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.

Alle Fische Arbeit wurde von der Institutional Animal Care und Use Committee der University of California-Berkeley (Protokollnummer R330) zugelassen. Euthanasie wurde mit dem Eintauchen in 0,025% Tricaine-S mit 0,1% Natriumbicarbonat 39 gepuffert durchgeführt. Alle Schritte werden bei Raumtemperatur durchgeführt. 1. Vorbereitung Hinweis: Führen Sie die Schritte 1,1-1,5 in konische Röhrchen oder Szintillationsgefäße, die dicht verschließen kann und horizontal verlegt werden. durch vorsichtiges Umdrehen oder Schütteln des Rack von Rohren oder Fläschchen zu belichten alle Seiten des Fisches auf der Färbungslösung und ermöglichen Fleck eindringen das Gewebe gleichmäßig wie möglich Fische brauchen nicht ständig geschüttelt werden, sondern versuchen, so oft die Lösung zu mischen. Sie nicht einen großen Stapel von Fisch auf einem Plattformschüttler platzieren, wie das hohe Gewicht der Flüssigkeit den Shaker brechen. <ol><li> Fix entweder frisch getöteten Fisch oder Fisch gelagert in Ethanol mit 10% neutral gepuffertem Formalin (NBF) über Nacht. Alternativ können Sie auch 4% ParaAldehyd in 1x PBS-Lösung anstelle von 10% NBF. Hinweis: Wenn DNA-Extraktion, befestigen einen kleinen Teil der Schwanz oder Brustflossen vor der Fixierung und lagern in Ethanol. </li><li> Entsorgen Sie Fix ordnungsgemäß in einer chemischen Haube und ersetzen mit Leitungswasser (dh ~ pH 7,0) für 2 Stunden. Vermeiden Sie die Verwendung de-ionisiertes Wasser, wie es oft sauer sein können und Knochen entkalken kann. </li><li> Entfernen Sie das Wasser und Flecken Fisch mit 0,008% Alizarin Red S in 1% KOH in Wasser für 24 Stunden. Für Fische weniger als 20 mm Standardlänge verwenden 0,004% Alizarin Red S. (Einen 100x (0,8%) Stammlösung von Alizarin Red S, die können dann verdünnt). </li><li> Entfernen Fleck (in geeigneten Abfallbehälter in der Haube setzen) und legen Fisch in Leitungswasser für ein paar Stunden. Wechseln Sie das Wasser nach Bedarf, bis Wasserspülung meist klar. </li><li> Entfernen Sie das Wasser und den Ort Fisch in 50% Glycerin, 0,25% KOH für leichte Reinigung und anschließender Trennung. Hinweis: Dieses Färbeprotokoll von zuvor beschriebenen Methoden modifiziert 40,41. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Präparation Hinweis: Siehe Abbildung 1 für eine Überprüfung der relevanten Kopfskelettmorphologie. <img alt="Abbildung 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> Abb . 1: Stickleback Kopf Skelettmorphologie Alizarin - Rot gefärbt Dreistachliger Stichling Kopf mit Fluoreszenz unter einem Satz Rhodamin B - Filter abgebildet wird . Nützliche Morphologie ist beschriftet: Op = Kiemendeckel, Subop = subopercle, BSR = Kiemenstrahlen, Preop = preopercle, Infraorb 1-3 = Infraorbital- 1-3 (auch genannt circumorbitals oder suborbitals), Dent = dentary, Premax = Prämaxillare, Max = Maxilla Nas, = nasal, Lat. ETHM = seitliche ethmoid, PSPH = Parasphenoid, Fron = Stirnbein. Für eine detailliertere Beschreibung der Kopfskelett stickleback siehe Anker (1974) 31. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">eine größere Version zu sehen</a>diese Figur. <ol><li> Legen Sie den Fisch flach (2A) und legen Sie scharfe # 5 Uhrmacherzange in die Seite des Auges bei einer ~ 45 ° Winkel der Membran bedeckt , das Auge zu durchstechen. </li><li> Ziehen Sie die Membran vom Auge weg, ähnlich wie ein Joghurt Deckel (2B) Peeling. </li><li> Legen Sie offene Zange hinter dem Auge, halten den Sehnerv hinter dem Auge fassen, und entfernen Sie das Auge (Abbildung 2C). Das Auge nicht durchstechen, wie es Melanin undicht wird. Wenn punktiert, kann Melanin während weg späteren Schritten gewaschen werden. </li><li> Wiederholen Sie auf der anderen Seite. </li><li> Ausgehend von der hinteren, legen Sie eine kleine Sezieren Scherenblatt unter dem Kiemendeckel Klappe, ziehen Scherenblatt vom Rücken her über Kiemendeckel, dann schneiden Weichgewebe bis in die Augenhöhle (2D). Schneiden Sie dorsal der