Name: inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis
Uploaded: 2017-01-14T13:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

Este trabajo fue apoyado por una de Células Madre de Nueva York Ciencia (NYSTEM C028107) Subvención a GHT.

1. Acondicionamiento de animales con la temperatura, la nutrición y la Luz del ciclo oscuro / <ol><li> Obtener adultos Nematostella vectensis de uno de los numerosos laboratorios de todo el mundo Nematostella, o un proveedor sin fines de lucro (Tabla 1) </li><li> Mantener Nematostella a temperatura constante (típicamente entre 18 y 21 ° C) en la oscuridad, en &quot;1 / 3x&quot; Artificial Agua de Mar (ASW) a una salinidad de 12 partes por mil (ppt). Mantener los cultivos en placas de cultivo simples vidrio de sosa-cal, por lo general de 250 ml o 1,5 capacidad de 11 l. Nota: Estas condiciones de cultivo simples se usan comúnmente entre los laboratorios que estudian Nematostella, pero el cuidado de cultivo también pueden automatizarse 19. </li><li> Alimentar Nematostella Artemia recién nacidas nauplios 2 - 4x por semana. Hatch Artemia quistes en toda su fuerza (36 ppt) o 1 / 3x ASW a 30 ° C, en un recipiente de vidrio rectangular poco profunda 20 o encualquiera de una serie de pequeña escala, los criaderos de camarón de salmuera comerciales o caseros. Si una incubadora no está disponible, camarones eclosionan a temperatura ambiente, pero lo hacen más lentamente. Nota: Esto requiere a menudo más de 24 horas para su finalización. </li><li> Cambie el agua de cultivo anémona al menos una vez a la semana. Para obtener la mejor salud de los adultos, completamente limpio (sin jabón), el cultivo cuencos una vez por semana de las secreciones mucosas acumuladas, que cubra el recipiente y pueden atrapar los alimentos no consumidos y los residuos, y enredar a los animales. </li></ol> 2. Selección y relajación de los Animales Nutricionalmente acondicionado <ol><li> Seleccione los pólipos de tamaño con ajuste de aproximadamente la misma longitud (3 - 5 cm, cuando, naturalmente, relajado) y colocarlos en un recipiente separado de la colonia durante tres días antes de la amputación. Nota: El número de animales seleccionados para el corte será determinado por el experimento está llevando a cabo, por supuesto, pero en general se recomienda un mínimo de cinco animales por punto de muestreo con seis repeticiones. Así,En un experimento típico de un mínimo de 30 animales se preselecciona. En general, es aconsejable seleccionar más que el número mínimo (30), ya que las amputaciones que son irregulares (véase más adelante) puede afectar a más de puntuación. </li><li> Retirar la cápsula de animales seleccionados de la incubadora en la luz de la habitación al menos una hora antes de la amputación. Nota: La exposición a la luz de la sala y las vibraciones de la manipulación probablemente hará que los animales se contraigan, por lo que necesitan ser adaptados o &quot;relajado&quot; por incubación en la mesa de laboratorio. Los animales serán refractarios al tacto ya la exposición a la luz y en ese momento puede ser movido por pipeteo suave. </li><li> Opcional: Anestesie los animales mediante la adición de 7,5% de MgCl2 (en 1 / 3x ASW). Añadir suavemente la solución de MgCl2 en el recipiente con un plástico de 5 ml pipeta estándar. Nota: Aunque los animales con el tiempo habituado a la luz y a la manipulación física, puede ser ventajoso para anestesiar a los animales para mantener o &quot;arreglar&quot; tse relajó estado después de que se han convertido en 16,21,22 alargada. </li><li> Utiliza una amplia perforación (&gt; 0,5 cm) pipeta de plástico