Nos gustaría agradecer a centro Caenorhabditis genética de las cepas, las ciencias naturales y Consejo de investigación Ingeniería (NSERC) y la Fundación de Canadá para la innovación (CFI) para la financiación. Nos gustaría agradecer a Dr. Lizhen Chen (Departamento de sistemas celular y anatomía, UT salud San Antonio) para permitir el uso irrestricto de sus instalaciones de laboratorio para todos los experimentos de C. elegans , así como el Dr. Exing Wang (Director asociado, centro de la proyección de imagen óptica UT salud San Antonio) para obtener ayuda con microscopía confocal. También nos gustaría agradecer a Dr. Myron Ignatius para proporcionar apoyo y aliento para facilitar la sesión de video.

1. Mancha y lisosomas intestinales de la imagen
<ol><li>Medios de cultivo nematodos de semilla (NGM) placas con OP50
	<ol>
<li>Preparar platos para NGM según el protocolo recomendado19 y permita que las placas cerradas secar por 2 días a temperatura ambiente.</li>
		<li>Inocular las bacterias OP50 en caldo estéril caldo de Luria (LB) y crecen en un baño incubador o agitación a 37 ° C durante 36 h o hasta que el OD es entre 0.2 y 0.4. Evitar el uso de un cultivo bacteriano con OD > 1 o OD < 0,2.</li>
		<li>Una vez seco planchas NGM, coloque una gota (30 μL) del inóculo OP50 en el centro de la placa y luego la gota en un parche más o menos que es de 2 cm x 2 cm de tamaño. Nota: Cualquier inóculo OP50 restantes puede almacenarse a 4 ° C por hasta un mes.</li>
		<li>Invertir las placas OP50 una vez que el inóculo ha secado (aproximadamente 10 o 15 min) y luego incubar las placas a 37 ° C durante 36 horas.</li>
		<li>Después de 36 h, quitar las placas de la incubadora y almacenar a 4 ° C hasta su uso posterior. Nota: Las placas deben ser buenas para un máximo de 1 mes a 4 ° C.</li>
	</ol></li>
	<li>CDCFDA que suple a OP50
	<ol>
<li>Para complementar cDCFDA sobre placas OP50, preparar una acción de trabajo de cDCFDA de 10 mM en dimetil sulfóxido (DMSO). Mantener la solución de cDCFDA protegida de la luz y almacenar a-20 ° C para su uso posterior.</li>
		<li>Suavemente coloque 100 μl de solución de cDCFDA de 10 mM sobre la superficie del parche OP50 2 cm x 2 cm, tal que la totalidad del parche está uniformemente cubierto con cDCFDA. Nota: Es muy importante que la totalidad del parche de OP50 está cubierto con cDCFDA. Si es necesario, utilizar hasta 150 μL de cDCFDA para cada placa. También es imprescindible que la solución de cDCFDA no se extiende más allá de las fronteras del parche OP50, puesto que cualquier cDCFDA que sale el parche OP50 no será incorporado a los alimentos y se traducirá en menor intensidad de la tinción.</li>
		<li>Coloque las placas OP50 en una caja oscura en la mesa hasta que todos los de la solución cDCFDA es absorbido en el parche bacteriano (generalmente alrededor de 25 a 30 minutos).</li>
	</ol></li>
	<li>Tinción de gusanos usando cDCFDA
	<ol>
<li>Una vez que el cDCFDA ha permeado totalmente el parche OP50, colocar no más de 20 gusanos por placa e incubar las placas invertidas a 20 ° C durante un mínimo de 14 h. Nota: Se recomienda colocar las placas de cDCFDA en una caja opaca para minimizar la exposición a la luz.</li>
		<li>Controlar por las diferencias de consumo entre experimentos y normalizar las señales cDCFDA, (recomiendan) Mancha Co con () 3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato) Difluoro) boro (2,5 μl de una solución recién hecho de 1 mM), un colorante de acidotropic amina débil cuya fluorescencia es en gran parte no variante sobre el espectro de pH ácido (véase Tabla de materiales para los nombres comunes de reactivos). Nota: No mancha gusanos de menos de 14 h, ya que esto puede proporcionar la intensidad de la tinción inadecuada cDCFDA y dar una falsa interpretación del pH lisosomal.</li>
	</ol></li>
	<li>Preparación de portaobjetos para microscopía
	<ol>
<li>Coloque dos tiras paralelas de etiquetado cinta unas 3 pulgadas aparte en una superficie plana lisa, como un espejo (figura 1).</li>
		<li>Capa de la superficie del espejo con spray repelente al agua y limpie el exceso de líquido.</li>
		<li>Derretir la agarosa al 2% (disuelto en agua destilada H2O) en el microondas hasta que totalmente licuado, luego coloque una gota 50 de μl de agarosa entre las dos tiras de cinta adhesiva y cubrir rápidamente la gota con un portaobjetos de microscopio, que es perpendicular a las tiras de la diapositiva cinta. Después de la agarosa ha solidificado, formando una almohadilla circular debajo de la diapositiva, voltee la diapositiva y agregar 10-15 μl de azida de sodio 5mM (NaN3) en la almohadilla de agarosa. Nota: NaN3 es un inhibidor del citocromo C que cuando se utiliza en bajas concentraciones, anestesia reversible gusanos para que no se mueva durante la proyección de imagen.</li>
