Nos gustaría agradecer a Alejandro Sánchez Alvarado, quien apoyó la optimización inicial y el desarrollo de esta técnica. Trabajo en laboratorio de Carolyn Adler está financiado por fondos de puesta en marcha de la Universidad de Cornell.

1. preparación
<ol><li>
Preparación de agua de la planaria 17<ol>
<li>Mantener planarias en una solución de sal de X Montjuïc 1. Para preparar el planarian agua, hacer soluciones stock individuales de 1 M CaCl2, 1 M MgSO4, 1 M de MgCl2, 1 M KCl y 5 M de NaCl en agua ultrapura. Filtro-esterilizar con un almacenamiento a largo plazo de la filterfor superior de la botella de 0.2 μm. Nota: Utilizar sólo agua desionizado ultrapura (con una resistividad de 18,2 MΩ a 25 ° C) para preparar Montjuïc sales.</li>
		<li>Para preparar un stock de 1 L de solución salina de 5 X, mezclar 5 mL de 1 M CaCl2, 5 mL de 1 M MgSO4, 0,5 mL de MgCl2, 0.5 mL de KCl y 1,6 mL de soluciones de NaCl de 5 M en 900 mL de agua ultrapura. A esta solución, Añadir 0,504 g de NaHCO3 y revuelva para mezclar. Ajustar el pH a 7.0 con ácido clorhídrico.</li>
		<li>Diluir este 5 X solución madre a un 1 X concentración de trabajo en agua ultrapura en un recipiente estéril como una Damajuana de gran capacidad (véase la Tabla de materiales). Utilice esta solución de X 1 para el mantenimiento de planarias asexuales. Nota: Como alternativa a la solución de sal de Montjuïc, use agua de manantial local comprado o agua ultrapura que contiene una sal de acuario disponible comercialmente (véase Tabla de materiales) de la mezcla en una concentración of0.5 g/L18.</li>
		<li>Para evitar la infección bacteriana en cultura estática19, mantener planarias en agua que contenga un antibiótico. Prepare un 50 mg/mL solución de sulfato de gentamicina en agua ultrapura y esterilizar por el filtro. Contenedores donde se mantienen animales, añadir sulfato de gentamicina a una concentración final de 50 μg/mL (dilución 1: 1000).</li>
	</ol>
</li>
	<li>
Preparación de pasta de hígado
	<ol>
<li>Como planarias prosperan en una dieta orgánica, alimentada con pasto de carne de vaca hígado, hígado fresco de compra y proceso dentro de 24 horas retire la cápsula membranosa encapsulando el hígado desprendiendo suavemente. Cortar el hígado en cubos de ~ 1 cm y use una hoja para raspar y eliminar todas las arterias y las venas hepáticas.</li>
		<li>Macerar el hígado piezas usando un pasapurés o una batidora y luego pasarlo por un colador de alambre malla de alimentos. Se combinan en bolsas de pasta y dispensan en jeringas o platos de Petri de 35 mm. Antes de la alimentación, centrifugar suavemente para eliminar las burbujas de aire.</li>
		<li>Guardar alícuotas de hígado a-80 ° C por hasta un año y descongelar antes de usar. Después de descongelar, volver a congelar cualquier hígado sobrante una vez, o almacenar a 4 ° C por 24 h.</li>
	</ol>
</li>
	<li>
Mantenimiento de los animales
	<ol>
<li>Planarias se crecen y decrecen en varios tamaños dependiendo de la frecuencia de la alimentación. Alimentación de planarias cada dos semanas (una vez cada 14 días). Para cultivos a largo plazo de a granel, use recipientes de plástico de varios tamaños (véase Tabla de materiales).</li>
		<li>Para alimentar animales, utilice una espátula de metal o pipeta de transferencia plástica para colocar una gota del tamaño de un guisante de hígado en una caja. Permiten animales comer de 1-2 h. retirar la comida que quedaba antes de limpiar la caja.</li>
