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.

Todo el trabajo de los peces fue aprobado por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de California en Berkeley (número de protocolo R330). La eutanasia se realizó mediante inmersión en 0,025% tricaína-S tamponada con bicarbonato de sodio 0,1% 39. Todas las etapas se realizaron a temperatura ambiente. 1. Preparación Nota: Realice los pasos 1.1-1.5 en tubos cónicos o viales de centelleo que puede sellar herméticamente y mantenerse en posición horizontal. Los peces no deben ser agitadas constantemente, pero tratan de mezclar la solución tan a menudo como sea posible invirtiendo suavemente o se agita el bastidor de tubos o viales para exponer todos los lados de los peces a la solución de tinción y permitir que la mancha penetre en el tejido de manera uniforme. No coloque un gran lote de pescado en un agitador de plataforma, como el peso pesado del líquido romperá el agitador. <ol><li> Fijar bien el pescado recién sacrificados o pescado almacenado en etanol con 10% de formalina tamponada neutra (NBF) durante la noche. Como alternativa, utilice el 4% paraformaldehído en solución 1x PBS en lugar de 10% NBF. Nota: Si la extracción de ADN, clip de una pequeña porción de las aletas caudales o pectorales antes de la fijación y almacenar en etanol. </li><li> Disponer de corrección adecuadamente en una campana química y reemplazar con agua del grifo (es decir ~ pH 7,0) durante 2 horas. Evitar el uso de agua desionizada, ya que a menudo puede ser ácida y puede descalcificar el hueso. </li><li> Eliminar el agua y los peces mancha con 0,008% rojo de alizarina S en el 1% de KOH en agua durante 24 horas. Para los peces de menos de 20 mm de longitud estándar, utilice 0,004% rojo de alizarina S. (Haga una solución de reserva de 100x (0,8%) de rojo de alizarina S que a continuación se puede diluir). </li><li> Retirar la mancha (poner en un recipiente apropiado para desechos en la campana) y el lugar de peces en el agua del grifo para unos pocos horas. Cambiar el agua según sea necesario hasta que el agua de enjuague es mayormente despejado. </li><li> Eliminar el agua y el pescado en lugar de 50% de glicerol, 0,25% de KOH para la limpieza suave y posterior disección. Nota: Este protocolo de tinción es una modificación de los métodos descritos anteriormente 40,41. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Disección Nota: Véase la Figura 1 para una revisión de la morfología del esqueleto cabeza relevante. <img alt="Figura 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> Figura 1:. Stickleback cabeza de la morfología del esqueleto rojo de alizarina manchada cabeza espinoso de tres espinas fotografiado con fluorescencia bajo un conjunto de filtro de rodamina B. la morfología de utilidad tiene la etiqueta: OP = opérculo, SubOp = subopérculo, BSR = branquiostegales rayos, Preop = preopérculo, Infraorb 1-3 = 1-3 infraorbitarios (también llamados circumorbitals o suborbitales), Dent = dentario, Premax = premaxilar, maxilar = Max , NAS = nasal, Lat. ETHM = etmoidal lateral, PSPH = parasphenoid, Fron = hueso frontal. Para una descripción más detallada del esqueleto de la cabeza del espinoso, véase Anker (1974) 31. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de</a>esta figura. <ol><li> Coloque el pescado plano (Figura 2A) e insertar una pinza afilada # 5 de relojero en el lado del ojo en un ángulo de 45 ° ~ para perforar la membrana que cubre el ojo. </li><li> Pelar la membrana lejos del ojo, similar a la descamación una tapa yogur (Figura 2B). </li><li> Inserte fórceps abierto detrás del ojo, agarrar el nervio óptico detrás del ojo, y quitar el ojo (Figura 2C). No perforar el ojo, ya que se fuga de melanina. En caso de pinchazo, la melanina se puede lavar durante las etapas posteriores. </li><li> Repita del otro lado. </li><li> A partir de la parte posterior, coloque una pequeña cuchilla de disección tijera debajo de la solapa opérculo, la cuchilla de arrastre de tijera dorsal por encima de opérculo, luego se corta el tejido blando a través de la cuenca del ojo (Figura 2D). Cortar dorsal de la médula opérculo. </li><li> Cortar el hueso frontal (dorsal a la cuenca del ojo) (Figura 2E). </li><li> Cortar el hueso alrededor de la línea media parasphenoid tque el centro de las cuencas de los ojos (Figura 2F). </li><li> corte opérculo Repita en el lado opuesto. </li><li> Inserte fórceps bajo el opérculo