Name: dissection and flat-mounting of the threespine stickleback branchial skeleton
Uploaded: 2016-05-07T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.

Tutti i lavori di pesce è stato approvato dal Comitato Istituzionale cura degli animali e l&#39;uso della University of California-Berkeley (numero di protocollo R330). L&#39;eutanasia è stata effettuata utilizzando immersione in 0,025% tricaine-S tamponata con 0,1% di bicarbonato di sodio 39. Tutte le fasi sono eseguite a temperatura ambiente. 1. Preparazione Nota: Eseguire i passi 1.1-1.5 in tubi conici o fiale di scintillazione in grado di sigillare ermeticamente ed essere fissate in orizzontale. I pesci non devono essere costantemente scosso, ma cercare di miscelare la soluzione più spesso possibile capovolgendo delicatamente o agitando il rack di tubi o fiale per esporre tutti i lati del pesce alla soluzione colorante e consentire macchia di penetrare il tessuto in modo uniforme. Non posizionare una grande partita di pesce su un agitatore piattaforma, come il peso del liquido si romperà lo shaker. <ol><li> Fissare o pesce appena eutanasia o pesce conservato in etanolo al 10% neutra tamponata formalina (NBF) durante la notte. In alternativa, utilizzare il 4% Paraformaldeide in soluzione PBS 1x invece del 10% NBF. Nota: se l&#39;estrazione del DNA, clip di una piccola porzione di caudale o pinne pettorali prima della fissazione e conservare in etanolo. </li><li> Smaltire correttamente fix in una cappa chimica e sostituire con acqua di rubinetto (cioè ~ pH 7,0) per 2 ore. Evitare di utilizzare acqua deionizzata come spesso può essere acido e può decalcificazione ossea. </li><li> Rimuovere l&#39;acqua e pesce macchia con 0,008% Alizarina Rossa S in 1% KOH in acqua per 24 ore. Per il pesce meno di 20 mm di lunghezza standard, utilizzare 0.004% Alizarina Rossa S. (Fai un 100x (0,8%) soluzione madre di Alizarina Rossa S che possono poi essere diluita). </li><li> Rimuovere macchia (mettendo in idoneo contenitore per rifiuti in cappuccio) e il luogo di pesce in acqua di rubinetto per pochi ore. Cambiare l&#39;acqua, se necessario fino a quando l&#39;acqua di risciacquo è per lo più chiara. </li><li> Rimuovere l&#39;acqua e il luogo di pesce in glicerolo al 50%, 0,25% KOH per la compensazione mite e la successiva dissezione. Nota: Questo protocollo di colorazione viene modificato dai metodi descritti in precedenza 40,41. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Dissezione Nota: Vedere la Figura 1 per una revisione delle pertinenti testa morfologia scheletrica. <img alt="Figura 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> Figura 1:. Stickleback testa morfologia scheletrica Alizarina Rossa macchiato testa Spinarello ripreso con fluorescenza sotto un set di filtri rodamina B. morfologia Utile è etichettato: Op = opercolo, Subop = subopercle, BSRS = branchiostegi raggi, preop = preopercolo, Infraorb 1-3 = infraorbitale 1-3 (chiamati anche circumorbitals o suborbitals), Dent = dentario, Premax = premaxilla, Max = mascellare , Nas = nasale, Lat. ETHM = etmoide laterale, Psph = parasphenoid, Fron = osso frontale. Per una descrizione più dettagliata della testa di scheletro spinarello, vedere Anker (1974) 31. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande</a>questa figura. <ol><li> Appoggiare il pesce piatto (Figura 2A) e inserire pinze taglienti # 5 orologiai nel lato dell&#39;occhio ad un angolo di ~ 45 ° per forare la membrana che ricopre l&#39;occhio. </li><li> Peel la membrana distanza dall&#39;occhio, simile alla pelatura un coperchio yogurt (Figura 2B). </li><li> Inserire una pinza aperta dietro l&#39;occhio, afferrare del nervo ottico dietro l&#39;occhio, e rimuovere l&#39;occhio (Figura 2C). Non forare l&#39;occhio in quanto perderà melanina. Se forato, la melanina può essere lavato via durante le fasi successive. </li><li> Ripetere sull&#39;altro lato. </li><li> A partire dal posteriore, posizionare una piccola lama da dissezione forbice sotto il lembo opercolo, lama trascinare forbice dorsale sopra opercolo, quindi tagliare i tessuti molli fino alla cavità oculare (Figura 2D). Tagliare dorsale all&#39;osso opercolo. </li><li> Tagliare l&#39;osso frontale (dorsale alla cavità oculare) (Figura 2E). </li><li> Tagliare l&#39;osso parasphenoid linea mediana intorno tegli centro delle orbite (Figura 2F). </li><li> taglio opercolo Ripetere sul lato opposto. </li><li> Inserire una pinza sotto l&#39;opercolo e sbucciare lentamente la faccia lontano dal corpo, tagliare qualsiasi tessuto molle ancora attaccato (Figura 2G - H). Fare attenzione a non interrompere la prima fila di branchiospine. <ol><li> Con una pinza, staccare i ceratohyals su entrambi i lati del basihyal linea mediana, mentre staccando e rimuovendo lo scheletro cranio anteriore (l&#39;intera mascella compreso il dentario, premaxilla, e della mascella, l&#39;intero scheletro ioide compreso il opercolo dermico esterno, preopercolo, subopercle, e raggi branchiostegi e dorsale alla base e gli elementi endochondral ventrali, e la parte anteriore del cranio, compresi il naso, etmoide laterali e ossa infraorbitale, vedi figure 1 e 2I). </li><li> spine pelvico possono essere ripiegati fuori dal corpo, e può servire come una maniglia per pinze per afferrare hold of quando presente. Spine bloccano in posizione. Per sbloccare, tirare delicatamente colonna vertebrale con una pinza direttamente via dal corpo di pesce, poi flettere leggermente posteriormente a premere schiena piatta contro il pesce. </li></ol></li><li> Inserire chiuso pinza posteriore e ventrale di scheletro branchiale (appena al di sotto del tubo intestinale) e pinze trascinare anteriormente, prendere in giro a parte i restanti muscoli e legamenti attaccati allo scheletro branchiale (Figura 2J - K). </li><li> Utilizzando la punta delle pinze chiuse, raschiare via i muscoli connessi dorsale scheletro branchiale alla scatola cranica ventrale in un posteriore in direzione anteriore (Figura 2L). </li><li> Ripetere 2.9 e 2.10 sul lato opposto. </li><li> Afferrare la base del tubo intestinale e tirare anteriormente per rimuovere lo scheletro e intestino branchiale tubo (Figura 2 M - N). </li><li> Separare il tubo intestinale effettuando un taglio perpendicolare posteriore alla fine del quinto ceratobranchial (Figura 2O </strong&gt;). </li><li> Dopo aver rimosso eventuali frammenti ossei rimanenti dalla scatola cranica sul lato dorsale dello scheletro branchiale, inserire le forbici nel cestello branchiale per fare un taglio dorsale (taglio anteriore a posteriore) tra i set bilaterali di piatti dorsale dei denti (Figura 3A - D). Assicurarsi taglio è centrato per evitare di danneggiare le piastre dorsali dei denti. </li><li> Fare due tagli laterali poco profonde nel lume intestinale gommose alla fine posteriore dello scheletro branchiale (estremità anteriore del tubo intestinale) per assistere con l&#39;apertura dello scheletro branchiale (Figura 3E). </li><li> Mettere pesce e tutti i pezzi di tessuto in un vaso e posizionare lo scheletro branchiale in una provetta con il 50% glicerolo, 0,25% KOH continuare compensazione dolce, o 100% glicerolo, se non è necessaria un&#39;ulteriore compensazione. vasetti di etichette e tubi con un identificatore univoco in modo che possano essere monitorati. La quantità di compensazione previsto è in gran parte una funzione della dimensione del pesce, grandi pesci adulti (oltre 40 mm di lunghezza standard) in genere richiedono ulteriore compensazione. </li></ol><img alt="figura 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> Figura 2:. Stickleback branchiale scheletro dissezione Alizarina Rossa macchiato pesci Spinarello pronto per la dissezione. L&#39;occhio è depigmented da un&#39;ampia radura. Le frecce blu indicano la direzione del moto. (A) Vista laterale della testa spinarello, anteriore è a destra. (B) La rimozione della membrana che ricopre l&#39;occhio. (C) La rimozione dell&#39;occhio. (D) dorsale taglio sopra il opercolo. Taglio dell&#39;osso (E) frontale. (F) Parasphenoid tagliato. (G - I) Rimozione dello scheletro facciale. (J) Rimozione delle connessioni dei tessuti molli scheletro branchiale ventrali. (K - L) Rimozione di dorsali connessioni scheletro branchiali. (&lt;strong&gt; M - N) Rimozione dello scheletro branchiale. (O) Separare il tubo intestinale dallo scheletro branchiale. Vedere i punti da 2.1 a 2.16 per maggiori dettagli. Barra di scala = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 3. branchiale scheletro Re-colorazione (se necessario) <ol><li> Per colorare la più scura scheletro branchiale o tessuto chiaro più, rimuovere il 50% glicerolo, soluzione di KOH 0,25% e lavare