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Neurosci. 14, 3475-3486 (1994).</li><li>Alexander, J., Rothenberg, M., Henry, G. L., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=casanova+plays+an+early+and+essential+role+in+endoderm+formation+in+zebrafish.">casanova plays an early and essential role in endoderm formation in zebrafish.</a> Dev. Biol. 215, 343-357 (1999).</li><li>Milewski, W. M., Duguay, S. J., Chan, S. J., Steiner, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+PDX-1+structure,+function,+and+expression+in+zebrafish.">Conservation of PDX-1 structure, function, and expression in zebrafish.</a> Endocrinology. 139, 1440-1449 (1998).</li><li>Argenton, F., Walker, M. 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Biol. 230, 189-203 (2001).</li><li>Thisse, C., Thisse, B., Schilling, T. F., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+zebrafish+snail1+gene+and+its+expression+in+wild-type,+spadetail+and+no+tail+mutant+embryos.">Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.</a> Development. 119, 1203-1215 (1993).</li><li>Cadieux, B., Chitramuthu, B. P., Baranowski, D., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+progranulin+gene+family+and+antisense+transcripts.">The zebrafish progranulin gene family and antisense transcripts.</a> BMC Genomics. 6, 156 (2005).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+and+knockdown+technology+to+define+proprotein+convertase+activity.">Use of zebrafish and knockdown technology to define proprotein convertase activity.</a> Methods Mol. Biol. 768, 273-296 (2011).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+situ+hybridization+on+whole-mount+zebrafish+embryo.">High resolution in situ hybridization on whole-mount zebrafish embryo.</a> Nat. Protoc. 3, 59-69 (2008).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Abbiamo sviluppato metodi migliori per visualizzare l&#39;RNA con alta risoluzione. Il nelle procedure di ibridazione in situ sono effettuate utilizzando semplici cesti su misura con fondi porosi (Figura 1). Gli embrioni sono trattati con soluzioni RNase-free in piastre da 6 pozzetti a temperatura ambiente in condizioni sterili. Per segregare gli embrioni cestini sono realizzati in diversi tubi colorati Eppendorf. Cestini con o senza cerchio vengono utilizzati per una facile orientamento e di identificare gli embrioni all&#39;interno del cestello come corrispondente ad una particolare sonda. Prehybridization a 65 ° C e ibridazione a 70 ° C con 50% formammide deionizzata è risultato essere particolarmente importante per ottenere segnali ad alta risoluzione. Inoltre incubando embrioni due volte con tampone Tris alcalina per 15 min alberino PBST lava dopo incubazione con anticorpo anti-DIG e prima dell&#39;incubazione in presenza di BCIP eNBT facilita la comparsa di segnali chiari di alta qualità. Nelle nostre mani abbiamo sperimentato che la permeabilizzazione e fissaggio di embrioni per il tempo ottimale e ibridazione a 70 ° C erano molto critico per ottenere segnali chiari. Permeabilizzazione inadeguata produce segnali ad alta sfondo. Permeabilizzazione estesa o di fissaggio inadeguato provocato il degrado di embrioni mentre fissaggio estesa inibito il rilevamento del segnale. Ibridazione tra 55-60 ° C producono anche segnali di fondo alti (Figura 7). La sonda senso rileverà un segnale per la trascrizione AS se il gene di interesse codifica antisenso (AS) trascritti. Dopo la reazione di colorazione viene arrestata ponendo embrioni in metanolo per 30 minuti o più converte il segnale ad un vero colore viola, e rimuove la colorazione non specifica. Embrioni fisse possono essere conservati al buio in soluzione di stop a 4 ° C per diversi mesi. I vantaggi e disadvantages di DESIDERIO Desiderio è una tecnica efficace per caratterizzare sia l&#39;espressione temporale e spaziale dei geni target durante l&#39;embriogenesi e stadi larvali. Questo protocollo permette anche la visualizzazione di espressione dell&#39;RNA dei due geni o co-localizzazione di RNA e proteina espressione al tempo stesso utilizzando opportune modifiche a seconda degli obiettivi sperimentali. Questo protocollo alta risoluzione può essere usato per rilevare l&#39;espressione di RNA in molti tessuti e tipi di cellule. Può essere usato per la rivelazione di specie molto basse di RNA abbondanti con minime segnali di sfondo. Tuttavia, per visualizzare l&#39;espressione precisa di geni all&#39;interno di scomparti interni di tessuti richiede l&#39;ibridazione in situ utilizzando sezioni di tessuto.

