scientific&#160;</td>
			<td>1014356190</td>
			<td>for collection of tissue sections</td>
		</tr>
		<tr>
			<td>Stereo fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>for fluorescence-based genotyping</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>e.g. Biorad, Applied Biosystems</td>
			<td>varies with the manufacturer</td>
			<td>for PCR-based genotyping</td>
		</tr>
		<tr>
			<td>Tissue freezing medium (TFM)</td>
			<td>Triangel Biomedical Sciences</td>
			<td>TFM-C</td>
			<td>for embedding of tissue samples</td>
		</tr>
		<tr>
			<td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>for anesthesia 
			use at 1 mg/ml in E3 embryo medium</td>
		</tr>
	</tbody>
</table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

لإنشاء نظام تيتون وظيفية لالأنسجة محددة التعبير الجيني محرض، TetActivator المعدلة وراثيا وخطوط TetResponder تحتاج إلى أن تتولد (الشكل 1A). يتم ذلك عن طريق microinjecting TetActivator (الشكل 1B - C) أو TetResponder (الشكل 1E) يبني في الأجنة الزرد المبكرة واللاحقة التك...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

استخدام نظام تيتون لدراسة الآليات الجزيئية لتجديد الزرد

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

use-teton-system-to-study-molecular-mechanisms-zebrafish

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JoVE Journal

Developmental Biology

10.6K Views.  Ulm University. Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Video: Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

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

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

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

تذرية الكبدية محددة في الزرد لدراسة يحركها صفراوية تجديد الكبد

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

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

تتبع الخلايا في الزرد-GFP المعدلة وراثيا باستخدام نظام Photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

تطوير متليفة الكبد نموذج الناجم عن الإيثانول في الزرد لدراسة السلف بوساطة الخلايا الكبدية التجديد

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

استخدام إيمونولابيلينج لتحليل Microtubules مستقرة ودينامية، والوليدة في الجنين الزرد

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

أساليب لدراسة التجديد في ستنتور

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

التهجين الكامل في الموقع في أجنة حمار وحشي وأنبوب تشكيل اساي في iPSC-ECs لدراسة دور Endoglin في التنمية الوعائية

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

فحص تجديد العضلات في نماذج حمار وحشي من أمراض العضلات

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

تعبير GFP الناجم عن الضوء في أجنة حمار وحشي باستخدام نظام TAEL/C120 Optogenetic

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

إزالة العين في يرقات الزرد الحية لفحص النمو والتطور المعتمد على التعصيب للنظام البصري

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

Primary Cell Cultures to Study the Regeneration Potential of Murine M&amp;#252;ller Glia after MicroRNA Treatment

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

مزارع الخلايا الأولية لدراسة إمكانات تجديد مورين مولر غليا بعد علاج الحمض النووي الريبي الميكروي

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...