con 1% KOH due volte (un lavaggio cinque minute seguito da un secondo lavaggio 24 ore agitando orizzontalmente su un agitatore piattaforma). </li><li> Rimuovere 1% KOH e ri-macchia con 0,008% Alizarina Rossa S in 1% KOH per 24 ore. </li><li> Rimuovere macchia e sostituirlo con 1% KOH per 24 ore. </li><li> Rimuovere la soluzione KOH e sostituirla con glicerolo al 50%, 0,25% KOH. </li></ol> 4. Montaggio branchiale scheletro <ol><li> Rimuovere scheletro branchialedal 50% glicerolo, 0,25% KOH o 100% glicerolo e posto vicino al fondo di un 22 mm x 60 millimetri copertura in vetro antiscivolo con lato dorsale rivolto verso l&#39;alto (Figura 3F). Aggiungere qualche goccia di glicerolo al 50%, 0,25% KOH o 100% glicerolo in cima scheletro branchiale. Se la transizione dal 50% glicerolo, 0,25% KOH al 100% glicerolo, soluzione cambiamento in una provetta e agitare per&gt; 5 min prima del montaggio per equilibrare il tessuto. </li><li> Stendete due piccole sfere di plastilina e posto su una delle estremità del vetrino di agire come distanziatori. </li><li> Liberamente posizionare un secondo vetrino in cima con una pressione sufficiente per appiattire lo scheletro branchiale anteriore (Figura 3G). </li><li> Peel aprire lo sportello dorsale sinistra tra cui lastre dei denti dorsali, appiattire, e scorrevole tra le lamelle (Figura 3H). </li><li> Ripetere la tecnica con diritto lembo dorsale e spingere intero scheletro branchiale lontano dal bordo del vetrino (Figura 3I). <ol><li> alterarenativamente, tenere entrambi i lembi dorsali aperti con una pinza e con attenzione inserire il vetrino su, appiattendo lo scheletro branchiale in un unico movimento fluido. </li><li> In alternativa, montare lo scheletro branchiale a testa in giù su un vetrino, dispiegamento ogni lato dorsale lateralmente in modo gravità non permette lo scheletro branchiale per chiudere il backup. Poi coprire con il secondo 22 mm x 60 millimetri copertura in vetro antiscivolo e capovolgere prep. Nota: diverse tecniche di montaggio tendono a lavorare meglio o peggio per ogni individuo. Prova ogni e vedere che cosa si sente più comodo. </li></ol></li><li> Premere leggermente sulla parte superiore vetrino per appiattire le palle d&#39;argilla abbastanza per mantenere lo scheletro branchiale montato piatta, ma fare attenzione a non schiacciare il campione. <ol><li> Durante il processo di montaggio, ceratobranchials possono ruotare e oscurare una fila di rakers. Porre rimedio a questo da una pinza tra i coprioggetti e ri-orientare il ceratobranchials o l&#39;intero scheletro branchiale scorrevole. </li></ol></li><li> Conservare prepara piatto in vassoi di diapositive a rotemperatura om. Montato in 100% glicerolo, preparazioni possono essere memorizzati tra lamelle a ponte per almeno un decennio. pinze pulite e forbici con isopropanolo o etanolo e suggerimenti copertura. </li></ol><img alt="Figura 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> Figura 3:. Piatto di montaggio branchiale scheletro di manipolazione e di montaggio dello scheletro branchiale è mostrato. Le frecce blu indicano la direzione del moto. (A) branchiale scheletro dorsale verso l&#39;alto. (B - D) Rotazione e incisione tra le piastre dorsali dente. (E) taglio laterale in tessuto molle per aprire ulteriormente la base del tubo intestinale. (F) branchiale scheletro posta sul fondo di un coprioggetto pronto per il montaggio. (G) Seconda coprioggetto posto sulla metà anteriore dello scheletro branchiale (sopra piastre dente dorsali). (H - I)montaggio piatto dello scheletro branchiale aprendo sportelli piastra dorsale denti e scorrevole tra due vetrini. Vedere i passaggi 4.1 tramite 4.6 per maggiori dettagli. Barra di scala = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+development+of+complex+biomechanical+systems:+300+million+years+of+fish+jaws.">Evolution and development of complex biomechanical systems: 300 million years of fish jaws.</a> Zebrafish. 2 (4), 243-257 (2005).</li><li>Lauder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+design+and+evolution+of+the+pharyngeal+jaw+apparatus+in+euteleostean+fishes.">Functional design and evolution of the pharyngeal jaw apparatus in euteleostean fishes.</a> Zool. J. Linn. Soc. 77, 1-38 (1983).