Il pesce zebra (Danio rerio) è emerso come un modello animale potente per lo studio dello sviluppo dei vertebrati, la malattia, il comportamento, e in-droga screening di 1-3. Zebrafish embrioni possono essere ottenuti in gran numero da un singolo incrocio. Fecondazione e sviluppo si verifica ex utero e gli embrioni otticamente chiare sviluppano rapidamente. Eventi critici evolutivi si verificano durante le prime 48 ore dopo la fecondazione (HPF). Ciò include la comparsa di organo primordi e l&#39;avvio di citodifferenziazione. Knockdown o sovra-espressione di proteine ​​può essere raggiunto attraverso la microiniezione di embrioni con antisenso morfolino (MO) oligonucleotidi o mRNA rispettivamente nella fase uno o due celle (0,75 HPF). Il desiderio di embrioni di zebrafish facilita lo studio della espressione spazio-temporale di specifici geni di interesse. Applicazione della tecnica WISH dopo microiniezione di embrioni con mRNA o MO a un eccesso di express o livelli di proteine ​​specifiche atterramento rivela regolazione differenziale di altri geni. Cambiamenti nei modelli di espressione genica possono essere correlati con i cambiamenti fenotipici e rivelano la funzione di geni bersaglio durante la prima organogenesi. Poiché sonde possono essere preparate e conservate in anticipo, la tecnica ISH può essere applicata a rivelare modelli di espressione genica per almeno 20 geni per volta utilizzando piastre a sei pozzetti e cestini misura.

<table><tbody><tr><td>Taq Polymerase</td><td>Invitrogen</td><td>10342053</td><td></td></tr><tr><td>TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td><td>Invitrogen</td><td>K4650-01</td><td></td></tr><tr><td>HQ Mini Plasmid Purification Kit</td><td>Invitrogen</td><td>K2100-01</td><td></td></tr><tr><td>Agarose</td><td>Roche</td><td>11 685 660 001</td><td></td></tr><tr><td>Not1 and HindIII Restriction Enzymes</td><td>Invitrogen</td><td>15441-025, 15207-012</td><td></td></tr><tr><td>Buffer Saturated Phenol, ultrapure</td><td>Invitrogen</td><td>15513039</td><td>Store at 4 &amp;deg;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td></tr><tr><td>Chloroform</td><td>Fisher</td><td>C607-1</td><td>Toxic and suspected carcinogen. Work under fume hood.</td></tr><tr><td>RNase-free DNAse I</td><td>Roche</td><td>4716728001</td><td>Prepare small aliquots and store at -20&amp;deg;C.</td></tr><tr><td>DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td><td>Roche</td><td>11277073910</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Roche</td><td>10777-019</td><td></td></tr><tr><td>3.0 M Sodium Acetate (pH 5.5)</td><td>Fisher</td><td>50-751-7355</td><td></td></tr><tr><td>100% Ethanol</td><td>Commercial alcohols</td><td></td><td></td></tr><tr><td>Diethyl Pyrocarbonate (DEPC)</td><td>Sigma</td><td>40718-25ML</td><td>DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS255-1</td><td></td></tr><tr><td>EDTA 0.5 M (pH 8.0)</td><td>Fisher</td><td>50-751-7404</td><td></td></tr><tr><td>Instant Ocean Salt</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher</td><td>FL-03-0900</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148-1KG</td><td>Prepare and store at -20&amp;deg;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td></tr><tr><td>Phenylthiocarbomide (PTC)</td><td>Sigma</td><td>P7629-25G</td><td>PTC is highly toxic. Use safety glasses, gloves, and dust mask.</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A947-4</td><td></td></tr><tr><td>Dumont (Watchmaker&amp;rsquo;s) Forceps pattern no. 5</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Six-well Cell Culture Cluster</td><td>Sarstedt</td><td>83.1839</td><td></td></tr><tr><td>Baskets are made of nylon mesh and Eppendorf tubes</td><td>In-house preparation</td><td></td><td>To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of&amp;nbsp;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&amp;nbsp;hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used.</td></tr><tr><td>Polyoxyethylenesorbitan Monolaurate (Tween 20)</td><td>Sigma</td><td>P1379-500ML</td><td></td></tr><tr><td>Proteinase K</td><td>Fermentas</td><td>EO0491</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma</td><td>242845</td><td></td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>90279-100ML</td><td></td></tr><tr><td>Formamide, high purity grade</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>AG501-X8 Resin</td><td>Bio Rad</td><td>142-6424</td><td></td></tr><tr><td>Citric Acid monohydrate</td><td>Sigma</td><td>C0706-500G</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Sigma</td><td>H3393-25KU</td><td></td></tr><tr><td>Saline-sodium Citrate Buffer (SSC)</td><td>Sigma</td><td>S6639</td><td></td></tr><tr><td>tRNA from bakers yeast type X</td><td>Sigma</td><td>R5636-1ML</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S640-500</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher</td><td>BP153-1</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>S25403</td><td></td></tr><tr><td>Levamisole Hydrochloride</td><td>Sigma</td><td>31742</td><td></td></tr><tr><td>N,N-Dimethylformamide&lt;br/&gt; anhydrous (DMF)</td><td>Sigma</td><td>227056</td><td>Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td></tr><tr><td>5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td><td>Sigma</td><td>N6639</td><td>Protect this solution from light.</td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Sigma</td><td>840W</td><td>Protect this solution from light.</td></tr><tr><td>Albumin from Bovine Serum, purified fraction V (BSA)</td><td>Sigma</td><td>A8806</td><td></td></tr><tr><td>Sheep Anti-digoxigenin-AP Fab Fragments</td><td>Roche</td><td>1093274910</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Sarstedt</td><td>83.1835</td><td></td></tr><tr><td>Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td><td>ZIRC</td><td>Tg(mnx1:GFP)ml2</td><td></td></tr><tr><td>Instant Ocean</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr></tbody></table>

