Los autores desean agradecer la GWU Harlan Becas para el apoyo de Paaqua Grant y la GWU Luther Rice Becas para el apoyo de la Mona Herold. Este trabajo fue financiado por la National Science Foundation de subvención MCB-1121711.

1. Preparación de los medios de comunicación, Cultura Instrumentos y platos <ol><li> Enfocar cuatro pinzas con película abrasivo de alúmina. Haga esto bajo un microscopio de disección para controlar el tamaño de la punta. Un par de pinzas sirve como una copia de seguridad en caso de que una punta se daña durante el procedimiento. Autoclave las cuatro pinzas y almacenar en un recipiente estéril. </li><li> Hacer cada 500 ml de medio de cultivo 0,1 X 1,0 X y (ya sea modificado de Marc Ringers [MMR] o salinos Modificado Barth [MBS]; recetas en 15) y el filtro de esterilización. Habitualmente usamos MBS (1X = 88 mM NaCl, 1 mM KCl, 0,7 mM CaCl 2, 1 mM MgSO 4, 5 mM HEPES [pH 7,8], 2,5 mM NaHCO 3). Almacenar a 14-18 ° C durante meses. </li><li> Haga una solución de agarosa al 2% en medio de cultivo (2 g electroforesis en agarosa al grado en 100 ml de medio de cultivo 1X en una botella de vidrio tornillo de la tapa). Esterilizar en autoclave para disolver la agarosa. Esto se puede almacenar a 4 ° C durante meses, y calentados para licuar la agarosa cuando se necesitaba siguiente. </li><li> Mientras que la agarosa es líquido, se vierte sobre 0,5 ml en el fondo de cada pocillo de una placa de cultivo estéril 24-así. Esto evitará que los explantes se pegue al plástico. </li><li> Mientras que la agarosa es líquido, se vierte sobre 2-3 ml en el fondo de dos a tres 60 mm placas de Petri, y agitar suavemente para asegurarse de que el fondo está cubierto. Estos servirán como platos de disección y la agarosa impide embriones se pegue al plástico una vez que sus membranas se retiran. </li><li> Cuando la agarosa se ha enfriado y endurecido, la llama de la punta de una pipeta 6 &quot;Pasteur hasta que se funde en una bola, y ligeramente toque en la superficie de la agarosa en cada pocillo de la placa de cultivo. Esto creará una depresión poco profunda en la que el explante se coloca. </li><li> Llenar cada pocillo de la placa de cultivo con medio de cultivo estéril 1X. </li><li> Cuando la agarosa se haya enfriado y endurecido, llenar el plato de disección con medio de cultivo diluido (0,1 X MMR o MBS 0.1x) para facilitar la separación de blastómeros. </li&gt; </ol> 2. Selección de embriones <ol><li> Obtención de huevos fertilizados y eliminar capas de la jalea de acuerdo con protocolos estándar de 15. Transferirlos a un medio de cultivo en una placa de 0,5 X 100 mm Petri. </li><li> Cuando los embriones llegar a la etapa 2-células, ordenar aquellos en los que el surco de división divide la primera área ligeramente pigmentado en el hemisferio animal (Figura 1) en un plato separado. Estos serán los donantes de los explantes. Mantenga las restantes celdas de 2 embriones en un plato aparte junto a los embriones de donantes de todo el procedimiento para servir como controles de hermanos para organizar los explantes. </li></ol> 3. Preparación de explantes <ol><li> Coloque 5-10 embriones en un plato de disección, pero sólo funcionan en un embrión en un momento. </li><li> Coloque el embrión por lo que la primera transparente membrana vitelina se puede ver, que está separada por un espacio libre (espacio perivitelino) por encima de la superficie del polo animal del embrión. Uso de una fuerza de afiladops en la mano subdominante (mano izquierda si es diestro), sujete la membrana vitelina por encima del espacio perivitelino. Usando unas pinzas afiladas en la otra mano, sujete la membrana cerca de la punta de unas pinzas en primer lugar, y tire suavemente en dirección opuesta a la cáscara de la membrana de distancia. Hacer el alcance inicial a una distancia desde la célula que va a ser disecado, de modo que la célula diana no está dañado durante la extracción de la membrana. Uno puede decir que la membrana se ha eliminado debido a que el embrión se aplanan. </li><li> Agarre una célula vecina con las pinzas en la mano subdominante y utilizar esta celda como un &quot;mango&quot; así que la célula a ser disecado no se toca directamente. Con las pinzas en la otra mano, tire suavemente las células vecinas restantes lejos de la blastómero deseado. Si las células de la línea media, que comparten el mismo destino, se contemplan, se pueden eliminar juntos como una pareja. Por último, diseccionar la &quot;manija&quot; célula. </li><li> Levante el blastómeros (o par de blastómeros), con una estéril, Pipeta Pasteur de vidrio, evitando burbujas de aire y de aspiración excesiva. La pipeta puede ser pulida al fuego para eliminar los bordes afilados que pueden dañar las células, pero no suelen hacerlo. Colocar la punta de la pipeta Pasteur bajo la superficie del medio de cultivo en un pocillo de la placa de cultivo de explante, y expulsar