Damos las gracias a Shlomi Krispin para la imagen del tubo neural. En particular, reconocemos Yuval Canela para iniciar la técnica de transfección de GFP. Este trabajo fue apoyado por becas de la Fundación Ciencias de Israel, Association Française contre les Miopatías, y Forschungsgemeinshaft Deutche, Centro de Investigación Cooperativa 488 a CK

1. Preparación de Micropipetas Sacamos nuestras pipetas en un estándar Narishige extractor pp-83 pipetas con un ajuste de calor de 13,5. La configuración precisa debe ser calibrado, con miras a un diámetro de punta de la pipeta de entre 8 y 10 micras. Usamos filamento que contienen los tubos de vidrio de borosilicato con un diámetro exterior de 1,0 mm y diámetro interior de 0,75-0,78 mm. Las pipetas, en consecuencia, almacenados en un plato de plástico con las puntas protegidas del contacto con los bordes del plato. 2. Preparación de ADN El plásmido de interés se transfecta en DH5a E. coli utilizando procedimientos estándar. Cultivos de una noche de las colonias solo se utilizan para purificar el plásmido con preparación maxi-kits (cualquier kit comercial debe hacer). Nosotros utilizamos una concentración del plásmido de 1,0 mg / l para los no clonal inyecciones. Las concentraciones más altas aumentan la obstrucción de las puntas de pipeta. Para aplicaciones clonal, las concentraciones de plásmido son mucho más bajos y se deben ajustar (ver más abajo). Para pCAGG-GFP, la concentración óptima producción clonal resultados se encontró que alrededor de 0,05-0,1 mg / l. Uno a 2 l de plásmido se utiliza normalmente por experimento, y una cantidad mínima de fast-verde en polvo (la adhesión a la punta de una pipeta seca, vidrio filoso) se añade a la plásmido antes de que el experimento comienza. El plásmido se centrifuga en una centrífuga de mesa durante 2 minutos a máxima velocidad. Este procedimiento parece reducir la obstrucción pipeta problemas. 3. La inyección de embriones 3.1 Preparación de los embriones Usamos la codorniz, así como huevos de gallina para preparaciones inyectables. Los huevos se incuban acostado sobre su eje longitudinal hasta la etapa de desarrollo deseado. Los huevos se limpiaron con etanol 70% y se deja secar. La punta del huevo se perfora con una tijera quirúrgica (esto es necesario sólo en huevos de gallina) y 1 ml (en codornices) o 2 ml (pollo) de albúmina se extrae del óvulo con una jeringa de 10 ml (19G aguja). Los agujeros se sellan con parafina caliente. Antes de la inyección actual, se abre una ventana en la parte superior de la cáscara de huevo con unas tijeras quirúrgicas. La colocación de cinta adhesiva sobre la cáscara y corte a través de ella puede ayudar a reducir los residuos de la cáscara. Tampón PBS que contiene antibióticos es una pipeta de inmediato en el óvulo. Para acceder a los embriones, la vitelina y / o membranas amnióticas se cortan y se desvía con 0,14 mm alfileres de insectos montados sobre un soporte de aguja. No es tóxico tinta (Pelican # 17) se inyecta debajo de la blastodermo para ayudar a visualizar el embrión, con un fuego tirado capilar montado en un aspirador. 3.2 Carga y montaje de micropipetas El extractor de micropipetas se puede cargar con el plásmido de fuego tirado por los capilares de diámetro adecuado, montado sobre un aspirador. Una mínima cantidad de plásmido se dibuja con el capilar y se cargan en la pipeta de lo contrario la apertura a la punta afilada, y acercándose a la punta desde el interior tanto como sea posible. La pipeta, en consecuencia, cargado en una presión de aire inyector manual montado en un micromanipulador. Presión mínima se aplica, si es necesario, a fin de permitir la solución de plásmido para llegar a la punta de la pipeta. 3.3 de inyección de plásmido La pipeta se manipula en el medio líquido del huevo mientras se aplica una presión de aire para evitar la obstrucción de la punta de la pipeta. El dominio del tejido relevante es traspasado. El cambio en la presión como la punta entra en el tejido es a menudo suficiente para liberar el plásmido con el verde rápido visible. Si este no es el caso, aplicar una ligera presión a la jeringa hasta que el verde plásmido / rápido son expulsados. Este proceso se puede repetir a lo largo del eje del embrión, por ejemplo, en somitas adyacentes oa lo largo de la extensión del tubo neural. Después de las inyecciones, PBS se puede añadir. La ventana está sellado con cinta adhesiva (parafina no es deseable, especialmente para los huevos de codorniz, que aumenta las tasas de muerte de embriones, probablemente mediante el bloqueo de intercambio de oxígeno a través de la cáscara). Los embriones tratados se devuelven a la incubadora. Evitar la ventilación y colocar pequeños recipientes con agua en la incubadora ya que ayudan a mantener los rendimientos y aumentar la humedad. 