y lentamente la cáscara de la cara lejos del cuerpo, el recorte de cualquier tejido blando todavía unido (Figura 2 G - H). Tenga cuidado de no perturbar la primera fila de hendiduras branquiales. <ol><li> Con unas pinzas, separar los ceratohyals en ambos lados de la basihyal línea media, mientras que pelar y retirar el esqueleto craneofacial anterior (toda la mandíbula incluyendo el dentario, premaxilar y maxilar; todo el esqueleto hioides incluyendo el opérculo dérmica exterior, preopérculo, subopérculo, y branquiostegales rayos y los dorsales y elementos que subyace endochondral ventral, y la parte anterior del cráneo, incluidos el nasal, etmoidal lateral, y los huesos infraorbitarios, véanse las figuras 1 y 2I). </li><li> espinas pélvicas se pueden plegar hacia fuera del cuerpo, y pueden servir como un asa para unas pinzas para agarrar of cuando está presente. Espinas encajen en su sitio. Para desbloquear, tirar suavemente de la columna vertebral con una pinza en dirección opuesta a cuerpo de los peces, y doblar suavemente en sentido posterior para presionar contra la columna vertebral plana peces. </li></ol></li><li> Insertar cerrado fórceps posterior y ventral al esqueleto branquial (justo debajo de tubo intestinal) y las pinzas de arrastre en sentido anterior, las burlas aparte los músculos y ligamentos que quedan unidos al esqueleto branquial (Figura 2J - K). </li><li> Usando la punta de unas pinzas cerradas, raspar los músculos que sujetan el dorsal esqueleto branquial a la caja craneana ventral en una posterior a la dirección anterior (Figura 2 l). </li><li> Repetir 2,9 y 2,10 en el lado opuesto. </li><li> Agarre la base del tubo intestinal y tire hacia delante para eliminar el esqueleto y el intestino branquial tubo (Figura 2 M - N). </li><li> Separar el tubo intestinal haciendo una posterior corte perpendicular al final de la quinta ceratobranquial (Figura 2O </strong&gt;). </li><li> Después de eliminar cualquier fragmento de hueso restante de la caja craneal en el lado dorsal del esqueleto branquial, insertar tijeras en la canasta branquial para hacer un corte dorsal (corte de anterior a posterior) entre los conjuntos bilaterales de placas de dientes dorsal (Figura 3A - D). corte se centra garantizar para evitar dañar las placas de los dientes dorsal. </li><li> Haga dos cortes laterales poco profundas en la luz del intestino de caucho en el extremo posterior del esqueleto branquial (extremo anterior del tubo intestinal) para ayudar en la apertura de la esqueleto branquial (Figura 3E). </li><li> Coloque el pescado y todas las piezas de tejido en un frasco y colocar el esqueleto branquial en un tubo de microcentrífuga con 50% de glicerol, 0,25% de KOH para continuar limpieza suave, o el 100% de glicerol, si se requiere ninguna otra compensación. frascos y tubos de etiquetas con un identificador único para que puedan ser rastreados. La cantidad de aclaramiento requerido es en gran parte una función del tamaño de los peces, peces grandes adulto (mayor de 40 mm de longitud estándar) requieren normalmente de compensación adicional. </li></ol><img alt="Figura 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> Figura 2:. Stickleback disección esqueleto branquial manchado rojo de alizarina pez espinoso de tres espinas listo para la disección. El ojo está despigmentada de amplio legrado. Las flechas azules indican la dirección del movimiento. (A) Vista lateral de la cabeza del espinoso, es anterior a la derecha. (B) La eliminación de la membrana que cubre el ojo. (C) La eliminación del ojo. (D) Dorsal cortado por encima del opérculo. Corte del hueso (E) frontal. (F) Parasphenoid cortar. (G - I) La eliminación del esqueleto facial. (J) La eliminación de las conexiones de los tejidos blandos esqueleto branquial ventrales. (K - L) La eliminación de las conexiones esqueleto branquial dorsales. (&lt;strong&gt; M - N) La eliminación del esqueleto branquial. (O) Separar el tubo intestinal del esqueleto branquial. Consulte los pasos 2.1 a 2.16 para obtener más detalles. Barra de escala = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> 3. branquial Esqueleto Re-tinción (si es necesario) <ol><li> Para teñir el más oscuro esqueleto branquial o tejido claro más, retire 50% de glicerol, solución de KOH 0,25% y se lava con 1% KOH dos veces (un lavado de 5 minutos seguido de un segundo lavado 24 hr mientras se agita horizontalmente en un agitador de