		<li>Limpiar gusanos dos veces a la semana si (una vez directamente después de la alimentación y una vez dos días más tarde), o una vez por semana si no es alimentado. Para limpiar, drenar el agua en un vaso de plástico por verter con cuidado el agua conservando planarias en el cuadro. Limpie la superficie de la caja con una toalla de papel, luego repetir por todos lados hasta que la caja esté limpia.</li>
		<li>Agua (aproximadamente 3/4 del volumen de caja) y se añade gentamicina a una concentración final de 50 μg/mL. Tienda animales en un gabinete oscuro o en una incubadora de 20 ° C.</li>
	</ol>
</li>
	<li>
Selección de gusanos
	<ol>
<li>Seleccione gusanos que se alimentaron con 5-7 días antes. Nota: Es significativamente más difícil que amputarle musculo de gusanos que han sido alimentados recientemente (1-2 días antes de la amputación). Animales alimentados también son más sensibles a la azida sódica.</li>
		<li>Seleccione gusanos que miden aproximadamente 6 mm de longitud. Para estimar el tamaño del gusano, transfiéralo a un plato de Petri utilizando una pipeta de transferencia plástica. Coloque una regla plana 6 - en o papel cuadriculado de 5 mm x 5 mm por debajo de la caja Petri y medir la longitud de gusano mientras arrastra. Nota: Gusanos más pequeños de 6 mm de longitud son más difíciles de amputar.</li>
	</ol>
</li>
	<li>
Preparación de la azida de sodio
	<ol>
<li>Preparar una solución de 100 mM disolviendo polvo de azida de sodio en agua planaria. PRECAUCIÓN: Azida sódica es tóxica y debe manipularse con cuidado. No se deshaga de azida de sodio por el desagüe. Después de su utilización, recoger por separado para su eliminación como residuos peligrosos.</li>
	</ol>
</li>
</ol>2. faringe amputación
<ol><li>Lugar los gusanos en un diámetro de 35 mm placa de Petri. Retire con cuidado toda el agua planarian del plato utilizando una pipeta de transferencia plástica. Nota: Un máximo de 20 gusanos de 6 mm puede ser alojado cómodamente en un plato de este tamaño.</li>
	<li>Coloque el plato bajo el microscopio y ajustar magnificación para que varios gusanos pueden verse simultáneamente (por ejemplo 10 aumentos). Reemplace planarian agua con solución de azida de sodio 100 mM. Nota: Para un plato de Petri de 35 mm, aproximadamente 5 mL de azida de sodio es suficiente. La solución debe sumergir completamente planarias. Ajustar el volumen de solución para los platos más grandes según sea necesario.</li>
	<li>Monitorear los animales bajo el microscopio y abstenerse de mover el plato para los primeros 3-4 minutos.</li>
	<li>Observar la extrusión de la faringe a través del microscopio; la faringe se sobresale de la bolsa faríngea y extienda completamente (aproximadamente 1 mm de longitud) como se muestra en la figura 1B. Nota: Es importante esperar hasta que haya terminado la faringe que se extiende completamente antes de continuar. Una vez que el animal se desprende de la faringe, extensión total toma aproximadamente 1 minuto.</li>
	<li>Utilice una pipeta de transferencia plástica para squirt animales alrededor del plato. Aspirar los animales en la pipeta y liberarlos por la fuerza en el plato un par de veces. Si la faringe está completamente extendida, este vigoroso uso hará que se suelte.</li>
	<li>Como alternativa, tome la faringe verticalmente a lo largo de su longitud, u horizontalmente a lo largo de su circunferencia usando un par de Pinzas finas (véase Tabla de materiales) como se muestra en la figura 1. La faringe atrapada en las pinzas, levantar el animal hacia arriba hacia el menisco. Mientras que el animal se levanta, tensión superficial hará que la faringe para desprenderse del animal. Nota: Este método puede utilizarse para eliminar el musculo de animales más pequeños (1-3 mm de longitud) donde se arremolinan contundente no es el adecuado. Evitar meter o herida de lo contrario el cuerpo del animal.</li>