high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function

Questo articolo si concentra su tutto il montaggio di ibridazione in situ (WISH) di embrioni di zebrafish. La tecnologia WISH facilita la valutazione dell&#39;espressione genica sia in termini di distribuzione tissutale e stadio di sviluppo. Protocolli sono descritti per l&#39;uso di DESIDERIO di embrioni di zebrafish utilizzando RNA antisenso sonde marcate con digossigenina. Le sonde sono generati da incorporare nucleotidi digoxigenin-linked attraverso la trascrizione in vitro su modelli genetici che sono stati clonati e linearizzato. Le chorions di embrioni raccolte a stadi di sviluppo definiti vengono rimossi prima dell&#39;incubazione con sonde specifiche. A seguito di una procedura di lavaggio per rimuovere la sonda in eccesso, gli embrioni vengono incubate con anticorpo anti-digossigenina coniugata con fosfatasi alcalina. Impiegando un substrato cromogenico della fosfatasi alcalina, specifica espressione genica può essere valutata. A seconda del livello di espressione genica l&#39;intera procedura può essere completata entro 2-3 giorni.

Utilizzando il protocollo con 50 embrioni per cesto (per gene / per condizione sperimentale) il pattern di espressione di tale gene può essere ottenuto in un esperimento. Quasi tutti gli embrioni presentano profili di espressione simili per un gene particolare. Esempi rappresentativi di ibridazione in situ colorazione sono mostrati nelle Figure 3-6. Sia il senso e anti-senso ribosonde stati sintetizzati dai cDNA corrispondenti a PC5.1 PC5.2, 6, 7, SCL/tal-1 gata-1 8,9, FLK / kdrl 10, shh 11, dlx2 12, fkd7 / FOXA1 13, 14,15 insulina, tripsina e 16 sono stati costruiti amplificando un segmento del mRNA full-length usando Taq DNA polimerasi, e clonato in pCRII-TOPO (Invitrogen). L&#39;autenticità dei singoli ampliconi è stata confermata mediante sequenziamento. Dopo la verifica dell&#39;orientamento clone, corrispondenti ribosonde antisenso sono stati sintetizzatiutilizzando i protocolli 17 con modifiche 18,19 come qui descritte utilizzando sia la Sp6 o T7 RNA polimerasi. Fasi coinvolte in tutto il montaggio ibridazione in situ vengono brevemente illustrate in Figura 2. Viola colorazione osservata dopo ibridazione con riboprobe di interesse rappresenta sia l&#39;abbondanza e siti di espressione del gene particolare. Per esempio PC5.1 mostra un&#39;espressione molto discreta all&#39;interno della parte anteriore e posteriore della vescicola otica e linea primordio laterale al 24hpf ed è fortemente localizzati entro i neuromasti linea laterale anteriore e posteriore da 72 HPF (Figura 3). Contrariamente PC5.2 spettacoli espressione ubiquitaria nel SNC con regionalizzazione distinto all&#39;interno somiti, vescicola otica e condotto pronephric ed è altamente espresso nel fegato e nell&#39;intestino a 96 HPF (Figura 4). Assenza di espressione genica utilizzando rispettivi riboprobes senso serve da controllo negativo. ( <strong&gt; Figure 3b e 4b). Analisi di espressione di marcatori del sangue hanno mostrato colorazione SCL/tal-1 all&#39;interno delle cellule cranici bilaterali e nella massa cellulare intermedia (ICM). Anche se la colorazione per gata-1 è anche visto nel ICM il pattern di espressione è diverso. L&#39;espressione di FLK / kdrl stata osservata entro hindbrain, principali e intersegmentale vasi e nella ICM (Figura 5). La colorazione per shh è stato osservato nel notocorda (Figura 5). L&#39;espressione di dlx2 a 34 hpf è localizzato nella telencefalo, diencefalo, l&#39;ipotalamo e cranici archi ectomesenchymal e l&#39;espressione fkd7/foxa1 a 24 HPF è visto soprattutto nel piatto piano e hypochord. Pattern di espressione dei marcatori pancreatici mostravano colorazione insulina nel pancreas endocrino e tripsina nel pancreas esocrino (Figura 6). Questi risultati mostrano pattern di espressione con alta risoluzione rappresenta il successo del protocollo descrittoper la rilevazione dell&#39;espressione di entrambi i geni abbondanti alta e bassa. Per ulteriori immagini chiarezza può essere preso usando la microscopia confocale dopo il montaggio degli embrioni su un vetrino da microscopio 20. Alcuni degli esempi dei risultati ottenuti durante la sperimentazione è stata effettuata, in condizioni sub-ottimali durante l&#39;ottimizzazione del protocollo sono mostrati in figura 7. Segnale di alta sfondo con scarsa espressione di Shh è stato notato sotto permeabilizzazione inadeguata e ibridazione tra 55-60 ° C. <img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig1large.jpg" src="/files/ftp_upload/50644/50644fig1.jpg" width="500px" /> Figura 1. Preparazione e utilizzo di Eppendorf-nylon cestelli a rete per tenere in scena embrioni. Un bene può contenere sei