plataforma). </li><li> Retire 1% KOH y re-mancha con 0.008% rojo de alizarina S en 1% de KOH durante 24 h. </li><li> Retire las manchas y reemplazar con 1% de KOH durante 24 horas. </li><li> Retire la solución de KOH y reemplazar con 50% de glicerol, 0,25% de KOH. </li></ol> 4. Montaje branquial Esqueleto <ol><li> Retire esqueleto branquialde 50% de glicerol, 0,25% de KOH o 100% de glicerol y el lugar cerca de la parte inferior de un 22 mm x 60 mm cubreobjetos de vidrio con el lado dorsal hacia arriba (Figura 3F). Añadir unas gotas de glicerina al 50%, 0,25% KOH o 100% de glicerol en la parte superior del esqueleto branquial. Si la transición de 50% de glicerol, 0,25% de KOH al 100% de glicerol, solución de cambio en un tubo de microcentrífuga y agitar durante&gt; 5 min antes de montar para equilibrar los tejidos. </li><li> Estirar dos pequeñas bolas de arcilla de modelar y colocar en cada extremo de la hoja de la cubierta para actuar como espaciadores. </li><li> Sin apretar colocar un segundo cubreobjetos en la parte superior con la presión suficiente para aplanar el esqueleto branquial anterior (Figura 3G). </li><li> Abra retirando la tapa dorsal izquierda que incluye placas de dientes dorsales, aplanar, y se deslizan entre los cubreobjetos (Figura 3H). </li><li> Repita técnica con la aleta dorsal derecha y empujar toda esqueleto branquial de distancia desde el borde del cubreobjetos (Figura 3I). <ol><li> Alterarde forma nativa, mantenga ambas aletas dorsales abiertas con unas pinzas y colocar con cuidado el cubreobjetos sobre, aplanando el esqueleto branquial con un movimiento suave. </li><li> Como alternativa, montar el esqueleto branquial al revés sobre una hoja de la cubierta, extendiendo cada lado dorsal lateralmente para que la gravedad no permite que el esqueleto branquial para cerrar una copia de seguridad. A continuación, cubrir con la segunda x 60 mm cubreobjetos de vidrio de 22 mm e invierta preparación. Nota: Las diferentes técnicas de montaje tienden a funcionar mejor o peor para cada individuo. Pruebe cada uno y ver lo que se siente más cómodo. </li></ol></li><li> Presione ligeramente en la parte superior cubreobjetos para aplanar las bolas de arcilla suficientes para mantener el esqueleto branquial montado plana, pero tenga cuidado de no aplastar el espécimen. <ol><li> Durante el proceso de montaje, ceratobranchials pueden rotar y oscurecer una fila de rastrillos. Remediar esto mediante una pinza entre el cubreobjetos y reorientando los ceratobranchials o de todo el esqueleto branquial deslizante. </li></ol></li><li> Tienda prepara plana en bandejas deslizantes en rotemperatura om. Montado en el 100% de glicerol, preparaciones se pueden almacenar entre cubreobjetos puenteados durante al menos una década. pinzas limpias y tijeras con isopropanol o etanol y consejos de cubierta. </li></ol><img alt="figura 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> Se muestra de montaje plana del esqueleto branquial manipulación y el montaje del esqueleto branquial: Figura 3.. Las flechas azules indican la dirección del movimiento. (A) branquial dorsal esqueleto hacia arriba. (B - D) La rotación y la incisión entre las placas dentales dorsal. (E) de corte lateral en el tejido blando para abrir aún más la base del tubo intestinal. (F) branquial esqueleto colocado en la parte inferior de un cubreobjetos listo para el montaje. (G) En segundo cubreobjetos colocado en la mitad anterior del esqueleto branquial (por encima de las placas dorsales de los dientes). (H - I)montaje plano del esqueleto branquial mediante la apertura de las aletas de la placa de los dientes dorsales y deslizamiento entre dos hojas de la cubierta. Consulte los pasos 4.1 a 4.6 para más detalles. Barra de escala = 5 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+kinetics+of+the+head+of+the+stickleback,+Gasterosteus+aculeatus.">Morphology and kinetics of the head of the stickleback, Gasterosteus aculeatus.</a> Trans. Zool. Soc. London. 32 (5), 311-416 (1974).</li><li>Meyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometrics+and+allometry+in+the+trophically+polymorphic+cichlid+fish,+Cichlusomu+citrinelfum:+Alternative+adaptations+and+ontogenetic+changes+in+shape.">Morphometrics and allometry in the trophically polymorphic cichlid fish, Cichlusomu citrinelfum: Alternative adaptations and ontogenetic changes in shape.</a> J. 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...