para transferir (en 1 / 3x ASW) de cinco animales de la piscina a ser amputada, en el fondo de un plato de corte de vidrio estéril de 100 mm de diámetro que contiene 12 partes por mil ASW. Coloque el plato en el escenario de un microscopio estereoscópico con aumento variable entre 10 - 40X. Nota: Si los animales no han sido anestesiado y relajado para el corte, aún pueden responder al tacto y la iluminación estereoscopio y por lo tanto puede necesitar unos minutos para convertirse relajado de nuevo. </li></ol> 3. La amputación <ol><li> Con un bisturí estéril, amputar la Physa aboral de cada pólipo, con el objetivo de obtener una sección de la Physa que es aproximadamente el tiempo que es ancho y que no contiene mesenterio. Nota: El sitio de corte ideal es sólo aboral a la terminación del mesenterio. En el plano de corte hay una transición de mesenterio adecuada para diluir lines correspondiente a cada inserción mesenterial (véase la Figura 1, las flechas). La ausencia de mesenterio es crítica, ya que produce mucosa que pueden facilitar &#39;tapar&#39; el agujero 17,30. <ol><li> Coloque la hoja de bisturí en contacto con el animal en el sitio deseado de la amputación. Para ello, sin necesidad de asistencia (a pulso), o agarrando suavemente el cuerpo del animal con unos fórceps # 5 (estilo Dumont o similares). </li><li> Cortar a través del tejido mediante el aprovechamiento de la hoja curva del bisturí en un movimiento de &quot;oscilación&quot; a través del cuerpo. Nota: El tejido debe cortar limpiamente como el bisturí se sacudió y liberar la sección deseada de Physa del donante. Sin embargo, si una pequeña porción de tejido sigue conectando el cuerpo y el Physa, cortar con el bisturí. No intente separar las piezas conectadas tirando, ya que esto puede dañar el Physa. </li></ol></li><li> Retire cada pólipo &quot;donante&quot; amputada del plato y devolverlo a una SEPAtazón dosis de etiqueta &#39;amputados puestas en común »; la fecha, el cuenco y devolverlo al cultivo de reserva. Nota: Los pólipos amputados se cura la herida aboral en un día y luego pueden ser alimentados normalmente. Ellos se regenerarán un Physa aspecto normal dentro de dos semanas y en ese momento la Physa puede ser amputada de nuevo si lo desea. </li><li> Enjuague el Physa extirpado que permanecen en el plato de corte en 12 ASW ppt, a continuación, transferir cada Physa a un pocillo estéril independiente en una placa de cultivo celular de múltiples pozos que ya cuenta con 10 ml de 12 partes por mil ASW en cada pocillo. Nota: En este ejemplo se utiliza una placa de seis pocillos, cada pocillo con la celebración de 10 ml de agua de mar y cinco Physa extirpado. En general, el agua de mar que debe cubrir los Physa lo suficiente como para evitar la exposición al aire debido al movimiento en el manejo y la evaporación potencial. La placa o pozos deben tener una tapa. </li><li> Repita los pasos 03.01 a 03.03 para recoger al menos 5 Physa en cada pocillo reservada para cada tratamiento experimental. </li><li> Incubar la Physa a una temperatura que provide la mejor tasa de regeneración de los interrogatorios experimentales planificadas. Colocar la placa que contiene el Physa en una incubadora de temperatura regulada, a una temperatura fija determinada por la velocidad deseada de regeneración. Nota: El Physa regenerará tejidos que faltan y formar un pólipo completo cuando se incuban a temperaturas entre 15 y 27 ° C. La tasa de regeneración es dependiente de la temperatura a excepción de las dos primeras etapas. El día medio para llegar a la etapa 4 para todas las temperaturas es de 7 d después