</li><li>Fraser, G. J., 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=An+ancient+gene+network+is+co-opted+for+teeth+on+old+and+new+jaws.">An ancient gene network is co-opted for teeth on old and new jaws.</a> PLoS Biol. 7 (2), e1000031 (2009).</li><li>Stock, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dentition+in+comparative+context.">Zebrafish dentition in comparative context.</a> J. Exp. Zool. B. Mol. Dev. Evol. 308, 523-549 (2007).</li><li>Liem, K., Greenwood, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+approach+to+the+phylogeny+of+the+pharyngognath+teleosts.">A functional approach to the phylogeny of the pharyngognath teleosts.</a> Am. Zool. 21 (1), 83-101 (1981).</li><li>Anker, G. 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Zool., Lond. 221, 237-260 (1990).</li><li>Huysseune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+plasticity+in+the+lower+pharyngeal+jaw+dentition+of+Astatoreochromis+alluaudi+(Teleostei:+Cichlidae).">Phenotypic plasticity in the lower pharyngeal jaw dentition of Astatoreochromis alluaudi (Teleostei: Cichlidae).</a> Arch. Oral Biol. 40 (11), 1005-1014 (1995).</li><li>Muschick, M., Indermaur, A., Salzburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+Evolution+within+an+adaptive+radiation+of+cichlid+fishes.">Convergent Evolution within an adaptive radiation of cichlid fishes.</a> Curr. Biol. 22 (24), 2362-2368 (2012).</li><li>Cleves, P. A., 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=Evolved+tooth+gain+in+sticklebacks+is+associated+with+a+cis-regulatory+allele+of+Bmp6.">Evolved tooth gain in sticklebacks is associated with a cis-regulatory allele of Bmp6.</a> Proc. Natl. Acad. Sci. 111 (38), 13912-13917 (2014).</li><li>Ellis, N. A., 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=Distinct+developmental+and+genetic+mechanisms+underlie+convergently+evolved+tooth+gain+in+sticklebacks.">Distinct developmental and genetic mechanisms underlie convergently evolved tooth gain in sticklebacks.</a> Development. (142), 2442-2451 (2015).</li><li>Tucker, A. S., Fraser, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+developmental+diversity+of+tooth+regeneration.">Evolution and developmental diversity of tooth regeneration.</a> Semin. Cell Dev. Biol. 25-26, 71-80 (2014).</li><li>Erickson, P. A., Glazer, A. M., Cleves, P. A., Smith, A. S., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+developmentally+temporal+quantitative+trait+loci+underlie+convergent+evolution+of+increased+branchial+bone+length+in+sticklebacks.">Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.</a> Proc. R. Soc. B. 281, (2014).</li><li>Leary, S., 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=.">.</a> AVMA Guidelines for the Euthanasia of Animals. , (2013).</li><li>Bell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+phenetics+and+genetics.">Evolutionary phenetics and genetics.</a> Evol. Genet. Fishes. , 431-528 (1984).</li><li>Taylor, W. R., Van Dyke, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+procedures+for+staining+and+clearing+small+fishes+and+other+vertebrates+for+bone+and+cartilage+study.">Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study.</a> Cybium. 9 (2), 107-119 (1985).</li><li>Erickson, P. A., 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=A+190+base+pair,+TGF-&#946;+responsive+tooth+and+fin+enhancer+is+required+for+stickleback+Bmp6+expression.">A 190 base pair, TGF-&#946; responsive tooth and fin enhancer is required for stickleback Bmp6 expression.</a> Dev. Biol. 401 (2), 310-323 (2015).</li><li>Miller, C. T., 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=cis-Regulatory+changes+in+Kit+ligand+expression+and+parallel+evolution+of+pigmentation+in+sticklebacks+and+humans.">cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans.</a> Cell. 131 (6), 1179-1189 (2007).</li><li>Aigler, S. R., Jandzik, D., Hatta, K., Uesugi, K., Stock, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+constraint+underlie+irreversibility+of+tooth+loss+in+cypriniform+fishes.">Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.</a> Proc. Natl. Acad. Sci. U. S. A. 111 (21), 7707-7712 (2014).</li><li>Pasco-Viel, E., 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=Evolutionary+trends+of+the+pharyngeal+dentition+in+Cypriniformes+(Actinopterygii+Ostariophysi).">Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii Ostariophysi).</a> PLoS One. 5 (6), e11293 (2010).</li></ol>