Una cantidad increíble de la diversidad existente en el esqueleto de la cabeza entre los vertebrados, en especial entre los peces. En muchos casos esta diversidad facilita diferentes estrategias de alimentación 1-4, y puede implicar cambios importantes en tanto patrón craneofacial externa e interna. El esqueleto branquial está situado internamente en la garganta de un pez y rodea la mayor parte de la cavidad bucal. El esqueleto branquial se compone de 5 segmentos serie homóloga, la parte anterior de cuatro de los cuales apoyan las branquias. En conjunto, estos cinco segmentos funcionan como una interfaz entre el pescado y sus alimentos 5. La variación en una multitud de rasgos incluyendo branquiespinas, dientes faríngeos, y los huesos branquiales contribuyen a forrajeo eficiente en diferentes tipos de alimentos. Espinosos han sido sometidos a una radiación adaptativa después de formas ancestrales oceánicas colonizados lagos de agua dulce y arroyos en todo el hemisferio norte. El cambio en la dietadel zooplancton pequeño en el océano para una presa más grande en el agua dulce se ha traducido en la variación trófica dramático en varios rasgos craneofaciales 6. Mientras que muchos estudios se han centrado en las diferencias craneofaciales externos en los espinosos 7 - 13, los cambios craneofaciales importantes evolucionan en repetidas ocasiones en el esqueleto branquial interno. La capacidad de crear híbridos fértiles entre poblaciones de espinosos morfológicamente distintos proporciona una excelente oportunidad para mapear la base genética de los cambios evolucionado hasta el esqueleto branquial. Un rasgo trófica de importancia ecológica es el patrón de hendiduras branquiales, los huesos dérmicos periódicas que recubren las caras anterior y posterior de los huesos branquiales y se utilizan para filtrar los elementos de presa. Los peces que normalmente se alimentan de pequeñas presas tienden a tener más largo y más densamente espaciados Gill RäKERS en comparación con los peces que se alimentan de presas más grandes 14,15. La variación en branquiespinas ha informado tanto wentro de las especies y entre 14-19, y los aspectos de enmalle de profundidad de corte de patrones contribuyen a nichos tróficos y gimnasio 16. Décadas de investigación han documentado ampliamente el número de enmalle de profundidad de corte y la variación de la longitud de los peces espinosos de tres espinas 17 - 21; Sin embargo, estos estudios suelen centrarse en la primera fila de hendiduras branquiales. Trabajos recientes han demostrado la modularidad en el control genético del número de enmalle de profundidad de corte a través del esqueleto branquial 22,23 ya través de una sola fila en la profundidad de corte de enmalle espaciamiento 23 y 24 de longitud que destaca la importancia de estudiar más de la fila uno o un solo diente de ataque de enmalle para entender la base genética del desarrollo de la reducción de enmalle de profundidad de corte. Un segundo rasgo trófica de tanto significado ecológico y biomédica es el patrón de los dientes faríngeos. Los dientes en los peces pueden estar situados tanto en la mandíbula oral y en el esqueleto branquial, conocido como dientes faríngeos. dientes orales se utilizan principalmente para pRey capturar mientras que los dientes faríngeos se utilizan para la masticación y la presa de manipulación 25 - 27. Ambos conjuntos se forman a través de los mecanismos de desarrollo compartidos y se consideran homólogas de desarrollo 28. Modularidad interesante se produce mediante el cual algunas especies, como el pez cebra, la falta de dientes de la faringe oral y dorsal 29, mientras que otras especies tienen múltiples ceratobranchials dentadas, pharyngobranchials y, a veces dentadas basihyal y hypobranchials 30. En los espinosos, dientes faríngeos se encuentran ventralmente en la quinta ceratobranquial y dorsalmente en la parte anterior y posterior pharyngobranchials 31. Cinemática sobre la alimentación del pez espinoso muestran la mandíbula oral se utiliza principalmente para la captura de presas y facilitar la alimentación de aspiración 9 dejando la masticación de la mandíbula faríngea. En cíclidos, menor morfología de la mandíbula faríngea varía dramáticamente 32,33 y se ha demostrado que ser adaptable y se correlacionó con nicho trófico 34. Multipoblaciones de espinosos de agua dulce ples han evolucionado aumentos espectaculares en la faringe ventral número de dientes 23,35,36. Trabajos recientes han demostrado que la base genética del desarrollo de esta ganancia diente evolucionado en gran medida es distinta en dos poblaciones derivadas independientemente de espinosos de agua dulce 36. A diferencia de los dientes de mamíferos, peces regeneran sus dientes de forma continua durante la vida adulta 37. Ambas poblaciones de agua dulce dentadas altas anteriormente descritos han evolucionado una tasa de reemplazo de dientes acelerado, proporcionando un sistema de vertebrados rara para estudiar la base genética de regeneración 36. Un tercer rasgo trófico que se ha desarrollado en varias ocasiones en los espinosos de agua dulce es más largo y huesos epibranchial ceratobranquiales, los homólogos de arco rebajado branquiales de la mandíbula superior e inferior, respectivamente 38. Más largos huesos branquiales confieren una cavidad bucal grande y probablemente son adaptativos para permitir que las presas más grandes para ser Consumed. Por otra parte, en otro pescado, huesos epibranchial son importantes para la depresión de las placas de dientes faríngeos dorsal 25. Al igual que branquiespinas y dientes faríngeos, los huesos branquiales son internos y, por tanto, difíciles de visualizar con facilidad o cuantificar. Aquí se presenta un protocolo detallado para diseccionar y de montaje en el esqueleto branquial plana, lo que permite una fácil visualización y cuantificación de una variedad de importantes rasgos craneofaciales. Si bien este protocolo describe una disección stickleback, este mismo método funciona en una variedad de otros peces.