del corte y esto también coincide con la regeneración a 21 ° C. A 27 ° C, Etapa 4 se alcanza alrededor de 3 días antes y a 15 ° C, Etapa 4 se retrasa en aproximadamente 3 días, en comparación con la regeneración a 21 ° C (también véase la referencia 10). </li></ol> 4. La evaluación de la regeneración con el sistema de clasificación de Nematostella Regeneración (NRSS) <ol><li> Anotar el Physa usando un microscopio estéreo-compuesto con magnífica variablesación (10 - 80X). Anotar el Physa Nematostella recién cortada que en la Etapa 0 y continuar anotando al mismo tiempo cada día después de la amputación (dpa) utilizando el NRSS 10. Nota: Para los criterios clave de parada y los detalles se refieren referencia 10. <ol><li> Puntuación Physa que en la Etapa 0 (herida abierta) si un Physa recién cortada aparece como una masa en forma de copa se asemeja a un globo flácida, con una zona de la herida abierta es probable visible. Nota: Los bordes de la herida también podrían permanecer juntos desde el principio, pero estarán colapsado el tejido y la falta de rigidez. Los bordes de la herida abierta se pueden observar de someterse a la contracción radial como la herida se cura. </li><li> Puntuación Physa que en la Etapa 1 (herida cerrada) si la herida de la amputación aparece cerrado. Nota: La ubicación de la herida se corresponderá con el futuro polo oral. La superficie exterior en torno al futuro polo oral puede comenzar a mostrar arcos distintos que corresponden a las inserciones subyacentes mesenterial endodermales radialmente simétrica. </li><li> ph puntajeYSA que en la Etapa 2 (de los arcos radiales) si la superficie del polo oral parece infla, revelando ocho arcos elevadas dispuestas en un patrón radialmente simétrica y separadas por ranuras. Observe ampollas pequeñas, hemisféricas en el vértice de los arcos. Ellos serán casi tan alto como ancho transitoria, probablemente, e inicialmente compuesto por una sola capa de células ectodérmicas. Nota: En algunos casos, las vesículas de doble capa pueden estabilizarse. Nota: En esta etapa hacia adelante una capa mucosa puede parecer que encapsular el Physa (Figura 2) en una &#39;vaina&#39; membranosa. Este material de encapsulación se debe quitar para facilitar la puntuación. </li><li> Puntuación Physa que en la Etapa 3 (tentáculo) si los brotes de los tentáculos que contienen capas de tejido endodérmico y ectodérmicas se forma estable formado en el extremo oral de al menos algunos arcos radiales. Nota: Los tentáculos son más largos que anchos y están mínimamente móviles. El Physa mostrarán un aumento, pero la inflación variable de modo que rudimentos mesenterio puede llegar a ser visIble que se extiende desde la inserción mesenterial en la cavidad corporal (celenteron). </li><li> Puntuación Physa que en la Etapa 4 (mesenterios lineal) si el Physa contiene ocho mesenterios distintas, visibles que se extienden en la celenteron de inserciones en la pared del cuerpo, con longitudes orales-aboral que son más del doble de su anchura radial medida desde donde aparecen para conectar a la faringe en su extremo aboral (enterostome). Nota: Cuatro o menos mesentenos tienen bordes libres internos &quot;plisados&quot;. La faringe es visible. Más de ocho tentáculos son visibles, móviles y, a veces se contraen en el cuerpo. </li><li> Puntuación Physa que en la Etapa 5 (Predominio plisados ​​mesenterio) si el Physa tiene más de cuatro mesenterio con plisado, y el plisado es más completa y sinuoso que en la Etapa 4. El animal tiene una apariencia casi adulta &quot;normal&quot;, pero no hay visibles células gonadales. </li></ol></li></ol>