	<li>Utilizando una pipeta de transferencia, hacia animales amputados un nuevo plato que contenga agua fresca de la planaria.</li>
	<li>Una vez transferidos, enjuague animales amputados completamente en el agua de la planaria, cambio totalmente el agua tres veces. Traslado a un nuevo plato que contiene agua de planaria con 50 μg/mL de gentamicina. Nota: Cambio de buffer completo durante estos tres lavados es esencial para asegurar el retiro de azida de sodio.</li>
	<li>Al día siguiente, reemplace el agua en el plato con agua fresca planaria 50 μg/ml de gentamicina. Repita todos los días después de eso.</li>
</ol>3. evaluación de la eliminación de la faringe después de la amputación
<ol><li>Examinar los animales bajo el microscopio (con aumentos de 10-20) para determinar si una mancha oscura ha aparecido después de la eliminación exitosa de la faringe, como se muestra en la figura 2A.</li>
	<li>Por otra parte, fijar y bleach animales según el protocolo descrito por Pearson et al. 20. animales de remojo durante la noche en 4', 6-diamidino-2-phenylindole (DAPI) o fluorescente conjugado estreptavidina (1 mg/mL diluido 1: 500 en el 1 X de tampón de fosfato salino que contiene 0.3% Triton-X). Montar animales de diapositivas glycerol/20% 80% 1 X solución salina tamponada de fosfato y la imagen bajo un estereomicroscopio fluorescente (figura 2B). Nota: Todas las imágenes de este manuscrito fueron capturadas en un estereomicroscopio fluorescente con un objetivo X 1,0.</li>
</ol>4. evaluación de la regeneración de la faringe mediante la medición de comportamiento alimentario
<ol><li>
Preparación de hígado
	<ol>
<li>Transferir la cantidad necesaria de pasta de hígado en un tubo cónico o microcentrífuga. Girar brevemente para eliminar burbujas de aire. Estimar el volumen del hígado y luego añadir planaria de agua a 1/5 de su volumen y 2% del volumen total de colorante de alimento rojo. Por ejemplo, a 1000 μl de hígado pegar, añadir 200 μL de agua de la planaria y 24 μl de colorante de alimento.</li>
		<li>Con un mortero plástico, pipeta o espátula de metal, mezcle bien hasta que el colorante se distribuya uniformemente la pasta de hígado. Vuelta otra vez brevemente. Pasta de hígado puede ser almacenado a 4 ° C durante 24 h o congelada en alícuotas a-80 ° C para almacenamiento a largo plazo.</li>
		<li>Si la prueba hasta 25 animales, preparar 25 μl de pasta de hígado por plato (aproximadamente 1 μl de pasta de hígado por animal).</li>
	</ol>
</li>
	<li>
Alimentación de animales
	<ol>
<li>Siete días después de la amputación química, transferencia de animales a una nueva placa de Petri. Si pruebas el 10-15 animales, utilizar un plato de 35 mm; más de 15 animales deben analizarse en un plato de Petri de 60 mm. Mantener los animales en la oscuridad, sin perturbaciones, de aproximadamente 1 h antes de la alimentación.</li>
		<li>Usando las tijeras, aumentar el ancho de una punta de pipeta P200 de ajuste aproximadamente 1/2 cm en el extremo estrecho. Pipetear 25 μl de rojo pasta de hígado en el plato. Nota: Recorte el extremo hace uso la pasta viscosa de hígado más fácil. Para evitar que el hígado flotando en la superficie, toque la punta en la parte inferior del plato mientras.</li>
		<li>Permiten animales alimentar durante 30 minutos.</li>
		<li>Anotar el número de animales que han comido por los animales la colocación sobre un fondo blanco o examinarlos bajo un microscopio con aumentos de 10-20.</li>
		<li>Después de alimentar, retirar el plato de hígado y limpiar.</li>
	</ol>
</li>
</ol>