		<li>Recoger gusanos de la placa OP50 complementado con cDCFDA y transferirlas a una placa limpia de NGM sin cualquier OP50. Que los gusanos a mover durante unos segundos permitir que la mayoría de los OP50 a eliminarse de la superficie de los gusanos y luego se transfieren los gusanos a la almohadilla de agarosa que contiene azida de sodio 5 mM.</li>
		<li>Cubra inmediatamente la almohadilla de agarosa con un cubreobjetos. Asegúrese de colocar suavemente el cubreobjetos sin aplicar demasiada presión, ya que esto puede resultar en explosión de gusanos. Nota: Bacterias OP50 complementadas con cDCFDA fluorescencia cuando es visualizado por microscopía, por lo tanto es importante limpiar los gusanos lo mejor posible antes de colocar sobre la almohadilla de agarosa que contiene azida de sodio. Si preparando más de 6 cepas de C. elegans (incluyendo N2, control de tipo salvaje), es recomendable preparar las muestras en lotes de 6, con el fin de asegurar que los gusanos no seque sobre la almohadilla de agarosa. En algunas cepas, la vulva comienza a romperse después de largos períodos de tiempo debido a la presión de la hoja de cubierta, por lo que es importante planificar en consecuencia.</li>
	</ol></li>
	<li>Microscopía confocal Nota: Algunos microscopios tienen una función incorporada que automáticamente aumenta la intensidad de fluorescencia para compensar niveles variables de fluorescencia. Estos microscopios pueden no funcionar bien para gusanos teñidas con cDCFDA, puesto que intrínsecamente aumentan la intensidad de fluorescencia de las muestras de la proyección de imagen.<ol>
<li>Realizar la proyección de imagen en un microscopio confocal estándar con longitudes de onda de excitación/emisión a 488/530 nm para cDCFDA. Solo plano imágenes (no Z-stacks) de lisosomas intestinales y utilizar sólo los lisosomas que se encuentran en foco (intensidad máxima) para la cuantificación de la intensidad. Nota: Tal vez debido a diferencias en la disponibilidad de tinte, lisosomas de las células intestinales directamente posteriores a la faringe mantienen cDCFDA tinción intensidad independientemente del genotipo o edad, por lo tanto debe tenerse cuidado para evitar imágenes de lisosomas de estas células.</li>
		<li>Imagen de la diapositiva que contiene 2 viejo tipo wild que N2 gusanos primero. Al hacerlo, ajustar los parámetros de proyección de imagen confocales como energía del laser, agujero de alfiler y apertura para garantizar que la cDCFDA intensidad de tinción no es sobresaturado. Nota: Una vez que estos ajustes se establecen, no cambiar para toda la duración de la proyección de imagen para ese día.</li>
		<li>Capturar imágenes de 1024 x 1024 píxeles de un solo plano del intestino (3 o 4 imágenes por gusano) usando un 60 X lente de aumento. Nota: El espectro de emisión de cDCFDA en gusanos producirá un solo pico prominente en 520 nm coincidiendo con su divulgado fluorescencia espectro20, proporcionando una lectura de la señal específica que es distinta de Autofluorescencia intestinal (figura 3).</li>
		<li>Exportar imagen confocal (ficheros .lsm) como archivos .tiff para cada canal.</li>
		<li>A continuación, abra ImageJ y haga clic en archivo | Abrir abrir el archivo de imagen para ser cuantificada. Una vez que se carga la imagen, utilice la herramienta de selección de forma ovalada en ImageJ para seleccionar una región de interés (ROI).</li>
		<li>Después de eso, haga clic en analizar | Medida para cuantificar la intensidad de la fluorescencia para ese retorno de la inversión. Seleccione 4 – 5 diferentes foco lisosomas por imagen y calcular la intensidad de fluorescencia relativa promedio de cada región de interés (ROI).</li>
		<li>Para cada imagen, medir la intensidad de la fluorescencia de fondo en una región del intestino entre lisosomas. Restar los valores de intensidad de fluorescencia de fondo de los valores de intensidad de fluorescencia lysosomal.</li>
		<li>En total, recogen alrededor de 30 a 50 valores normalizados individuales de intensidad cDCFDA (animales de 6 a 10) para cada cepa ensayada. Use estos valores para trazar un diagrama de caja y bigotes utilizando cualquier software estadístico apropiado. Nota: La coloración puede variar considerablemente dependiendo de factores como temperatura, tiempo de incubación y la salud general, edad de los animales que sólo son relevantes en proceso de muestras en el mismo experimento tinción comparaciones de intensidad de fluorescencia. Por lo tanto, es importante procesar controles en cada experimento de tinción. Calcular diferencias estadísticas (t-pruebas) utilizando cualquier software estadístico apropiado.</li>
		<li>Imagen todos las cepas del mismo experimento (teñido en el mismo día) en secuencia, teniendo cuidado de no para cambiar a cualquiera de los parámetros de la proyección de imagen.</li>
	</ol></li>
</ol>