<ol>
	<li>Lenhoff, S. G., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydra and the Birth of Experimental Biology: Abraham Trembley's Memoirs Concerning the Natural History of a Type of Freshwater Polyp with Arms Shaped like Horns. , (1986).</li><li>Trembley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> M&#233;moires pour servir &#224; l'histoire d'un genre de polypes d'eau douce, &#224; bras en forme de cornes. , (1744).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nat Rev Genet. 11 (10), 710-722 (2010).</li><li>Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydra,+a+fruitful+model+system+for+270+years.">Hydra, a fruitful model system for 270 years.</a> Int J Dev Biol. 56 (6-8), 411-423 (2012).</li><li>Gold, D. A., Jacobs, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+Cnidaria:+are+there+unifying+principles?.">Stem cell dynamics in Cnidaria: are there unifying principles?.</a> Dev Genes Evol. 233 (1-2), 53-66 (2013).</li><li>Holstein, T. W., Hobmayer, E., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnidarians:+an+evolutionarily+conserved+model+system+for+regeneration?.">Cnidarians: an evolutionarily conserved model system for regeneration?.</a> Dev Dyn. 226 (2), 257-267 (2003).</li><li>Amiel, A. R., 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=Characterization+of+Morphological+and+Cellular+Events+Underlying+Oral+Regeneration+in+the+Sea+Anemone,+Nematostella+vectensis.">Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis.</a> Int J Mol Sci. 16 (12), 28449-28471 (2015).</li><li>Warren, C. R., 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=Evolution+of+the+perlecan/HSPG2+gene+and+its+activation+in+regenerating+Nematostella+vectensis.">Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.</a> PLoS One. 10 (4), e0124578 (2015).</li><li>Gong, Q., 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=Integrins+of+the+starlet+sea+anemone+Nematostella+vectensis.">Integrins of the starlet sea anemone Nematostella vectensis.</a> Biol Bull. 227 (3), 211-220 (2014).</li><li>Bossert, P. E., Dunn, M. P., Thomsen, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+staging+system+for+the+regeneration+of+a+polyp+from+the+aboral+physa+of+the+anthozoan+Cnidarian+Nematostella+vectensis.">A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis.</a> Dev Dyn. 242 (11), 1320-1331 (2013).</li><li>Stefanik, D. J., Friedman, L. E., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting,+rearing,+spawning+and+inducing+regeneration+of+the+starlet+sea+anemone,+Nematostella+vectensis.">Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis.</a> Nat Protoc. 8 (5), 916-923 (2013).</li><li>Tucker, R. P., 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+thrombospondin+in+the+anthozoan+Nematostella+vectensis+is+associated+with+the+nervous+system+and+upregulated+during+regeneration.">A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.</a> Biol Open. 2 (2), 217-226 (2013).</li><li>Passamaneck, Y. J., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+is+necessary+for+the+regeneration+of+oral+structures+in+the+anthozoan+cnidarian+Nematostella+vectensis.">Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis.</a> BMC Dev Biol. 12, (2012).</li><li>Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S., Burton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+canonical+Wnt+signaling+by+alsterpaullone+is+sufficient+for+oral+tissue+fate+during+regeneration+and+embryogenesis+in+Nematostella+vectensis.">Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis.</a> Dev Dyn. 240 (12), 2673-2679 (2011).</li><li>Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+muscle-specific+transgenic+reporter+line+of+the+sea+anemone,+Nematostella+vectensis.">A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis.</a> Proc Natl Acad Sci U S A. 107 (1), 104-108 (2010).</li><li>Burton, P. M., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+novel+gene+expression+between+regeneration+and+asexual+fission+in+Nematostella+vectensis.">Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis.</a> Dev Genes Evol. 219 (2), 79-87 (2009).</li><li>DuBuc, T. Q., Traylor-Knowles, N., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+a+regenerative+response;+cellular+and+molecular+features+of+wound+healing+in+the+cnidarian+Nematostella+vectensis.">Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis.</a> BMC Biol. 12, (2014).</li><li>Hand, C., Uhlinger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+by+transverse+fission+and+some+anomalies+in+the+sea+anemone+Nematostella+vectensis.">Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis.</a> Invert Biol. 114, 9-18 (1995).</li><li>Fritzenwanker, J. H., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+gametogenesis+in+the+basal+cnidarian+Nematostella+vectensis(Anthozoa).">Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa).</a> Dev Genes Evol. 212 (2), 99-103 (2002).</li><li>Magie, C., Bossert, P., Aramli, L., Thomsen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science's+super+star:+The+starlet+sea+anemone+is+an+ideal+tool+for+student+inquiry.">Science's super star: The starlet sea anemone is an ideal tool for student inquiry.</a> The Science Teacher. 83 (3), 33-40 (2016).</li><li>Genikhovich, G., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+of+starlet+sea+anemone+(Nematostella+vectensis)+embryos,+larvae,+and+polyps.">In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps.</a> Cold Spring Harb Protoc. (9), (2009).</li><li>Magie, C. R., Pang, K., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+inventory+and+expression+of+Sox+and+Fox+genes+in+the+cnidarian+Nematostella+vectensis.">Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis.</a> Dev Genes Evol. 215 (12), 618-630 (2005).</li><li>Chera, S., Kaloulis, K., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cAMP+response+element+binding+protein+(CREB)+as+an+integrative+HUB+selector+in+metazoans:+clues+from+the+hydra+model+system.">The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system.</a> Biosystems. 87 (2-3), 191-203 (2007).</li><li>Reitzel, A. M., Burton, P. M., Krone, C., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+developmental+trajectories+in+the+starlet+sea+anemone+Nematostella+vectensis:+embryogenesis,+regeneration,+and+two+forms+of+asexual+fission.">Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission.</a> Invertebr Biol. 126, 99-112 (2007).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nat Commun. 5, 5486 (2014).</li><li>Jahnel, S. M., Walzl, M., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+epithelial+organisation+of+muscle+cells+in+the+sea+anemone+Nematostella+vectensis.">Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.</a> Front Zool. 11, 44 (2014).</li><li>Kelava, I., Rentzsch, F., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+eumetazoan+nervous+systems:+insights+from+cnidarians.">Evolution of eumetazoan nervous systems: insights from cnidarians.</a> Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).</li><li>Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+systems+of+the+sea+anemone+Nematostella+vectensis+are+generated+by+ectoderm+and+endoderm+and+shaped+by+distinct+mechanisms.">Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms.</a> Development. 139 (2), 347-357 (2012).</li><li>Richards, G. S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. Q., 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=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. I., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+autophagy:+cross-talk+and+co-operation+to+control+cellular+homeostasis.">Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.</a> Amino Acids. 47 (10), 2065-2088 (2015).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Rodolfo, C., Di Bartolomeo, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+and+progenitor+cells.">Autophagy in stem and progenitor cells.</a> Cell Mol Life Sci. 73 (3), 475-496 (2016).</li><li>Guan, J. L., 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=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9 (6), 830-849 (2013).</li><li>Phadwal, K., Watson, A. S., Simon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightrope+act:+autophagy+in+stem+cell+renewal,+differentiation,+proliferation,+and+aging.">Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.</a> Cell Mol Life Sci. 70 (1), 89-103 (2013).</li><li>Varga, M., Fodor, E., Vellai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+zebrafish.">Autophagy in zebrafish.</a> Methods. 75, 172-180 (2015).</li><li>Varga, M., 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=Autophagy+is+required+for+zebrafish+caudal+fin+regeneration.">Autophagy is required for zebrafish caudal fin regeneration.</a> Cell Death Differ. 21 (4), 547-556 (2014).</li><li>Gonzalez-Estevez, C., Salo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+apoptosis+in+planarians.">Autophagy and apoptosis in planarians.</a> Apoptosis. 15 (3), 279-292 (2010).</li><li>Buzgariu, W., Chera, S., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+investigate+autophagy+during+starvation+and+regeneration+in+hydra.">Methods to investigate autophagy during starvation and regeneration in hydra.</a> Methods Enzymol. 451, 409-437 (2008).</li><li>Tettamanti, G., 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=Autophagy+in+invertebrates:+insights+into+development,+regeneration+and+body+remodeling.">Autophagy in invertebrates: insights into development, regeneration and body remodeling.</a> Curr Pharm Des. 14 (2), 116-125 (2008).</li></ol>