Eppendorf-nylon cestelli a rete. L&#39;immagine mostra le piastre sterili 6 pozzetti contenenti sei Eppendorf-nylon cestelli a reteutilizzato per diluizioni successive di metanolo in PBS. <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig2large.jpg" src="/files/ftp_upload/50644/50644fig2.jpg" width="500px" /> Figura 2. Diagramma che mostra i passaggi necessari per il tutto il montaggio in tecnica di ibridazione in situ. Dopo fissazione in scena embrioni possono essere conservati in metanolo a -20 ° C fino all&#39;uso. Le sonde possono essere sintetizzati in anticipo e conservati a -80 ° C. Memorizzazione di sonde in piccole aliquote è consigliabile, se si vuole utilizzare per lunghi periodi. In generale tutto il montaggio in esperimenti di ibridazione in situ può essere completato in 3 o 4 giorni. La reazione di colorazione per i geni espressi debolmente ci vorranno fino a 16 ore a temperatura ambiente o più a lungo quando la reazione di colorazione è effettuata a 4 ° C <a href="https://www.jove.com/files/ftp_upload/50644/50644fig2large.jpg" target="_blank">Clicca qui per ingrandire f</a>IGURA. <img alt="Figura 3" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig3large.jpg" src="/files/ftp_upload/50644/50644fig3.jpg" width="500px" /> Figura 3. L&#39;espressione di PC5.1 da tutto il montaggio nelle analisi di ibridazione in situ è stato effettuato con 24 HPF e 72 HPF in scena embrioni. (A) Vista laterale di 24 HPF embrioni mostrano un&#39;espressione molto discreta PC5.1 all&#39;interno anteriore e parte posteriore della vescicola otica e linea primordio laterale che utilizza PC5.1 antisenso riboprobe. Al 72 HPF colorazione specifica è stata osservata nei neuromasti linea laterale anteriore e posteriore. (B) Assenza di espressione genica utilizzando PC5.1 senso riboprobe serve come controllo negativo. <a href="https://www.jove.com/files/ftp_upload/50644/50644fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . gether.within-page = &quot;always&quot;&gt; <img alt="Figura 4" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig4large.jpg" src="/files/ftp_upload/50644/50644fig4.jpg" width="500px" /> Figura 4. Alle 24 HPF e 96 HPF tutto il montaggio nelle analisi di ibridazione in situ di PC5.2 è stata effettuata utilizzando PC5.2 anti-senso e ribosonde senso. (A) Vista laterale di 24 HPF embrioni mostrano espressione ubiquitaria all&#39;interno del sistema nervoso centrale, somiti vescicola otica e condotto pronephric. Colorazione specifica nel fegato e intestino è visto a 96 HPF utilizzando PC5.2 antisenso riboprobe. (B) Assenza di espressione genica utilizzando PC5.2 senso riboprobe serve come controllo negativo. <a href="https://www.jove.com/files/ftp_upload/50644/50644fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . oad/50644/50644fig5.jpg &quot;width =&quot; 500px &quot;/&gt; . Figura 5 Whole-mount in analisi di ibridazione in situ a 24 HPF utilizzando SCL/tal-1 riboprobe mostrava colorazione all&#39;interno delle cellule cranici bilaterali e nella massa cellulare intermedia (ICM), Gata-1 riboprobe colorazione è visto nel ICM; la flk espressione / kdrl all&#39;interno del cervello posteriore, principale, e vasi intersegmentale e l&#39;ICM. La colorazione per shh è stato osservato nel notocorda. <img alt="Figura 6" fo:content-width="5in" fo:src="/files/ftp_upload/50644/50644fig6large.jpg" src="/files/ftp_upload/50644/50644fig6.jpg" width="500px" /> . Figura 6 Whole-mount in analisi di ibridazione in situ a 34 HPF utilizzando dlx2 riboprobe rivelato espressione dlx2 nel telencefalo, diencefalo, l&#39;ipotalamo, e cranici archi ectomesenchymal; l&#39;espressione fkd7/foxa1 a 24 HPF è visto soprattutto nel piano ptardi e hypochord; colorazione insulina è osservata nel pancreas endocrino ed espressione tripsina nel pancreas esocrino. <img alt="Figura 7" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig7large.jpg" src="/files/ftp_upload/50644/50644fig7.jpg" width="500px" /> Figura 7. Permeabilizzazione adeguata e appropriata temperatura di ibridazione sono fondamentali per una chiara in segnali di ibridazione in situ. Tutto il montaggio nelle analisi di ibridazione in situ a 24 HPF e 48 HPF utilizzando un riboprobe shh rivelato espressione all&#39;interno della notocorda e fondello senza segnale di fondo, quando gli embrioni sono permeabilizzate da digestione con proteinasi K per 12-15 min e ibridato a 70 ° C. Pattern di espressione incompleti e segnali di fondo alti sono stati osservati quando gli embrioni sono stati sottoposti a digestione insufficiente (5 min trattamento con proteinasi K) o ibridato a bassa temperatura (60 ° C). <a href="https://www.jove.com/files/ftp_upload/50644/50644fig7large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

