This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1. Desarrollo de embriones cigóticos <ol><li> Material Vegetal <ol><li> Crece la Medicago truncatula tipo salvaje Jemalong o su isogénica más cercana, altamente re-generable genotipo Jemalong 2HA 13 (conocido como 2HA) en un invernadero con un fotoperíodo de 14 horas y 23 ° C / 19 ° C de temperatura día / noche. <ol><li> Pierce la superficie de la cubierta de la semilla (con una aguja de 23 G) antes de sembrar la semilla de modo se permite que el agua para entrar en la semilla y en remojo en agua durante la noche. Agregar suficiente agua para cubrir la totalidad de la semilla. </li><li> Siembre 3 semillas en cada maceta 15 cm de diámetro (un total de 10 macetas) en la mezcla para macetas (gruesa arena, perlita, fibra de coco, turba (1: 1: 1) más de 5 g de Osmocote Estándar exacta: fertilizante de liberación lenta) y traslado al invernadero. Conserve la plántula más saludable después de la germinación por lo que no es de 1 planta por maceta y rodeado de un enrejado para la planta proviene de escalar. </li></ol></li></ol></li><li> Vainas de cosecha <ol><li> Recoger las vainas de la wTipo ild Jemalong o 2HA para obtener las fases de embrión temprano adecuadas como primera describe 9. NOTA: Las diferentes etapas de la vaina se muestran en la Figura 1 y las etapas de embriones correspondientes se muestran en la Figura 2. </li><li> Compruebe las diferentes etapas de la vaina en la cosecha utilizando criterios como por la Tabla 1. Medir el tiempo transcurrido desde la etapa 6 para ayudar etapas separadas 6 y 7 (Tabla 2). </li></ol></li><li> La disección de los óvulos de las vainas y la comprobación de la etapa de embrión <ol><li> Aislar óvulos de vainas utilizando unas pinzas finas sola o un microscopio de disección estéreo y pinzas si es necesario. Si se requiere la etapa de embrión se puede comprobar rápidamente usando un microscopio compuesto estándar (Figura 3B). </li><li> Prepárese modificado solución de Hoyer que contiene 7,5 g de goma árabe, 100 g de hidrato de cloral, 5 ml de glicerol, 60 ml de agua desionizada destilada. Disolver por agitación durante 3-5 horas o durante la noche. </li><li> Para obtener más refinard microscopía borrar los óvulos disecados en solución de 15,16 Hoyer modificado. Recoja cuidadosamente los óvulos intactos y el lugar en un pequeño volumen de solución de Hoyer (suficiente para cubrir el óvulo) a temperatura ambiente hasta que se aclare. </li><li> Ver las muestras con contraste de interferencia diferencial (DIC) óptica. Captura las imágenes iwth una cámara digital 9 (Figura 2). </li><li> Obtener los óvulos más uniformes de la región central de la vaina y acumular el número requerido. Un ejemplo se muestra en la Figura 3A. Recoge los óvulos en el hielo y almacenar a temperatura requerida (ácidos -80 ° C durante nucleicos) para su posterior análisis. NOTA: Para obtener más información sobre el análisis de las secciones histológicas, la microscopía electrónica de transmisión, y la hibridación in situ por favor vea los papeles 8,9. </li></ol></li></ol> 2. Desarrollo de embriones somáticos in vitro <ol><li> Utilice una M. truncatula cultivar tsombrero es capaz de formar fácilmente embriones en cultivo. Para este protocolo para explantes de hoja utilizar las plantas 2HA 13,17 que son 2-4 meses de edad. </li><li> Tome folletos de la última hoja trifoliada casi totalmente expandido de un vástago alargando. </li><li> Mantenga las hojas trifoliadas sobre toallas de papel húmedo en una olla de la cultura para evitar la deshidratación. NOTA: Una vez que las hojas trifoliadas se cosechan que necesitan para ser utilizado con la mínima demora. </li><li> Preparación del medio de cultivo <ol><li> Utilice el P4 10: 5 en medio de agar (0,8% w:: v) 4: 1 en 9 cm placas de Petri. NOTA: El P4 10: 4: 1: 5 medio consiste en el medio basal P4 18 más 10 mM de ácido 1-naftalenacético (NAA), 4 M 6-bencilaminopurina (BAP), 1 mM de ácido abscísico (ABA) y 5 mM giberélico ácido (GA). El uso de GA + ABA acelera la formación del embrión y provoca aumentos sustanciales en el número de embriones 14. </li><li> Conforman el medio basal P4 en 1 L; que consiste en sales principales (hacer calcium hasta por separado), sales menores y vitaminas (Tabla 3), de hierro quelado (compensar por separado), casamino ácidos (hidrolizado de caseína), 30 g de sacarosa, 8 g de agar. </li><li> Soluciones tienda del stock de las principales sales, calcio, casaminoácidos, sales menores y vitaminas a -20 ° C en alícuotas adecuadas. Guarde hierro quelado a 4 ° C. </li><li> Completar el hierro quelado (200x) disolviendo 7,44 g de Na 2 EDTA • 2H 2 O en aproximadamente 900 ml de agua destilada