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Los autores no declaran conflicto de intereses.

Una variedad de eventos celulares y moleculares que contribuyen al envejecimiento, influenciado por características de historia de vida y factores genéticos. Nuestro reciente estudio22 sugiere que el ciclo reproductivo juega un papel importante en el control de la aptitud del soma a través de la regulación de la dinámica de pH lisosomal. Hemos mostrado que proteolisis lisosomal mediada es promovido mientras que animales se reproducen activamente upregulation de la transcripción de la v-ATPas...

La aparición de agregados proteicos es ampliamente aceptada que un sello de envejecimiento en las células eucariotas1,2,3y la formación de los cuales se cree que entre los conductores de principio de senescencia celular4 , 5 , 6 , 7. Existe creciente evidencia que como células edad, catabolismo proteico se deteriora, provocando un aumento en la agregación de la proteína. El colapso de la proteólisis en el envejecimiento de las células conlleva una debilitación de la autofagia8 , así como de la degradación de proteínas mediada por proteasoma9. Finalmente, oxidación irreversible de la proteína aumenta en las células viejas, deteriorando aún más proteína catabolismo10.
Autofagia fue pensada inicialmente para ser un proceso no selectivo para la degradación de la mayor parte de proteínas dañadas, pero estudios recientes han indicado que la autofagia es altamente selectiva para el catabolismo de los agregados de proteínas y organelos disfuncionales que no son susceptibles a la degradación a través de otras proteínas separación mecanismos11. Durante el proceso de autofagia, proteínas dañadas y agregadas son secuestradas en una vesícula de membrana doble llamada el autophagosome. Este autophagosome entonces se funde con los ácidos organelos llamados lisosomas, que conduce a la degradación de la carga de autophagosome12. Lisosomas representan el punto final de la vía de la autofagia y participan en diversos procesos celulares tales como reparación de membrana, control transcripcional y detección de nutrientes; destacando su papel centralizado en la homeostasis celular (revisada en Ref. 13). Varios estudios han demostrado una asociación entre una disminución dependiente de la edad en la función lisosomal y de trastornos neurodegenerativos diversos13. Constantemente, restaurar la función lysosomal en células mayores puede retrasar la aparición de fenotipos relacionados con el envejecimiento14,15. Estudios de la composición del medio intralumen sugieren que el colapso de la función lysosomal en células mayores no es debido a una reducción en la producción de proteasas lisosomales16. Por otra parte, se ha propuesto que la pérdida de acidez produce, un requisito fundamental de su actividad enzimática, puede ser la base la disminución de la proteólisis mediada por el lisosoma17. Para poder explorar esta hipótesis, es esencial desarrollar los reactivos y protocolos para cambios dinámicos en pH lisosomal en células vivas de una manera coherente y replicable.
El intestino de C. elegans es el tejido metabólico importante en gusanos y es un regulador crítico de la homeostasis sistémica y la vida útil. Desarrollamos análisis para evaluar los cambios en la acidez del lumen intestinales lisosomas de gusanos para determinar cómo lisosoma-mediada de la proteólisis contribuye al envejecimiento. Aunque fluoróforos sensibles al pH han sido utilizados previamente en C. elegans para marcar lisosomas intestinales, sin embargo no ha habido un esfuerzo por establecer un protocolo exitoso que puede detectar pequeños incrementos en el pH lisosomal en vivo18. Presentamos un protocolo que puede utilizarse para detectar pérdida de acidez lisosomal de las células intestinales de C. elegans mediante un protocolo de alimentación sencillo y cómodo que incorpora un fluoróforo sensible al pH (cDCFDA) OP50 alimentos.