<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> Este protocolo da como resultado un esqueleto branquial montada diseccionado y plana (Figura 4), ​​donde una variedad de importantes rasgos tróficos se puede cuantificar. Desde una vista dorsal, todas las filas de hendiduras branquiales, todas las placas de dientes faríngeos, y casi todos los huesos branquiales pueden ser fácilmente visualizados y cuantificaron 22 - 24,35,36,38,42. Rojo de alizarina S también emite fluoresce...

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.

La disección y-montaje plana del esqueleto branquial de tres espinas Stickleback

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

Research

JoVE Journal

Biology

9.7K vistas.  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.

Expresión de proteínas fluorescentes en<em&gt; Lanceolatum Branchiostoma</em&gt; Por mRNA La inyección en ovocitos no fecundados

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

Investigación

Biología del Desarrollo

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.

La microinyección de transgénesis y Genoma edición en tres espinas espinosos

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.

La construcción de modelos de elementos finitos para investigar pez cebra mandíbula Biomecánica

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.

Caracterización de las escalas de peces de múltiples capas-(<em&gt; Atractosteus espátula</em&gt;) Usando Nanoindentación, X-ray CT, FTIR y SEM

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

Bioingeniería

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.

Secuencial Foto-blanqueamiento para Precisar las células individuales de Schwann en la unión neuromuscular

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 ...

Neurociencias

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.

La disección y de montaje lateral de embriones de pez cebra: Análisis para el Desarrollo de la médula espinal

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.

Un cultivo de explantes de cola 3-D para estudiar la segmentación de vertebrados en el pez cebra

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.

Enfoques de visualización y análisis tridimensionales y de cuatro dimensiones para estudiar la elongación y segmentación axial de vertebrados

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.

Modelo de lesión por puñalada del Tectum óptico adulto utilizando pez cebra y medaka para el análisis comparativo de la capacidad regenerativa

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.

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje<em&gt; In situ</em&gt; La hibridación

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

La disección y-montaje plana del esqueleto branquial de tres espinas Stickleback

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