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Los autores no tienen nada que revelar.

Este protocolo describe un método de ablación selectiva de la faringe con azida de sodio. Otros estudios de ablación dirigida en planarias han utilizado cirugía modificada para eliminar fotorreceptores21 o tratamiento farmacológico a la ablación de las neuronas dopaminérgicas22. Una ventaja importante de amputación química sobre los métodos existentes es que no requiere cirugía. La estructura rígida de la faringe en comparación con el resto del cuerpo planarian...

La regeneración es un fenómeno que ocurre en todo el reino animal, con capacidad regenerativa de la regeneración de todo el cuerpo de ciertos invertebrados a más facultades en vertebrados1. Sustitución de tejido funcional es un proceso complejo y a menudo implica la restauración simultánea de múltiples tipos de células. Por ejemplo regenerar el miembro de Salamandra, osteoblastos, condrocitos, neuronas, músculos y células epiteliales deben ser reemplazado2. Estos tipos de celda recién generado también tiene que organizarse adecuadamente para facilitar la nueva función del miembro. Comprensión de estos procesos complejos requiere de técnicas que se centran en la regeneración de tipos celulares específicos y su integración en órganos.
Una de las estrategias empleadas para simplificar el estudio de la respuesta regenerativa es la ablación dirigida de ciertos tipos celulares o colecciones más grandes de los tejidos. Por ejemplo, en el pez cebra, expresión de nitroreductase en tipos celulares específicos conduce a su destrucción después de la aplicación de metronidazol3,4. En larvas de Drosophila , expresando genes pro-apoptosis en promotores específicos de tejido selectivamente puede ablar regiones específicas del disco imaginal5,6. Dos de estas estrategias dañar rápido pero controlado y han sido utilizados para diseccionar los mecanismos moleculares y celulares para la regeneración.
En este manuscrito, se describe un método para extirpar selectivamente un órgano entero llamado la faringe en el planarian Schmidtea mediterranea. Planarias son un modelo clásico de la regeneración, conocido por su prolífica capacidad regenerativa, donde fragmentos incluso minutos pueden volver a crecer todo animales de 7,8. Tienen una población grande y heterogénea de las células madre de células pluripotentes y progenitores de linaje restringido9,10,11. Estas células proliferan y diferencian para reemplazar todos los tejidos faltantes, incluyendo la faringe, sistemas nerviosos, digestivos y excretorios y músculo y las células epiteliales9,10,12. Si bien sabemos que estas células madre iniciar la regeneración, no comprendemos plenamente los mecanismos moleculares que conducen a sustituir todos estos diferentes tipos de células. Definidos métodos heridos que provocan respuestas precisas células madre pueden ayudar a delinear este complejo proceso.
La faringe es un tubo cilíndrico, grande necesario para la alimentación y contiene neuronas, músculo, células epiteliales y secreción13,29. Normalmente oculto en una bolsa en la parte ventral del animal, se extiende por el cuerpo del animal una apertura sobre la detección de la presencia de alimentos. Para amputar selectivamente la faringe, remojan planarias en un químico llamado sódica, un anestésico utilizado en C. elegans14,15,16. Su uso en planarias primero fue divulgado por Adler et al., en el 201412. A minutos de exposición a la azida sódica, planarias sacan su musculo y con suave agitación, se separa la faringe del animal. Nos referimos a esta pérdida selectiva y completa de la faringe como "amputación química". Una semana después de la amputación, una faringe completamente funcional es restaurado12. Ya que la faringe se requiere para la alimentación, regeneración funcional puede medirse mediante el control de comportamiento de alimentación. A continuación, se describe el protocolo para amputación química y para evaluar la regeneración de la faringe y la restauración del comportamiento alimentario.