<table border="0" cellpadding="0" cellspacing="0" width="1166">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">OP50 (E. coli)</td>
			<td style="width:188px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td style="width:385px;">Order online at https://cgc.umn.edu/strain/OP50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">5(6)-carboxy-2&#8217;,7&#8217;-dichlorofluorescein diacetate&#160;</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">C369</td>
			<td>Commonly known as cDCFDA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:475px;">9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">L7528</td>
			<td>Commonly known as Lysotracker Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Confocal microscope (e.g. Zeiss LSM 510)</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">ImageJ</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td>Download for free from https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">LB Broth powder</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">22700041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Agar</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">NaCl</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Peptone</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:119px;">S71604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cholesterol powder</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">C3045&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">CaCl2</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">449709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">MgSO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">K3PO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">P5629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sodium Azide</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DMSO</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:119px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cover Slips</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">3406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Agarose</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Incubator</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mirror or other smooth flat surface</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

El nematodo Caenorhabditis elegans (C. elegans) es un modelo que es ampliamente utilizado para el estudio de longevidad y las vías de desarrollo. Tales estudios son facilitados por la transparencia de los animales, la capacidad de marcha adelante y atrás ensayos genéticos, la relativa facilidad de generar proteínas fluorescencia de etiquetado y el uso de tintes fluorescentes que tampoco puede ser microinyectados en el temprano embrión o incorporado en su alimento (e. coli cepa OP50) etiquetar organelos celulares (e.g. 9-dietilamino-5 H-benzo (a) fenoxazina-5-one y (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-yl-kappaN} - N - [2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Aquí, presentamos el uso de un fluorescente tinte pH-sensible que tiñe lisosomas intestinales, proporcionando una lectura visual de los cambios dinámicas, fisiológicas en acidez lysosomal en gusanos vivos. Este protocolo no mide pH lisosomal, sino más bien tiene por objeto establecer un método confiable de evaluación fisiológicas variaciones relevantes en acidez lysosomal. cDCFDA es un compuesto celular florescente que es convertido a fluorescente fluoróforo 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) al producirse la hidrólisis por esterasas intracelulares. Protonación dentro de lisosomas trampas cDCF en estos orgánulos, donde se acumula. Debido a su bajo pKa de 4.8, este colorante se ha utilizado como un sensor de pH en la levadura. Aquí describimos el uso de cDCFDA como suplemento del alimento para determinar la acidez de lisosomas intestinales en C. elegans. Esta técnica permite la detección de alcalinizantes lisosomas en animales vivos, y tiene una amplia gama de aplicaciones experimentales, incluyendo estudios sobre envejecimiento, autofagia y biogénesis lisosomal.

cDCFDA lisosomas tiñe de manera dependiente del pH y su pKa bajo y lista absorción en lisosomas es un sensor de pH ideal21. cDCFDA coloración de intensidad es inversamente proporcional al pH lisosomal (es decir, tinción intensidad los aumentos como disminuciones de pH)18,22. las señales cDCFDA son siempre débiles en los lisosomas de los animales tratados con 20 mM de la cloroquina, un inhibid...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Una guía paso a paso para la pérdida de acidez lysosomal en el intestino de C. elegans con el colorante vital sensible a pH 6 - carboxi - 2', 7'-Diclorofluoresceína diacetato (cDCFDA)

Evaluación de alcalinización Lysosomal en el intestino de vivir Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K vistas.  University of Saskatchewan. Una guía paso a paso para la pérdida de acidez lysosomal en el intestino de C. elegans con el colorante vital sensible a pH 6 - carboxi - 2', 7'-Diclorofluoresceína diacetato (cDCFDA)

Video: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Evaluación completa de la línea germinal Química de toxicidad de los nematodos<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Investigación

Biología del Desarrollo

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Los protocolos para analizar el papel de las células de Paneth en Regeneración del Murino Intestino utilizando condicional<em&gt; Cre-lox</em&gt; Ratón Modelos

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Análisis espacial y temporal de ERK en el Activo<em&gt; C. elegans</em&gt; línea germinal

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Cultivo de óvulos para la proyección de imagen de cigoto polarización de Live-celular y patrones del embrión en Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imágenes en vivo de la fusión secundaria del paladar del ratón

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

El intestino C. elegans como modelo una morfogénesis Lumen intercelular y En Vivo biogénesis de la membrana polarizada a nivel unicelular: etiquetado de anticuerpos, ARNi pérdida-de-función análisis y proyección de imagen de

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Inmovilización de Caenorhabditis elegans para analizar el transporte intracelular en las neuronas

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Aplicación de RNAi y expresión del Factor de transcripción inducida por choque térmico para reprogramar las células de germen para las neuronas en C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análisis computacional de lo elegans de Caenorhabditis del Germline para estudiar la distribución de núcleos, proteínas y citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Evaluación de alcalinización Lysosomal en el intestino de vivir Caenorhabditis elegans

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