The authors have nothing to disclose.

El uso de Nematostella como modelo de curación y regeneración de la herida se está convirtiendo cada vez más popular. Por lo tanto, es importante ser capaz de visualizar los patrones morfológicos de un protocolo particular antes de los análisis celulares y moleculares eficaces pueden ser asignados y comparación. Nematostella tener un alto grado de regeneración &quot;flexibilidad&quot;, siendo capaz de reformar casi cualquier estructura que falta amputada en cualquier lugar, en las etapas poster...

La observación de la regeneración en un solo hydra fue el evento seminal en el advenimiento de la biología como un 1,2 ciencia experimental. La regeneración sigue siendo un fenómeno extraordinariamente amplia de apelación al biólogo y laico por igual. La posibilidad de que los biólogos del desarrollo, médicos, científicos e ingenieros biomédicos de tejido para comprender y superar los límites de la regeneración humana hace que la biología de regeneración más intrínsecamente interesante. Ahora, con el uso de las tecnologías emergentes, tales como la secuenciación del genoma y la ganancia y pérdida de función de las herramientas, el campo está a punto de burlarse de los mecanismos regenerativos aparte y para entender en última instancia cómo varias especies pueden regenerarse mientras que otros no pueden. El grado de coincidencia en las respuestas moleculares, celulares y morfológicos que queda por esclarecer, pero hasta el momento parece que las respuestas básicas entre los animales que pueden regenerar es más similar que habría sido ImagiNed hace sólo una década 3. Cnidarios en particular, son fácil a la regeneración casi todas las partes del cuerpo en medio de un amplio espectro de diversidad morfológica. Desde el pólipo solitario agua dulce, Hydra junto con los pequeños pólipos marinos que construyen inmensos arrecifes de coral, a los sifonóforos coloniales complejos, tales como el portugués Man-O-War, la regeneración es a menudo un modo de reproducción, además de un mecanismo para la reparación o la reforma de las partes del cuerpo dañados o perdidos como resultado de la lesión y la depredación. Si las diversas especies de cnidarios utilizan mecanismos similares o diferentes para la regeneración es una pregunta interesante, fundamentalmente, 4-6. Nosotros, y otros han estado desarrollando la anthozoan, Nematostella vectensis como modelo para la regeneración 7-17. Recientemente hemos desarrollado un sistema de clasificación para describir la regeneración de un cuerpo entero de un pedazo de tejido morfológicamente uniforme dividido en dos de los ABORAl final del pólipo 10. Mientras que los pólipos Nematostella pueden regenerarse cuando queda dividido en dos en cualquier nivel, se optó por cortar los adultos en una posición aboral en la región más morfológicamente simple, el Physa, en parte porque este está cerca del plano normal de la fisión asexual natural de 18 años, y también porque permite la observación y análisis moleculares de cómo un cuerpo entero se vuelve a montar a partir de los componentes morfológicos simples. El sistema de estadificación Nematostella Regeneración (NRSS) proporciona un conjunto relativamente simple de los puntos de referencia morfológicas que podrían ser utilizados para marcar el progreso de cualquier aspecto de la regeneración por un Physa amputada, en condiciones de cultivo normales o experimentalmente perturbado situaciones tales como tratamientos de molécula pequeña, la manipulación genética o alteración ambiental. Como se preveía, el NRSS se está adoptado como un andamio morfológica en la que puede hacer referencia a los eventos celulares y moleculares de la regeneración10. Finalmente nuestro método de corte produce un enorme agujero de varios órdenes de magnitud mayor que la punción punto de terminales que se utiliza en un estudio reciente 17, sin embargo, ambas heridas se curan en torno a 6 horas. Documentación de las fases visualmente llamativas y distintas de cierre de la herida debe sugerir enfoques experimentales para explicar la aparente independencia del tamaño de una herida y el tiempo que toma para cerrar. Por lo tanto, una comprensión visual más profunda del proceso de amputación aboral, proporcionada por este protocolo, ayudará a nuevas investigaciones sobre este sistema de regeneración de modelo y de ampliar la aplicación de este sistema de estadificación usando Nematostella vectensis.