Kiemendeckel Knochen. </li><li> Schneiden Sie das Stirnbein (dorsal der Augenhöhle) (Abbildung 2E). </li><li> Schneiden Sie die Mittellinie Parasphenoid Knochen um ter Mitte der Augenhöhlen (2F). </li><li> Wiederholen Sie Kiemendeckel Schnitt auf der gegenüberliegenden Seite. </li><li> Setzen Sie Zange unter dem Kiemendeckel und ziehen Sie langsam das Gesicht weg vom Körper, jede Weichgewebe Trimmen noch angebracht (Abbildung 2G - H). Achten Sie darauf, nicht die erste Reihe der Kiemenreusen zu stören. <ol><li> Mit einer Pinzette lösen sich die ceratohyals auf beiden Seiten von der Mittellinie basihyal während Ablösen und Entfernen des vorderen kraniofazialen Skeletts (die gesamte Kiefer einschließlich der dentary, Prämaxillare und Oberkiefer, die gesamte hyoid Skelett einschließlich der Außenhautkiemendeckel, preopercle, subopercle und Kiemenstrahlen und die zugrunde liegenden dorsalen und ventralen endochondral Elemente, und der vordere Teil des Schädels einschließlich der nasalen, seitliche ethmoid und Infraorbital- Knochen, siehe Abbildungen 1 und 2 I). </li><li> Becken- Stacheln aus dem Körper herausgeklappt werden kann und als Griff dienen zur Zange halten o greifenf, wenn vorhanden. Spines einrasten. Um die Sperre aufzuheben, ziehen Sie vorsichtig Wirbelsäule mit einer Pinzette aus Fischkörper direkt weg, dann sanft nach hinten biegen Wirbelsäule flach gegen Fisch zu drücken. </li></ol></li><li> Insert geschlossen Zange posterior und ventralen zur Kiemenskelett (knapp unter Darmrohr) und ziehen Zange nach vorn, um die verbleibenden Muskeln und Bänder necken auseinander an den Kiemenskelett (2J - K). </li><li> Mit den Spitzen der geschlossenen Zange, kratzen die Muskeln Befestigung des dorsalen Kiemen Skelett ventralen Hirnschale in einer posterior anterior (Abbildung 2 l) entfernt. </li><li> Wiederholen 2.9 und 2.10 auf der gegenüberliegenden Seite. </li><li> Fassen Sie die Basis des Darmrohr und ziehen nach vorn die Kiemen Skelett und Darmrohr (Abbildung 2 M - N) zu entfernen. </li><li> Trennen Sie die Darmröhre durch einen senkrechten Schnitt hinter dem Ende des fünften ceratobranchial machen (Abbildung 2O </strong&gt;). </li><li> Nach dem Entfernen eines dorsalen Schnitt (Schneiden von vorne nach hinten) zwischen den bilateralen Sätze von Dorsalzahn Platten (- D 3A) alle verbleibenden Knochenfragmente aus der Hirnschale auf der Rückenseite des Kiemen Skelett, legen Schere in den Kiemenkorb zu machen. Stellen Sie sicher, Schnitt zentriert Platten eine Beschädigung der Dorsalzahn zu vermeiden. </li><li> Machen Sie zwei flache seitliche Schnitte in den gummiDarmLumen am hinteren Ende des Kiemen Skelett (vordere Ende der Darmrohr) mit dem Öffnen der Kiemen Skelett (Abbildung 3E) zu unterstützen. </li><li> Legen Sie Fisch und alle Gewebestücke in ein Gefäß und stellen Sie das Kiemen Skelett in einem Mikrozentrifugenröhrchen mit 50% Glycerin, 0,25% KOH auf sanfte Lichtung weiter, oder 100% Glycerin, wenn keine weiteren Clearing erforderlich ist. Label Gläser und Röhren mit einer eindeutigen Kennung, so dass sie verfolgt werden. Die erforderliche Menge des Clearing ist weitgehend abhängig von der Größe der Fische, große ausgewachsene Fische (über 40 mm in Standardlänge) benötigen in der Regel zusätzliche Clearing. </li></ol><img alt="Figur 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> Abbildung 2:. Stickleback Kiemen Skelett Dissektion Alizarin Red gefärbt Dreistachliger Stichling Fisch für die Präparation bereit. Das Auge wird von umfangreichen Clearing depigmented. Blaue Pfeile zeigen die Richtung der Bewegung. (A) Seitenansicht von stickleback Kopf, ist vorne nach rechts. (B) Entfernung von Membran , die das Auge abdeckt. (C) Entfernen des Auges. (D) dorsale Schnitt über dem Kiemendeckel. (E) Frontal Knochen geschnitten. (F) Parasphenoid geschnitten. (G - I) Die Entfernung des Gesichtsschädels. (J) Entfernung von ventralen Kiemen Skelett Weichgewebe - Verbindungen. (K - L) Entfernung von dorsalen Kiemen Skelett - Verbindungen. (&lt;strong&gt; M - N) Die Entfernung des Kiemen Skelett. (O) Trennen Sie das Darmrohr aus dem Kiemen Skelett. Siehe Schritte 2.1 bis 2.16 für weitere Details. Maßstabsbalken = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> 3. Branchiogene Skeleton Re-Färbung (falls erforderlich) <ol><li> Um die Kiemen Skelett dunkler oder klar Gewebe mehr Fleck, entfernen Sie 50% Glycerin, 0,25% KOH-Lösung und Waschen mit 1% KOH zweimal (eine 5 Minuten waschen, gefolgt von einer zweiten 24-Stunden-Wasch während sie horizontal auf einer Plattform Schüttler Schütteln). </li><li> Entfernen 1% KOH und re-Fleck mit 0,008% Alizarin Red S in 1% KOH für 24 Stunden. </li><li> Entfernen Fleck und ersetzen mit 1% KOH für 24 Stunden. </li><li> Entfernen KOH-Lösung und ersetzen mit 50% Glycerin, 0,25% KOH. </li></ol> 4. Montage Branchiogene Skeleton <ol><li> Entfernen Kiemenskelettvon 50% Glycerin, 0,25% KOH oder 100% Glycerin und Ort in der Nähe der Unterseite eines 22 mm x 60 mm Deckglas mit der Rückenseite nach oben (Abbildung 3F). Fügen Sie ein paar Tropfen 50% Glycerin, 0,25% KOH oder 100% Glycerin oben auf Kiemen Skelett. Wenn Übergang von 50% Glycerin, 0,25% KOH auf 100% Glycerin, ändern Lösung in einem Mikrozentrifugenröhrchen und schütteln&gt; 5 min vor der Montage Gewebe äquilibrieren. </li><li> Ausrollen zwei kleine Kugeln aus Modelliermasse und auf beiden Enden des Deckglases als Abstandshalter wirken. </li><li> Platz locker ein zweites Deckglas auf der Oberseite mit genügend Druck , um den vorderen Kiemen Skelett (Abbildung 3G) zu glätten. </li><li> Peel Öffnen Sie die linke Dorsallappen einschließlich Dorsalzahn Platten, glätten und Gleiten zwischen den Deckgläsern (3H). </li><li> Wiederholen Technik mit rechten dorsalen Klappe und schieben gesamten Kiemen Skelett weg vom Rand des Deckglases (Abbildung 3I). <ol><li> Ändernnativ, halten Sie beide Klappen dorsalen offen mit einer Pinzette vorsichtig das Deckglas legen auf, die Kiemen Skelett in einer geschmeidigen Bewegung Abflachung. </li><li> Alternativ montieren Sie die Kiemen Skelett Kopf nach unten auf ein Deckglas, jede Rückenseite Spreizen aus seitlich so die Schwerkraft ist nicht der Kiemen Skelett wieder nach oben zu schließen. Dann decken Sie mit dem zweiten 22 mm x 60 mm Deckglas und prep invertieren. Hinweis: Unterschiedliche Montagetechniken besser zu arbeiten neigen oder schlechter für jeden Einzelnen. Versuchen Sie, jeden und sehen, was am angenehmsten ist. </li></ol></li><li> Drücken Sie leicht auf Deck Lehmkugeln genug zu halten, die Kiemen Skelett montiert flach, aber darauf achten, nicht zu glätten um die Probe zu zerkleinern. <ol><li> Während des Montageprozesses kann ceratobranchials drehen und eine Reihe von rakers verschleiern. Abhilfe dies durch die Zange zwischen den Deck Gleiten und zum erneuten Ausrichten der ceratobranchials oder ganze Kiemen Skelett. </li></ol></li><li> Shop PREPS flach in Diamagazinen bei room Temperatur. Montiert in 100% Glycerin, kann preps zwischen überbrückten Deck für mindestens zehn Jahre gelagert werden. Saubere Pinzette und Schere mit Isopropanol oder Ethanol und Abdeckung Spitzen. </li></ol><img alt="Figur 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> Abbildung 3:. Flache Montage des Kiemen Skelett Manipulation und Montage des Kiemen Skelett dargestellt. Blaue Pfeile zeigen die Richtung der Bewegung. (A) Branchiogene Skelett Rückenseite nach oben. (B - D) Rotation und Einschnitt zwischen den Dorsalzahn Platten. (E) Lateral Schnitt in weichem Gewebe weiter zu öffnen Sie die Basis des Darmrohr. (F) Branchiogene Skelett am unteren Rand des Deckglases gelegt bereit für die Montage. (G) der zweiten Deckglas auf der vorderen Hälfte des Kiemen Skelett gelegt (über Dorsalzahn Platten). (H - I)Flache Montage des Kiemengerüst durch Öffnen Dorsalzahn Plattenklappen und Gleiten zwischen zwei Deckgläsern. Siehe Schritte 4.1 bis 4.6 für weitere Details. Maßstabsbalken = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