4. Ajuste de los parámetros de inyección y de seguimiento las tasas de éxito Asessing y calibración de los diferentes parámetros de la inyección es crítico para las aplicaciones destinadas a una sola célula de análisis. No todos los eventos de inyección llevará al etiquetado de éxito de una sola célula. Concentración de plásmido que es demasiado alto puede llevar a no clonal de etiquetado, mientras que el resultado concentraciones muy bajas en las tasas de fracaso. La concentración de la inyección del plásmido debe ser optimizado para inyecciones clonal. Se recomienda la dilución de la totalidad de acciones plásmido purificado en lugar de diluir preparaciones individuales a una concentración predeterminada. Este últimopodría introducir alguna variación de las tasas de éxito, probablemente debido a las variaciones en la pureza y otros parámetros tales como la proporción de plásmido superenrollado. 4.1 Evaluación de las tasas de éxito por toda la observación de montaje Evaluación inicial de los embriones inyectados pueden ser realizadas por todo el montaje de observación bajo un binocular fluorescentes. Seis a ocho horas después de la inyección son suficientes para evaluar las tasas de éxito con una construcción de buenas prácticas agrarias pCAGG mejorada. Esto es siempre que el sitio de destino es superficial suficiente para ser evidentes (por ejemplo, el somite dorsal, dorsal del tubo neural, etc ectodermo, ver Figura 1-C.). Este paso es especialmente útil para la evaluación de sus series experimentales exhiben ya sea células marcadas demasiados o, alternativamente, a falta de la presencia de células fluorescentes. Si se desea más de incubación, los antibióticos que contienen una solución de PBS, debe añadirse a la del huevo y después de ver la cinta de sellado debe ser reemplazado. 4.2 La evaluación de clonalidad mediante el análisis de serie de la sección El mejor medio para evaluar las tasas de éxito de las inyecciones es de serie-el corte embriones inyectados y el uso de inmunohistoquímica para visualizar las células marcadas. En estas condiciones, los tiempos de incubación puede ser tan corto como 4 horas después de la inyección. Los embriones son fijados y procesados ​​para la sección de parafina (o muestras criostáticas). Montado de vidrio secciones se de-paraffinized e inmuno-tinción para visualizar el reportero. Métodos de amplificación tales como biotina-estreptavidina que sería deseable. Finalmente, los cortes seriados se analizan para detectar células marcadas en los segmentos inyectados. Si después de varios ensayos iniciales en vez de células individuales se obtienen, las acciones plásmido debe ser diluido y el proceso se repite hasta que los resultados se logran adecuadas. De dos a 10 diluciones de la solución del plásmido puede ser necesario en los casos de las tasas de éxito son demasiado altos. Para cada plásmido se transfectadas, una calibración completa que asegura clonalidad se debe repetir. 5. Resultados representante Por lo general llevar a cabo una sola inyección de pCAGG-GFP por somite, mientras que 12.6 por dirigirse a segmentos de embriones 1 2. Bajo este contexto, una clara señal puede ser detectada por la observación todo el montaje 8 horas de la inyección, en al menos un segmento de un embrión de cada diez. En estas condiciones, el etiquetado se confirmó que se clonal (Figura 1 B, C). Cuando se utilizan altas concentraciones de ADN, la mayoría de los embriones pueden presentar al menos una inyección de éxito, aumentando la posibilidad de que varias celdas han sido transfectadas. Análisis de la sección de serie reveló que transfecciones clonal se alcanza cuando la tasa de éxito fue del 10%. Esto significa que a fin de lograr clonalidad, la técnica es por definición, altamente ineficiente. Por otro lado, en estas condiciones, más del 93% de los eventos etiquetado positivo se encontró que una clonal. <img src="/files/ftp_upload/2133/2133fig1.jpg" alt="Figura 1" /> Figura 1. Inyecciones clonal de pCAGGS-GFP en células progenitoras neurales y esquelético. (AC) que muestra las secciones transversales de etiquetado de la dorsal del tubo neural (NT) (A), el dominio ventrolateral de la dermomiotoma (DM) (B), o una hoja de DM central (C) , 6 h después de la microinyección de pCAGGS-GFP con un solo progenitor. (D) Dos días después de la transfección de clones de la central de progenitores DM, solo genera derivados, tanto en la dermis (D), así como el músculo (M) (ver ref. 1 para más detalles).