desionizada Mientras se agita, a continuación, llevar la solución a 98-99 ° C, mientras que la adición de 1,853 g de FeSO4 • 7H 2 O. Finalmente hacer hasta 1 L cuando se disuelven los ingredientes. Almacenar en color ámbar botellas a 4 ° C. </li><li> Completar el medio basal P4 con las soluciones madre como en la Tabla 4. Las hormonas en el medio son 10 M NAA, 4 BAP M, 1 ABA M, 5 M GA (ver Tabla 4 y Materiales Específicos Tabla). Añadir la NAAy BAP antes de tratamiento en autoclave y añadir ABA y GA después de tratamiento en autoclave y de enfriar a aproximadamente 55 ° C, utilizando esterilización por filtración con un filtro de 0,22 micras unida a una jeringa. Mezclar bien agitando suavemente. </li><li> Ajuste los medios a pH 5,8 con 1 M de KOH antes de autoclave. Autoclave a 121 ° C durante 20 min. </li><li> Después de autoclave agregar la ABA esterilizada filtro y GA, y vierta aproximadamente 25 ml de medio en 9 cm placas de Petri estériles en una campana de flujo laminar o gabinete de riesgo biológico. Enfriar y dejar que el agar establece con la tapa. A continuación, poner en la tapa y asegúrese de que no hay condensación en la tapa. Etiqueta según sea necesario y se almacena a 4 ° C (en una caja de cartón para evitar condensado tapa). </li></ol></li><li> La esterilización de la hoja de tejido <ol><li> Trabajar en una campana de flujo laminar UV-esterilizados o gabinete de riesgo biológico. </li><li> Coloque las hojas en la bola del acoplamiento de un infusor de té de primavera previamente tratada en autoclave. </li><li> Sumerja infusor de té que contiene las hojas en el 70% (v: v) de etanol en una callepote tura durante 30 segundos. NOTA: Para todos los pasos de este procedimiento utilizan 250 ml tornillo de cabeza macetas de cultivo de policarbonato que son tratados en autoclave. </li><li> Escurrir y luego transferir y sumergir el tejido de las hojas en 1: 8 (v: v) de blanqueador diluido (0,5% de cloro) durante 10 minutos. Agitar suavemente de vez en cuando. </li><li> Escurrir la lejía y transferir el sistema de infusión de té en una olla de cultivo que contiene agua destilada desionizada estéril. Hacer girar suavemente y drenar el exceso de agua. Repetir una vez más con una olla de cultivo fresco de agua destilada desionizada estéril. </li><li> Quite las hojas desde el infusor de té con unas pinzas estériles a una olla de cultivo fresco de agua destilada desionizada estéril. Tornillo en la tapa estéril y enjuague invirtiendo y remolinos. Deja las hojas que flotan en el agua estéril hasta que esté listo para preparar los explantes. </li></ol></li><li> Preparación y Plating explantes <ol><li> Usando técnicas estériles, recorte folletos esterilizados individuales desde el borde con un bisturí, y cortar el rectángulo restante en dos o three explantes rectangulares (8-10 x 3-5 mm) con la mitad de la vena en el centro de cada explante (Figura 5A). NOTA: El tamaño del explante es muy importante en la iniciación de la primera formación de callos. Llevar a cabo el corte en las tapas de los recipientes estériles comida para llevar. </li><li> Placa de 6 explantes en medio de cultivo agar-solidificado en 9 cm de diámetro placas de Petri (Figura 5B). Placa del lado abaxial explantes abajo para mantener la orientación habitual hoja. </li><li> Sellar las placas de Petri con Parafilm (Figura 5C) se extendía alrededor del plato. El tejido está ahora listo para la incubación. </li></ol></li><li> La incubación del tejido <ol><li> Incubar las placas en la oscuridad a 28 ° C en un ambiente de temperatura controlada o gabinete crecimiento en todo el período de cultivo. NOTA: Los explantes iniciarán desdiferenciación, someterse a la división celular, la proliferación (formación de callo) y la embriogénesis (diferenciación). </li><li> Subcultivar la EXPOSICIÓN diferenciadornoches después de 3 semanas a un medio fresco. En este subcultivo transferir todo el explante usando una espátula de acero inoxidable estéril y pinzas, en una campana de flujo laminar o un armario de riesgo biológico. Como se produce formación de callos y el callo supera el tamaño de la más grande en la Figura 5C, transferir 50% del callo individual cuando el subcultivo a un medio fresco. </li><li> Observe los primeros embriones en unos 4 semanas. Mientras que hay un grado de sincronía, recoger las diferentes etapas de embrión de más de un período de incubación de 4-8 semanas (Figura 6A, B). Reunir bajo un microscopio de disección y el proceso de acuerdo con los objetivos experimentales. </li><li> Subcultivar cada 3 semanas y eliminar periódicamente los embriones de modo que los explantes continuarán produciendo embriones durante 3 meses. </li></ol></li></ol>