The authors have nothing to disclose.

The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all branchial skeletal elements in a simple two-dimensional plane for ease of imaging and quantification of multiple traits. 
One limitation of this protocol is that although teeth, gill rakers, and most endochondral bones within this flat-mounted two-dimensional prep can be easily imaged and quantified, the second through fourth epibranchials, that have much more three-dimensional shapes, are not easily visualized and would require additional dissection to accurately measure. A second limitation of this protocol is the method is minimally destructive to the fish head. However, only two bones are damaged with a single cut each, the parasphenoid and the frontal bone. All other craniofacial skeletal elements can be returned to and measured at later points in time.
This protocol, although developed for sticklebacks, can be used in many other fish species, although sometimes modifications are necessary. For example, some fish species like zebrafish have a non-ossified dorsal chewing pad44 requiring the dorsal cut to be lateral, around this plate. The posterior end of the fifth ceratobranchial is curved is some species (e.g., zebrafish and other Cypriniformes) and must be trimmed or much larger clay spacers used to allow flat mounting. Particularly large specimens can be mounted between two pieces of glass or acrylic in the same manner. Species with a particularly thick epithelial layer covering tooth plates can be restained and cleared after dissection or more aggressively cleared with 2% KOH. Fish in this situation may also be presoaked in 1 or 2% KOH prior to Alizarin staining to increase dye penetrance. 
An alternative more technologically advanced method for imaging the branchial skeleton is Micro Computed Tomography (&#181;CT)23,45. However, &#181;CT requires an expensive scanner, is much more time consuming, and requires considerable training. Instead, this dissection protocol is inexpensive, quick, and amenable to high throughput studies required for quantitative studies such as quantitative trait loci (QTL) mapping genomic regions controlling evolved changes in craniofacial patterning. Additionally, these dissections can be performed at any larval or adult time point22,35,36,38 to study skeletal development. Although fish head anatomy is complex, this simple protocol allows easy visualization of the internal branchial skeleton, a critically important component of trophic morphology.