<ol>
	<li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+the+avian+dermomyotome+sheet+reveals+the+existence+of+single+cells+with+both+dermal+and+muscle+progenitor+fates.">Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates.</a> Development. 132, 689-701 (2005).</li><li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+and+bone+morphogenetic+protein+differentially+act+on+dermomyotome+cells+to+generate+endothelium,+smooth,+and+striated+muscle.">Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle.</a> J Cell Biol. 180, 607-618 (2008).</li><li>Cinnamon, Y., Kahane, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+development+of+specific+hypaxial+muscles+from+the+ventrolateral+myotome.+.">Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome. .</a> Development. 126, 4305-4315 (1999).</li><li>Stark, D. A., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+green+fluorescent+protein+as+a+single-cell+marker+in+living+embryos.">Photoactivatable green fluorescent protein as a single-cell marker in living embryos.</a> Dev Dyn. 233, 983-992 (2005).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation+in+vivo+within+the+intact+developing+brain.">Single cell electroporation in vivo within the intact developing brain.</a> J Vis Exp. , (2008).</li><li>Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat Biotechnol. 20, 87-90 (2002).</li><li>Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+integration+and+conditional+expression+of+electroporated+transgenes+in+chicken+embryos.">Stable integration and conditional expression of electroporated transgenes in chicken embryos.</a> Dev Biol. 305, 616-624 (2007).</li></ol>

El método descrito anteriormente es una modificación de una técnica descrita anteriormente para la entrega de colorantes lipofílicos a los subconjuntos de células pequeñas o progenitores solo 3. Tiene tres ventajas principales sobre la técnica de electroporación estándar. En primer lugar, proporciona un medio para el etiquetado de los sitios de confianza discreto en los tejidos, lo que permite una resolución mucho mayor espacio que la electroporación (véase, por ejemplo, la figura 1B, C). En segun...

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<td>Pipette tubes</td>
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<td>Drummond Scientific</td>
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<td>Insect needles</td>
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<td>Pen-Strep solution</td>
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single cell transfection in chick embryos

Un tema central en la biología del desarrollo es la diversificación de los linajes y la elucidación de los mecanismos subyacentes molecular. Esto implica un análisis exhaustivo de los destinos de las células individuales, en condiciones normales y experimentales. Con este fin, los métodos de transfección que se dirigen a los progenitores solo son un requisito previo. Se describe aquí un método técnicamente sencillo para la transfección de células individuales en los tejidos de pollo in ovo, lo que permite el rastreo fiable linaje, así como la manipulación genética. Dominios específicos de tejido son orientadas dentro de los somitas o del tubo neural, y el ADN se inyecta directamente en el epitelio de interés, lo que resulta en la transfección de células individuales esporádicos. Utilizando a los periodistas, poblaciones clonales en consecuencia, puede ser rastreado hasta por tres días, y el comportamiento de manipulados genéticamente poblaciones clonales se puede comparar con la de los controles. Este método tiene la ventaja de la accesibilidad del embrión de pollo, junto con nuevas herramientas para la manipulación genética. Comparar y discutir sus ventajas sobre el método de electroporación utilizadas, y las posibles aplicaciones y su uso en otros modelos in vivo también se proponen. Abogamos por el uso de este método como un complemento importante y se complementan para el linaje actual trazado y herramientas genéticas interferencia.

Watch this Scientific Journal Video about Single Cell Transfection in Chick Embryos at JoVE.com

Single Cell Transfection in Chick Embryos

Usando la punta fina micropipetas se inyecta el plásmido de ADN en subdominios de somitas pollo o tubos neurales. La concentración del plásmido se ajusta a generar solo las células transfectadas. A continuación, permitir que las células se desarrollan en poblaciones clonales.

Transfección de células individuales en embriones de pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

single-cell-transfection-in-chick-embryos

Research

JoVE Journal

Biology

11.0K vistas.  Hadassah Medical School - Hebrew University. Usando la punta fina micropipetas se inyecta el plásmido de ADN en subdominios de somitas pollo o tubos neurales. La concentración del plásmido se ajusta a generar solo las células transfectadas. A continuación, permitir que las células se desarrollan en poblaciones clonales.

Video: Single Cell Transfection in Chick Embryos

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

La disección de embriones de ratón 6,5 dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Investigación

Biología

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

En Electroporations Ovo de embriones de pollo HH Etapa 10

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Una sola célula grabaciones de succión de los conos del ratón

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

De dos fotones basado en fotoactivación en vivo embriones de pez cebra

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Celular imagen Cambiar forma de vida Drosophila Los embriones

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Sola célula de mapeado de destino en el pez cebra

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Los trasplantes faciales en<em&gt; Xenopus laevis</em&gt; Los embriones

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...