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The authors declare that they have no competing interests.

Los protocolos descritos son relativamente sencillo y permiten la investigación de la embriogénesis leguminosa con toda la célula contemporánea y técnicas moleculares. Reconocemos que hay ventajas y desventajas de ambos in vivo e in vitro enfoques. Ambos permiten un mayor enfoque en la embriogénesis temprana en comparación con la cultura de las semillas inmaduras 19. En el caso de los estudios in vivo lo que se describe es predominantemen...

Las legumbres son la tercera familia más grande de plantas superiores, con aproximadamente 20.000 especies y el (o Fabaceae) familia de las leguminosas son segundos a los cereales en la superficie cosechada y la producción total 1. La soja es el cultivo cultivado tercera más grande. Leguminosas de grano proporcionan alrededor de un tercio de la proteína dietética y un tercio de aceite vegetal para el consumo humano 2. Legumbres con su capacidad de fijación de N2 también contribuyen a los sistemas agrícolas sostenibles. Medicago truncatula, como la soja, las tiendas de proteína y aceite en los cotiledones de las semillas y es una leguminosa modelo genética y genómica con considerables recursos genéticos y genómicos 3,4. Mientras M. truncatula ha permitido avances en la comprensión de la leguminosa simbiosis Rhizobium 4 se ha empleado cada vez más para estudiar la biología de semillas de leguminosas 5-7 y embriogénesis 8,9. Embriogénesis Arabidopsis ha sido ampliamente estudiado 10,11 pero isa no leguminosas y los detalles de la embriogénesis no son idénticos a Medicago 8,10. Embriogénesis cigóticos en M. truncatula tiene características interesantes, con un hipófisis multicelular distintivo, un suspensor endoployploid y transferencia de células basales 8. La embriogénesis somática (SE) se utiliza comúnmente para la regeneración de plantas 12. En el modelo de leguminosas M. truncatula, la línea de semillas Jemalong 2HA (2HA) se ha desarrollado a partir de la matriz Jemalong tener altas tasas de embriogénesis somática 13. El número de embriones producidos recientemente se ha incrementado sustancialmente por la adición tanto de ácido giberélico (GA) y ácido abscísico (ABA) al medio de largo establecido 14. En este caso, GA y actuar sinérgicamente ABA, que es inusual dado que GA y ABA generalmente actúan antagónicamente 14. Los embriones producidos a partir de callos se desarrollan en la superficie que permite la etapa de la embriogénesis a ser determinada fácilmente Visually cosechado y fácilmente. Tener cerca de líneas isogénicas que son embriogénico (2HA) y no embriogénico (Jemalong) facilita la investigación de la embriogénesis somática y que tiene tanto in vivo como sistemas in vitro proporciona diferentes posibilidades experimentales. La comprensión de los mecanismos celulares y moleculares del desarrollo embrionario es esencial para la comprensión de semillas y desarrollo de la planta. En las legumbres, como en otras dicotiledóneas, son los cotiledones del embrión que almacenan los productos que se utilizan para la nutrición humana. Embriogénesis temprana implica la división de células, y los patrones de embriones correcta. En aproximadamente 8 días después de la fecundación, el M. truncatula embrión alcanza etapas tempranas de cotiledones. La caracterización morfológica no se indica exactamente por día después de la fecundación en condiciones de invernadero. Por lo tanto, un enfoque estandarizado eficiente para indicar la etapa de embriones en desarrollo es valioso en el estudio de la genética Regulación de principios de la embriogénesis cigóticos. En este trabajo, ofrecemos dos protocolos estandarizados para recoger embriones en desarrollo para estudios biológicos de la embriogénesis en el modelo de leguminosas M. truncatula. El primero es recoger embriones cigóticos asociando la embriogénesis y pod morfología mientras que la segunda es la embriogénesis somática a través de explantes de hoja de cultivo para proporcionar los números de embriones grandes de fácil acceso.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >P4 medium</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td>
		</tr>
		<tr >
			<td >Major salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Minor salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vitamins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Bacto Laboratories</td>
			<td>214010</td>
			<td>Bacto agar</td>
		</tr>
		<tr >
			<td >Plant hormones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1-Naphthaleneacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0640</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >Abscisic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A1049</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >6-Benzylaminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>B3274</td>
			<td>Dissolve in MQ water with heating and few drops 1N HCl</td>
		</tr>
		<tr >
			<td >Gibberellic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7645</td>
			<td>Dissolve in small amount of ethanol</td>
		</tr>
		<tr >
			<td >Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo dissecting microscope</td>
			<td>Leica</td>
			<td>MZFLIII</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Light microscope</td>
			<td>Zeiss</td>
			<td>Axiophot</td>
			<td>Or similar, with suitable optics</td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td>Zeiss</td>
			<td>AxioCam HRc</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Sterilising leaves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >250 mL screw cap polycarbonate container with polypropylene lid</td>
			<td>SARSTEDT</td>
			<td>75.9922.519</td>
			<td>Autoclavable</td>
		</tr>
	</tbody>
</table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