Una quantità incredibile di diversità esiste nello scheletro testa tra i vertebrati, soprattutto tra i pesci. In molti casi questa diversità facilita differenti strategie di alimentazione 1 - 4, e può comportare importanti modifiche sia modello craniofacciale esterna e interna. Lo scheletro branchiale è situato internamente nella gola di un pesce e circonda la maggior parte della cavità buccale. Lo scheletro branchiale comprende 5 segmenti omologhi serialmente, anteriore quattro delle quali supportano branchie. Insieme, queste cinque segmenti funzionano da interfaccia tra i pesci e il loro cibo 5. Variazione in una moltitudine di tratti, tra cui branchiospine, denti faringei, e le ossa branchiali contribuire a foraggiare efficace su diversi tipi di cibo. Spinarelli sono stati sottoposti a una radiazione adattativa dopo forme ancestrali oceaniche colonizzati laghi d&#39;acqua dolce e torrenti in tutto l&#39;emisfero settentrionale. Il cambiamento nella dietadal piccolo zooplancton nell&#39;oceano di grande preda in acqua dolce ha provocato drammatica variazione trofico in diversi tratti cranio-facciali 6. Mentre molti studi si sono concentrati sulle differenze cranio esterni in spinarelli 7 - 13, importanti cambiamenti cranio-facciali evolvono più volte nello scheletro branchiale interna. La possibilità di creare ibridi fecondi tra le popolazioni spinarello morfologicamente distinte offre un&#39;eccellente opportunità per mappare la base genetica delle modifiche si sono evoluti per lo scheletro branchiale. Una caratteristica trofico di importanza ecologica è il patterning di branchiospine, ossa dermiche periodici che costeggiano le facce anteriore e posteriore delle ossa branchiali e sono utilizzati per filtrare prede. I pesci che in genere si nutrono di piccoli oggetti preda tendono ad avere più a lungo e più densamente spaziati Gill rakers rispetto ai pesci che si nutrono sulla preda più grande 14,15. Variazione branchiospine stato segnalato sia wntro e tra le specie 14-19, e gli aspetti di Gill raker patterning contribuiscono a nicchie trofiche e fitness 16. Decenni di ricerche hanno ampiamente documentato numero Gill raker e variazione di lunghezza in spinarelli threespine 17 - 21; Tuttavia, questi studi in genere si concentrano sulla prima fila di branchiospine. Studi recenti hanno dimostrato modularità nel controllo genetico del numero branchiali raker attraverso lo scheletro branchiale 22,23 e attraverso una singola riga in branchiali raker spaziatura 23 e lunghezza 24 mettendo in evidenza l&#39;importanza di studiare più di remare uno o un singolo rastrello branchia per capire il sviluppo basi genetiche della riduzione Gill raker. Un secondo tratto trofico sia di importanza ecologica e biomedica è il patterning di denti faringei. Denti in pesci possono essere posizionati sia nella mascella orale e nello scheletro branchiale, noto come i denti faringei. denti orali sono utilizzati principalmente per prey catturare mentre denti faringei sono utilizzati per la masticazione e la preda manipolazione 25 - 27. Entrambi i gruppi si formano tramite meccanismi di sviluppo condivisi e sono considerati evolutivamente omologa 28. Modularità Interessante si verifica per cui alcune specie, come zebrafish, mancanza orale e della faringe dorsale denti 29, mentre le altre specie hanno più ceratobranchials dentate, pharyngobranchials, e talvolta dentate basihyal e hypobranchials 30. In spinarelli, denti faringei si trovano ventrale al quinto ceratobranchial e dorsalmente sul anteriore e posteriore pharyngobranchials 31. Cinematica di alimentazione stickleback mostrano la mascella orale viene utilizzato principalmente per la cattura la preda e facilitare l&#39;alimentazione di aspirazione 9 lasciando la masticazione alla mascella faringea. In ciclidi, inferiore faringeo mascella morfologia varia notevolmente 32,33 e ha dimostrato di essere adattivo e correlato con nicchia trofica 34. MultiPLE popolazioni stickleback d&#39;acqua dolce si sono evoluti gli aumenti drammatici nella ventrale faringea numero di denti 23,35,36. Studi recenti hanno dimostrato che la base genetica evolutiva di questo guadagno dente evoluto è in gran parte distinta in due popolazioni derivate indipendentemente spinarelli d&#39;acqua dolce 36. A differenza di denti di mammiferi, pesci rigenerare i denti continuamente per tutta la vita adulta 37. Entrambe queste popolazioni di acqua dolce dentate alti precedentemente descritti si sono evoluti un tasso di sostituzione dei denti accelerato, fornendo un sistema di vertebrati rara per studiare le basi genetiche della rigenerazione 36. Una terza caratteristica trofica che si è evoluta più volte in spinarelli d&#39;acqua dolce è più ossa epibranchial e ceratobranchial, le branchiali omologhi arco ribassato della mascella superiore e inferiore, rispettivamente 38. Più lunghe ossa branchiali conferiscono una grande cavità orale e probabilmente sono adattativi per consentire prede più grandi per essere Consumed. Inoltre, in altri pesci, ossa epibranchial sono importanti per la depressione delle lastre dei denti dorsale faringei 25. Come rastrelli branchiali e denti faringei, le ossa branchiali sono interni e, quindi, difficile da visualizzare o quantificare facilmente. Qui vi presentiamo un protocollo dettagliato per sezionare e TV a montare lo scheletro branchiale, consentendo una facile visualizzazione e la quantificazione di una varietà di importanti caratteristiche cranio-facciali. Mentre questo protocollo descrive una dissezione stickleback, questo stesso metodo funziona su una varietà di altri pesci.