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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.

Eine unglaubliche Menge an Vielfalt existiert im Kopf Skelett unter den Wirbeltieren, vor allem unter den Fischen. In vielen Fällen erleichtert diese Vielfalt verschiedene Fütterungsstrategien 1. - 4. und größere Änderungen an externen und internen craniofacial Strukturierung beinhalten können. Die Kiemen Skelett wird intern in der Kehle eines Fisches angeordnet und umgibt den größten Teil der Mundhöhle. Die Kiemen Skelett besteht aus 5 seriell homologe Segmente, die vordere, von denen vier die Kiemen unterstützen. Zusammen wirken diese fünf Segmente als Schnittstelle zwischen Fisch und ihre Nahrung 5. Variation in einer Vielzahl von Merkmalen, einschließlich Kiemenreusen, Schlundzähne und Kiemen Knochen tragen zur effizienten Nahrungssuche auf verschiedenen Arten von Lebensmitteln. Stichlinge haben eine adaptive Radiation nach Vorfahren ozeanischen Formen kolonisiert Süßwasserseen und Bäche in der gesamten nördlichen Hemisphäre erlebt. Die Verschiebung in der Ernährungvon kleinen Zooplankton im Ozean in Süßwasser auf größere Beute hat 6 in dramatischen trophic Variation in mehreren craniofacial Züge geführt. Während viele Studien über externe craniofacial Unterschiede in Stichlinge konzentriert haben 7-13, entwickeln wichtige craniofacial Änderungen wiederholt im internen Kiemen Skelett. Die Fähigkeit, zu schaffen fruchtbaren Hybriden zwischen morphologisch unterschiedlichen stickleback Populationen bietet eine hervorragende Gelegenheit, die genetische Grundlage entwickelte sich Änderungen an der Kiemen Skelett abzubilden. Ein trophic Merkmal ökologischer Bedeutung ist die Strukturierung von Kiemenreusen, regelmäßige Hautknochen, die die vorderen und hinteren Flächen der Kiemen Knochen Linie und werden verwendet, um Beutetiere zu filtern. Fische , die normalerweise ernähren sich von kleinen Beutetieren sind in der Regel mehr zu haben und dichter Abstand Kiemenreusen im Vergleich zu Fischen , die 14,15 auf größere Beutetiere ernähren. Variation in Kiemenreusen wurde berichtet, beide within und zwischen den Arten 14-19 und Aspekte der Kiemenreuse Muster tragen zu trophischen Nischen und Fitness - 16. Jahrzehnte der Forschung haben ausführlich Kiemenreuse Nummer und Längenänderung in threespine Stichlinge 17 dokumentiert - 21; Allerdings konzentrieren sich diese Studien typischerweise in der ersten Reihe von Kiemenreusen. Neuere Arbeiten über die Kiemen Skelett 22,23 und über eine einzelne Zeile in Kiemenreuse 23 Abstand Modularität in der genetischen Kontrolle Kiemenreuse Nummer angezeigt und Länge 24 Hervorhebung der Bedeutung mehr zu studieren , als Zeile ein oder eine einzelne Kiemenreuse zu verstehen , die Entwicklungs genetische Basis der Kiemenreuse Reduktion. Eine zweite trophische Merkmal sowohl ökologische und biomedizinische Bedeutung ist die Strukturierung von Schlundzähne. Zähne in Fischen kann sowohl in der Mundkiefer und im Kiemen Skelett, bekannt als Schlundzähne befinden. Oral Zähne sind in erster Linie für p verwendetrey erfassen , während Schlundzähne für Kauens und Beute Manipulation 25 verwendet werden - 27. Beide Sätze bilden über gemeinsame Entwicklungsmechanismen und sind entwicklungs homolog 28 betrachtet. Interessante Modularität tritt wobei einige Arten, wie Zebrabärbling, mangelnde Mund und Rückenschlundzähne 29 , während andere Spezies , die mehrere Zahn ceratobranchials haben, pharyngobranchials und manchmal basihyal verzahnten und hypobranchials 30. In Stichlinge sind Schlundzähne ventral am fünften ceratobranchial und dorsal auf den vorderen und hinteren pharyngobranchials 31 gefunden. Kinematics auf stickleback Fütterung zeigen die orale Kiefer ist in erster Linie für den Beutefang eingesetzt und erleichtert Saug Zuführung 9 Kauens zur Pharyngealia verlassen. In Cichliden, niedrigere Pharyngealia Morphologie variiert 32,33 dramatisch und wurde mit trophischen Nischen 34 adaptive und korreliert zu sein gezeigt. Multiweise Süßwasser stickleback Populationen dramatischen Anstieg der ventralen Schlundzähnezahl 23,35,36 entwickelt. Neuere Arbeiten haben gezeigt , dass die Entwicklung der genetischen Grundlage dieser entwickelte Zahn Verstärkung in zwei unabhängig voneinander abgeleitet Populationen von Süßwasser Stichlinge 36 weitgehend verschieden ist. Im Gegensatz zu Säugerzähne, regenerieren Fisch ihre Zähne kontinuierlich während des Erwachsenenlebens 37. Beide zuvor beschriebenen hohen Zahnsüßwasserpopulationen haben eine beschleunigte Zahnersatzrate entwickelt, ein seltenes wirbel System bietet die genetische Basis der Regeneration zu untersuchen 36. Eine dritte trophische Eigenschaft , die immer wieder in Süßwasser Stichlinge entwickelt hat , ist länger epibranchial und ceratobranchial Knochen, die Kiemenbogen segmentale Homologe des Ober- und Unterkiefer bzw. 38. Längere Kiemen Knochen verleihen einen größeren Mundhöhle und sind wahrscheinlich adaptive für so dass größere Beute c zu seinonsumed. Darüber hinaus in andere Fische sind epibranchial Knochen wichtig für die Vertiefung der Rückenschlundzahnplatten 25. Wie Kiemenreusen und Schlundzähne sind die Kiemen Knochen interne und somit schwer zu leicht zu visualisieren oder zu quantifizieren. Hier präsentieren wir ein ausführliches Protokoll zu sezieren und Flach montieren Kiemen Skelett, was eine einfache Visualisierung und Quantifizierung einer Vielzahl von wichtigen craniofacial Züge. Während dieses Protokoll eine stickleback dissection beschrieben, arbeitet dieses gleiche Verfahren auf einer Vielzahl von anderen Fischen.