Watch this Scientific Journal Video about High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function at JoVE.com

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Il pesce zebra, un piccolo pesce tropicale, è diventata un modello importante per studiare la funzione del gene durante lo sviluppo dei vertebrati e la malattia. L&#39;espressione temporale e spaziale dei geni bersaglio può essere determinata<em&gt; In situ</em&gt; Ibridazione. Nostra migliore protocollo permette l&#39;individuazione di bassi trascritti abbondanti con basso segnale sfondo non specifico.

Alta Risoluzione intero monte<em&gt; In Situ</em&gt; Ibridazione in embrioni di zebrafish per studiare l&#39;espressione genica e la funzione

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

high-resolution-whole-mount-situ-hybridization-within-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

22.1K Views.  Royal Victoria Hospital. Il pesce zebra, un piccolo pesce tropicale, è diventata un modello importante per studiare la funzione del gene durante lo sviluppo dei vertebrati e la malattia. L'espressione temporale e spaziale dei geni bersaglio può essere determinata In situ Ibridazione. Nostra migliore protocollo permette l'individuazione di bassi trascritti abbondanti con basso segnale sfondo non specifico.

Video: High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Immunocolorazione fosfo-epitopi in Ciliated Organi di monte intero embrioni di zebrafish

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Ricerca

Chimica

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualizzazione di cellule Multiciliated in Zebrafish attraverso un protocollo combinato di tutta Monte fluorescente ibridazione In Situ e immunofluorescenza

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Biologia dello sviluppo

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biologia

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotipizzazione e quantificazione della colorazione dell'ibridazione in situ nel pesce zebra

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Un metodo di montaggio a strati per la microscopia confocale Time-Lapse estesa di embrioni di pesci zebra interi

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Intero Monte Immunohistochimica in Embrioni e Larve di pesce zebra

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

Sul posto Ibridazione in larve di zebrafish e giovani durante lo sviluppo di Mesonephros

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Utilizzando il monte intero In situ Ibridazione a Link Biologia Molecolare e organismal

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Preparazione di montaggio schermo per l&#39;osservazione e l&#39;analisi di zebrafish campioni macchiato da Whole Monte<em&gt; In situ</em&gt; Ibridazione

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

Sul posto Ibridazione combinata con immunoistochimica in embrioni di zebrafish criosezionati

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

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