<table><tbody><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>C79-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Sulfate Anhydrous (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>M65-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Chloride Hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Acros/VWR</td><td>41341-5000</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Acros Organics/VWR</td><td>196770010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Acros Organics/VWR</td><td>207790050</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Acros Organics/VWR</td><td>123360010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Nalgene autoclavable polypropylene copolymer lowboy with spigot</td><td>ThermoFisher Scientific/VWR</td><td>2324-0015</td><td>Storing planaria water</td></tr><tr><td>Instant Ocean Sea Salt</td><td>Spectrum Brands</td><td>Amazon</td><td>Planaria water</td></tr><tr><td>Gentamicin Sulfate</td><td>Gemini Bio-products</td><td>400-100P</td><td>Planaria water</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>71970</td><td>Mincing liver&amp;nbsp;</td></tr><tr><td>Disposable pastry bags-16&amp;rdquo;, 12 pack</td><td>Wilton</td><td>Amazon</td><td>Liver aliquots</td></tr><tr><td>5 mL syringes</td><td>BD/VWR</td><td>309647</td><td>Liver aliquots</td></tr><tr><td>Petri dishes-35mm/60mm</td><td>Greiner Bio-One/VWR</td><td>82050-536/82050-544</td><td></td></tr><tr><td>Plastic containers (various sizes)</td><td>Ziploc</td><td>Amazon</td><td>Housing planarians in static culture</td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Transfer pipette</td><td>Globe Scientific</td><td>138030</td><td></td></tr><tr><td>Forceps - Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>72700-D (0203-5-PO)</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>ThermoFisher</td><td>62247</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>ThermoFisher</td><td>S11223</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP229-1</td><td></td></tr></tbody></table>

chemical amputation and regeneration of the pharynx in the planarian schmidtea mediterranea

Planarias son gusanos planos que son muy eficientes en la regeneración. Deben esta capacidad a un gran número de células madre que pueden responder rápidamente a cualquier tipo de lesión. Modelos de lesiones comunes en estos animales eliminar grandes cantidades de tejido, lo cual daña múltiples órganos. Para superar el daño de tejido amplia, se describe aquí un método para eliminar selectivamente un único órgano, la faringe, en el planarian Schmidtea mediterranea. Logramos esto por los animales remojo en una solución que contiene la azida de sodio inhibidor de la citocromo oxidasa. Breve exposición a azida de sodio provoca la protuberancia de la faringe del animal, que llamamos "química amputación." Química amputación elimina la faringe entera y genera una pequeña herida en la faringe se fija al intestino. Después de lavado amplia, todos los animales amputados regeneran una faringe completamente funcional en aproximadamente una semana. En el resto de la regeneración de la unidad de cuerpo de la faringe nuevas células. Presentamos un detallado protocolo de amputación química y describir los métodos histológicos y de comportamiento para evaluar éxito amputación y regeneración.

Exposición a la azida de sodio altera la motilidad normal de planarias, causando animales y writhe. Estos movimientos la fuerza la faringe de la parte ventral del animal, y después de aproximadamente 6 minutos en solución de azida de sodio, puede verse la punta blanca de la faringe (figura 1B-panel de la izquierda). Unos minutos más tarde, animales activamente contraen y extienden totalmente la faringe empujando con fuerza fuera del cuerp...

Watch this Scientific Journal Video about Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea at JoVE.com

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

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

El planarian Schmidtea mediterranea es un excelente modelo para estudiar las células madre y regeneración de los tejidos. Esta publicación describe un método para eliminar selectivamente un órgano, la faringe, por la observación de animales de la azida de sodio químicos. Este protocolo también describe métodos para el monitoreo de la regeneración de la faringe.

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 ...

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

Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

10.7K vistas.  Cornell University. El planarian Schmidtea mediterranea es un excelente modelo para estudiar las células madre y regeneración de los tejidos. Esta publicación describe un método para eliminar selectivamente un órgano, la faringe, por la observación de animales de la azida de sodio químicos. Este protocolo también describe métodos para el monitoreo de la regeneración de la faringe.

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

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

Analizando poblaciones de células sanguíneas de la<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Investigación

Biología del Desarrollo

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

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

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 ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

La ablación quirúrgica de ensayo para estudio de la regeneración de los ojos en planarias

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

Un ensayo de motilidad planarian para medir las propiedades biomodulantes de los productos naturales

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Bioquímica

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Amputación y regeneración de dígitos de ratón adultos: un modelo simple para investigar la formación de Blastema de mamíferos y la osificación intramembranosa

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

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

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 ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Exploración planarian como una lectura cuantitativa del comportamiento para la detección de estímulos nocivos

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. 
This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

<meta charset="utf8"></head><body>Disección de ovario planario para análisis ultraestructural y tinción de anticuerpos

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

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

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