<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> Dieses Protokoll führt zu einer seziert und flach montiert Kiemenskelett (Abbildung 4) , wo eine Vielzahl von wichtigen trophischen Merkmale quantifiziert werden können. Aus einer Rückenansicht werden alle Zeilen von Kiemenreusen, alle Zahnschlundplatten und fast alle Kiemen Knochen leicht sichtbar gemacht und quantifiziert 22 werden kann - 24,35,36,38,42. Alizarin Red S fluoresziert auch auf einem Rhodamin oder einem ähnlichen Rotfilter ermöglicht Doppelmarkierung mit anderen Markern (zB transgene GFP 42) und einem alternativen Verfahren zur Visualisierung. Die Fluoreszenz schnell verblasst im Licht, so speichern PREPS im Dunkeln, wenn Fluoreszenz-Bildgebung oder Phänotypisierung geplant. Aus einer ventralen Ansicht können die Kiemen sichtbar gemacht und ihre Pigmentierung 43 quantifiziert werden. Preps können in 100% Glycerin für Jahre gelagert werden. 4.jpg &quot;/&gt; Abbildung 4:. Repräsentative stickleback Kiemen Skelett Zwei Beispiele von gefärbten und gelöscht Kiemen Skelette gezeigt. (A) Hellfeldbild zeigt Knochen rot markiert. (B) Fluoreszenzbild unter einem Rhodamin B - Filtersatz. Beispiele für Reusen, Zähne und Knochen sind beschriftet mit Carets, Pfeilspitzen und Sternchen sind. Maßstabsbalken = 2 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</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.