<table><tbody><tr><td>&lt;em&gt;Nematostella vectensis,&lt;/em&gt; adults</td><td>Marine Biological Lab (MBL)</td><td></td><td>non-profit supplier</td></tr><tr><td>Glass Culture Dish, 250 mL</td><td>Carolina Biological Supply</td><td>741004</td><td>250 mL</td></tr><tr><td>Glass Culture Dish, 1,500 mL</td><td>Carolina Biological Supply</td><td>741006</td><td>1,500 mL</td></tr><tr><td>Polyethylene transfer pipette, 5&amp;nbsp;mL</td><td>USA Scientific&amp;nbsp;</td><td>1022-2500</td><td>narrow bore, graduated</td></tr><tr><td>Polyethylene transfer pipet, tapered</td><td>Samco</td><td>202-205</td><td>cut off 1 inch of tip to make wide bore</td></tr><tr><td>Disposable Scalpel</td><td>Feather Safety Razor Co. Ltd</td><td>no. 10</td><td>blade should be curved</td></tr><tr><td>#5 Dumont Fine point tweezers</td><td>Roboz</td><td>RS5045</td><td>alternative suppliers available</td></tr><tr><td>Pyrex Petri dish, 100 mm diameter</td><td>Corning</td><td>3160</td><td>can substitute other glass Petri plates</td></tr><tr><td>Sterile 6-well plate</td><td>Corning Falcon&amp;nbsp;</td><td>353046</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 12-well plate</td><td>Nunc&amp;nbsp;</td><td>150628</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 24-well plate</td><td>Cellstar, Greiner bio-one</td><td>662-160</td><td>or similar from other manufacturer</td></tr><tr><td>Brine shrimp hathery kit</td><td>San Francisco Bay; drsfostersmith.com</td><td>CD-154005</td><td>option for growing brine shrimp</td></tr><tr><td>pyrex baking dish</td><td>common in grocery stores</td><td></td><td>option for growing brine shrimp</td></tr><tr><td>artificial seawater mix 50 gal or more&amp;nbsp;</td><td>Instant Ocean; drsfoster-smith.com</td><td>CD-116528</td><td>others brands may suffice</td></tr><tr><td>Plastic tub for stock ASW preparation</td><td>various</td><td></td><td>common 25 gallon plastic trash can OK</td></tr><tr><td>Polypropylene Carboy</td><td>Carolina Biological Supply</td><td>716391</td><td>For working stock of ASW @ 12 ppt</td></tr><tr><td>Beaker, Graduated, 4,000 mL</td><td>PhytoTechnology Laboratories</td><td>B199</td><td>For dilution of 36 ppt ASW to 12 ppt</td></tr><tr><td>Stereomicroscope and light source</td><td>various&amp;nbsp;</td><td></td><td>with continuous 1 - 40X magnification</td></tr></tbody></table>

inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis

Cnidarios, y en concreto Hydra, fueron los primeros animales indicado para regenerar estructuras dañadas o cortadas, y de hecho podría decirse que este tipo de estudios en marcha la investigación biológica moderna a través de la obra de Trembley hace más de 250 años. En la actualidad el estudio de la regeneración ha visto un resurgimiento usando ambos organismos regenerativas &quot;clásicos&quot;, como la hidra, planaria y Urodeles, así como un espectro ensanchamiento de las especies que abarcan la gama de metazoos, a través de las esponjas mamíferos. Además de su interés intrínseco como un fenómeno biológico, la comprensión de cómo funciona la regeneración en una variedad de especies nos informará acerca de si los procesos regenerativos comparten características y / o especies comunes o mecanismos celulares y moleculares específicas del contexto. La anémona de mar estrella, Nematostella vectensis, es un organismo modelo emergente para la regeneración. Al igual que Hydra, Nematostella es un miembro de la antigua phylum, cnidarios, pero dentro de tque anthozoa clase, un clado hermano a la hidrozoos que es evolutivamente más basal. Así, los aspectos de la regeneración en Nematostella será interesante comparar y contrastar con los de Hydra y otros cnidarios. En este artículo, presentamos un método para dividir en dos, observar y clasificar a la regeneración del extremo aboral del adulto Nematostella, que se llama el Physa. El Physa sufre naturalmente fisión como medio de reproducción asexual, y, o bien la fisión natural o amputación manual de la Physa desencadena re-crecimiento y la reforma de morfologías complejas. Aquí hemos codificado estos cambios morfológicos simples en un Sistema de estadificación Nematostella Regeneración (la NRSS). Usamos la NRSS para poner a prueba los efectos de la cloroquina, un inhibidor de la función lisosomal que bloquea la autofagia. Los resultados muestran que la regeneración de estructuras de pólipos, en particular los mesenterios, es anormal cuando se inhibe la autofagia.

La progresión de los acontecimientos morfológicos durante la regeneración en Physa cortada se muestra en la Figura 1A, que incluye vistas representativas de Physa en cada etapa NRSS. El sitio típico Physa corte se indica en el adulto (puntas de flecha). Las fotografías muestran en la Figura 1A regeneración progresiva de las estructuras orales y corporales de Physa recién cortada a través de pólipos completamente formado. Figura 1B, C</st...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Inducir completa Pólipo regeneración de la aboral Physa de la anémona de mar Starlet<em&gt; Vectensis Nematostella</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

inducing-complete-polyp-regeneration-from-aboral-physa-starlet-sea

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

8.5K vistas.  Stony Brook University. Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Video: Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Química la amputación y la regeneración de la faringe en el Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

Investigación

Biología del Desarrollo

Methods for the Study of Regeneration in Stentor

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

Methods for the Study of Regeneration in Stentor

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

Métodos para el estudio de la regeneración en Stentor

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

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

Genotipado de anémona sin mar durante el desarrollo temprano

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Identificación de la actividad hemolítica y fosfolipasa en extractos crudos de anémonas de mar mediante bioensayos sencillos

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Bioquímica

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biología

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generación y Mantenimiento a Largo Plazo de Nervios<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Neurociencias

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

La inmovilización de las planarias, irradiación parcial, y el trasplante de tejido

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Un método para la microinyección de<em&gt; Patiria minata</em&gt; Los cigotos

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Inducción del rescate de pólipos en colonias de coral para obtener micropropagados individualizados para uso experimental de laboratorio

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

Inducir completa Pólipo regeneración de la aboral Physa de la anémona de mar Starlet

Resumen

Explorar más videos

Capítulos en este video

Química la amputación y la regeneración de la faringe en el Planarian Schmidtea mediterranea

Métodos para el estudio de la regeneración en Stentor

Genotipado de anémona sin mar durante el desarrollo temprano

Identificación de la actividad hemolítica y fosfolipasa en extractos crudos de anémonas de mar mediante bioensayos sencillos

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia

Generación y Mantenimiento a Largo Plazo de Nervios

La inmovilización de las planarias, irradiación parcial, y el trasplante de tejido

Un método para la microinyección de

Inducción del rescate de pólipos en colonias de coral para obtener micropropagados individualizados para uso experimental de laboratorio

Caracterización de la cicatrización de heridas epiteliales in vivo utilizando el organismo modelo cnidario Clytia hemisphaerica