La embriogénesis temprana a partir de una sola célula cigoto pasa a través de una rápida división celular y la morfogénesis, y se caracteriza morfológicamente por etapas pre-globular, globulares, corazón, de torpedos y de cotiledones. Este desarrollo progresivo está bajo la regulación estricta de una red molecular compleja. La recolección de suficientes embriones tempranos en una etapa similar de desarrollo es esencial para la investigación de la regulación celular y molecular de la embriogénesis temprana. Esto no es directo desde la embriogénesis temprana sufre morfogénesis rápido en un corto periodo de tiempo, por ejemplo 8 días para Medicago truncatula para llegar al estadio de cotiledones temprano. Aquí, abordamos el tema por dos enfoques. El primero establece una vinculación entre el desarrollo del embrión y la morfología pod en ayudar a indicar la etapa del embrión cigóticos. Esto se basa sobre todo en el número de espirales de la vaina y el desarrollo de las espinas. Una forma alternativa para complementar la in vivo sos estudios es a través de explantes de hoja de cultivo para producir embriones somáticos. El medio incluye una combinación de hormonas inusual - una auxina (ácido 1-naftalenacético), una citocinina (6-bencilaminopurina), el ácido abscísico y ácido giberélico. Las diferentes etapas se pueden discernir que crece fuera del callo sin disección.

Para embriogénesis diferentes estructuras vaina cigóticos correspondientes a las diferentes etapas de embriones se muestra en la Figura 1A - F, mientras que las diferentes etapas de embriones se muestran en la Figura 2A - F. Mediante la selección de las vainas en la misma etapa, las muestras de óvulos que son bastante uniforme puede obtenerse (Figura 3A). Mediante el uso de RT-qPCR embrión genes específicos se pueden detectar fác...

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protocolos para la obtención de embriones somáticos y cigóticos para el estudio de la regulación del desarrollo embrionario temprano en el Modelo de Leguminosas<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Research

JoVE Journal

Developmental Biology

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

13.8K vistas.  University of Newcastle, Callaghan, New South Wales, Australia. The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided. 

Video: Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Investigación

Biología del Desarrollo

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptoma de perfiles de<em&gt; In-Vivo</em&gt; Productos fabricados preimplantación de embriones bovinos mediante plataforma de microarrays de dos colores

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualización de la actividad eléctrica celular en embriones tempranos de pez cebra y tumores

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Transferencia de embriones mínimamente invasiva y vitrificación de embriones en la etapa óptima del embrión en el modelo Rabbit

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Preparación de pequeñas bibliotecas de ARN para la secuenciación de embriones de ratón tempranos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Un protocolo para la inmunohistoquímica y la distribución in situ de ARN dentro del embrión temprano de Drosophila

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Un dispositivo de trasplante simple y efectivo para embriones de pez cebra

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Aislamiento de células madre mesenquimales del tejido adiposo de rata para la diferenciación en células productoras de insulina

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face Preparación del cojín endocárdico para el análisis de morfogénesis plana en embriones de ratón

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...