Dissection und Flach Montage des Dreistachliger Stichling Branchiogene 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 ...

dissection-flat-mounting-threespine-stickleback-branchial

Nanyang Technological University

Research

JoVE Journal

Biology

9.7K Aufrufe.  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. 

Serie: 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.

Expression von fluoreszierenden Proteinen in<em&gt; Branchiostoma lanceolatum</em&gt; Durch mRNA Injektion in ungedüngte Eizellen

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

Forschung

Entwicklungsbiologie

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.

Mikroinjektions für Transgenese und Genome Editing in Threespine Stichlinge

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.

Der Aufbau Finite-Elemente-Modelle zu Zebrabärbling Jaw Biomechanik Untersuchen

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.

Charakterisierung von mehrschichtigen Fischschuppen (<em&gt; Atractosteus Spachtel</em&gt;) Verwendung Nanoindentierung, Röntgen-CT-, FTIR-und SEM

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

Biotechnik

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-Bleiche zur Einzel-Schwann-Zellen an der neuromuskulären Synapse Delineate

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 ...

Neurowissenschaften

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.

Dissection und seitlicher Anbau von Zebrafisch-Embryonen: Analyse der Rückenmarksentwicklung

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.

Eine 3D-Schwanz-Explant-Kultur zur Untersuchung der Wirbeltiersegmentierung bei Zebrafischen

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.

Drei- und vierdimensionale Visualisierungs- und Analyseansätze zur Untersuchung der axialen Dehnung und Segmentierung von Wirbeltieren

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.

Stichwundenverletzungsmodell des adulten optischen Tektums unter Verwendung von Zebrafisch und Medaka zur vergleichenden Analyse der Regenerationsfähigkeit

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.

Wohnung Berge Vorbereitung für Beobachtung und Analyse der Zebrafisch-Embryo Proben von Bunt Ganze Berge<em&gt; In situ</em&gt; Hybridisierung

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

Dissection und Flach Montage des Dreistachliger Stichling Branchiogene Skeleton

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