<table><tbody><tr><td>Potassium Hydroxide (KOH)</td><td>EMD</td><td>PX1480-1</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893-4L</td><td></td></tr><tr><td>10% Neutral Buffered Formalin (NBF)</td><td>Azer Scientific</td><td>NBF-4-G</td><td></td></tr><tr><td>Alizarin Red S</td><td>EMD</td><td>AX0485-3</td><td></td></tr><tr><td>Microscope Cover Glasses 22 mm x 60 mm</td><td>VWR</td><td>16004-350</td><td></td></tr><tr><td>100 mm x 10 mm Glass Petri Dish</td><td>Kimble Chase</td><td>23064-10010</td><td>To dissect samples on</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>Can be poured into glass or plastic Petri dishes to make dissecting plates</td></tr><tr><td>Modeling Clay</td><td>Sargent Art</td><td>22-4000</td><td>1 lb cream</td></tr><tr><td>Scintillation Vials (case of 500)</td><td>Wheaton</td><td>986586</td><td>Borosilicate Glass with Screw Cap</td></tr><tr><td>Forceps-Dumont #5 Inox (Biologie tip)</td><td>FST</td><td>11252-20</td><td>Dumostars are an alternative</td></tr><tr><td>Dissecting Scissors&amp;nbsp;</td><td>FST</td><td>15003-08</td><td>Alternate sizes are available depending on size of sample</td></tr><tr><td>Dissecting Microscope</td><td>Leica</td><td>S6E with KL300 LED</td><td>Many other models work nicely, having a flat base helps</td></tr><tr><td>Microcentrifuge Tubes 1.7 ml</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Cardboard slide tray</td><td>Fisher</td><td>12-587-10</td><td></td></tr></tbody></table>

dissection and flat-mounting of the threespine stickleback branchial skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

<html> Questo protocollo si traduce in uno scheletro branchiale montato sezionato e piatta (Figura 4), ​​dove una serie di importanti caratteristiche trofiche può essere quantificato. Dal punto di vista dorsale, tutte le righe di branchiospine, tutte le lastre della faringe denti, e quasi tutte le ossa branchiali possono essere facilmente visualizzati e quantificati 22 - 24,35,36,38,42. Alizarina Rossa S reagisce anche su rodamina o filtro rosso simile permettendo doppia marcatura con altri marcatori (ad esempio, GFP transgenici 42) e un metodo alternativo di visualizzazione. Fluorescenza svanisce rapidamente in luce, in modo da conservare preparazioni al buio, se è prevista l&#39;imaging a fluorescenza o fenotipizzazione. Dal punto di vista ventrale, le branchie possono essere visualizzati e la loro pigmentazione quantificati 43. Preps possono essere memorizzati in 100% glicerolo per anni. 4.jpg &quot;/&gt; Figura 4:. Rappresentante spinarello scheletro branchiale Due esempi di scheletri branchiali macchiati e liquidati sono mostrati. (A) Brightfield immagine che mostra l&#39;osso etichetta rossa. (B) immagine fluorescente sotto un set di filtri rodamina B. Esempi di rastrelli, denti e le ossa sono etichettati con le parentesi acute, punte di freccia, e asterischi, rispettivamente. Barre di scala = 2 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

Dissezione e Flat-montaggio del Threespine Stickleback branchiale scheletro

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

dissection-flat-mounting-threespine-stickleback-branchial

Universidad San Sebastian

Research

JoVE Journal

Biology

9.7K Views.  University of California, Berkeley. The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species. 

Video: Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

L&#39;espressione di proteine ​​fluorescenti in<em&gt; Branchiostoma lanceolatum</em&gt; By mRNA iniezione in ovociti non fecondate

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Ricerca

Biologia dello sviluppo

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Costruire modelli ad elementi finiti per Indagare Zebrafish Jaw Biomeccanica

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

Caratterizzazione di Fish Scales Sovrapposizione (<em&gt; Atractosteus spatola</em&gt;) Usando Nanoindentazione, X-ray CT, FTIR e SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Bioingegneria

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Neuroscienze

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissezione e laterale Montaggio di embrioni di zebrafish: analisi dello sviluppo del midollo spinale

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

Una cultura di espianto della coda 3D per studiare la segmentazione dei vertebrati nel pesce zebra

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Approcci di visualizzazione e analisi tridimensionali e quadridimensionali per studiare l'allungamento e la segmentazione assiale dei vertebrati

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

Modello di lesione da coltellata del tectum ottico adulto utilizzando zebrafish e medaka per l'analisi comparativa della capacità rigenerativa

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

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 ...

Dissezione e Flat-montaggio del Threespine Stickleback branchiale scheletro

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine fluorescenti in

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

Costruire modelli ad elementi finiti per Indagare Zebrafish Jaw Biomeccanica

Caratterizzazione di Fish Scales Sovrapposizione (

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Dissezione e laterale Montaggio di embrioni di zebrafish: analisi dello sviluppo del midollo spinale

Una cultura di espianto della coda 3D per studiare la segmentazione dei vertebrati nel pesce zebra

Approcci di visualizzazione e analisi tridimensionali e quadridimensionali per studiare l'allungamento e la segmentazione assiale dei vertebrati

Modello di lesione da coltellata del tectum ottico adulto utilizzando zebrafish e medaka per l'analisi comparativa della capacità rigenerativa

Preparazione di montaggio schermo per l'osservazione e l'analisi di zebrafish campioni macchiato da Whole Monte

Dissezione e Flat-montaggio del Threespine Stickleback branchiale scheletro

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine ​​fluorescenti in

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

Costruire modelli ad elementi finiti per Indagare Zebrafish Jaw Biomeccanica

Caratterizzazione di Fish Scales Sovrapposizione (

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Dissezione e laterale Montaggio di embrioni di zebrafish: analisi dello sviluppo del midollo spinale

Una cultura di espianto della coda 3D per studiare la segmentazione dei vertebrati nel pesce zebra

Approcci di visualizzazione e analisi tridimensionali e quadridimensionali per studiare l'allungamento e la segmentazione assiale dei vertebrati

Modello di lesione da coltellata del tectum ottico adulto utilizzando zebrafish e medaka per l'analisi comparativa della capacità rigenerativa

Preparazione di montaggio schermo per l'osservazione e l'analisi di zebrafish campioni macchiato da Whole Monte

L'espressione di proteine fluorescenti in