suavemente el blastómero. Debe caer en la depresión baja por gravedad. El blastómero mismo de más de un embrión se pueden combinar en un solo explante. </li><li> Repita este procedimiento con los embriones restantes en el plato de disección, uno por uno. Después de aproximadamente 10 embriones, el plato de disección se llena de restos celulares. Cuando esto ocurre, cambiar a un plato fresco disección. </li><li> Después de todas las disecciones terminado, comprobar si los explantes se encuentran en las depresiones poco profundas. Si no, pueden colocarse suavemente en la depresión con un bucle de pelo que ha sido esterilizado en 70% de etanol y se secó al aire. </li><li> Alrededor de una hora después de la disección último, retirar escombros surrouhallando los explantes curadas con una pipeta estéril, vidrio Pasteur. </li><li> Cultura de la placa de los explantes a 14-20 ° C al lado de la placa de Petri que contiene el hermano, los embriones de control. Embriones de hermanos indicará la etapa de desarrollo de los explantes blastómero. </li></ol> 4. Los explantes de cosecha para el Análisis de <ol><li> Cosechar los explantes cuando los hermanos llegan a la etapa de desarrollo deseado. Estos explantes sobreviven muy bien, incluso si algunas de las células se desintegran. Por lo tanto, si hay una masa nublado en el pocillo de cultivo, que explorar con un bucle de pelo o fórceps para determinar si un explante sano está enterrado dentro. </li><li> Recoger explantes en un pequeño volumen de solución de cultivo con una pipeta Pasteur de vidrio, y suavemente expulsarlos en un tampón de lisis adecuado o fijador para que el ensayo se llevó a cabo. </li></ol>

<ol>
	<li>Dale, L., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+32-cell+stage+of+Xenopus+laevis.">Fate map of the 32-cell stage of Xenopus laevis.</a> Development. 100, 279-295 (1987).</li><li>Masho, R., Kubota, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+fates+of+blastomeres+of+the+eight-cell+stage+Xenopus+embryo.">Developmental fates of blastomeres of the eight-cell stage Xenopus embryo.</a> Dev. Growth, Diff. 30, 347-359 (1988).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+16-cell+stage+Xenopus+embryo.">Fates of the blastomeres of the 16-cell stage Xenopus embryo.</a> Dev. Biol. 119, 560-578 (1987).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell+stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell stage Xenopus embryo.</a> Dev. Biol. 122, 300-319 (1987).</li><li>Moody, S. A., Kline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+fate+during+cleavage+of+frog+(Xenopus+laevis)+blastomeres.">Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.</a> Anat. Embryol. 182, 347-362 (1990).</li><li>Takasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+roles+of+the+presumptive+organizer+region+in+the+32-cell+embryos+in+normal+development+of+Xenopus+laevis.">Fates and roles of the presumptive organizer region in the 32-cell embryos in normal development of Xenopus laevis.</a> Dev. Growth, Diff. 29, 141-152 (1987).</li><li>Sullivan, S. A., Moore, K. B., Moody, S. A., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20+Early+events+in+frog+blastomere+fate+determination.">Chapter 20 Early events in frog blastomere fate determination.</a> Cell Fate and Lineage Determination. , 297-321 (1999).</li><li>Moore, K. B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal-vegetal+asymmetries+influence+the+earliest+steps+in+retinal+fate+commitment+in+Xenopus.">Animal-vegetal asymmetries influence the earliest steps in retinal fate commitment in Xenopus.</a> Dev. Biol. 212, 25-41 (1999).</li><li>Yan, B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competence+of+Xenopus+blastomeres+to+produce+neural+and+retinal+progeny+is+repressed+by+two+endo-mesoderm+promoting+pathways.">The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways.</a> Dev. Biol. 305, 103-119 (2007).</li><li>Gallagher, B. C., Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+differentiation+of+dorsal+axial+structures+from+an+animal+cap+cleavage+stage+blastomere+in+Xenopus.">Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus.</a> Development. 112, 1103-1114 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+maternal+mRNAs+from+a+dorsal+animal+blastomere+induce+a+secondary+axis+in+host+embryos.">Xenopus maternal mRNAs from a dorsal animal blastomere induce a secondary axis in host embryos.</a> Development. 116, 347-3345 (1992).</li><li>Godsave, S. F., Slack, J. M. <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+analysis+of+mesoderm+formation+in+the+Xenopus+embryo.">Single cell analysis of mesoderm formation in the Xenopus embryo.</a> Development. 111, 523-530 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activin-like+signal+activates+a+dorsal-specifying+RNA+between+the+8-+and+16-cell+stages+of+Xenopus.">An activin-like signal activates a dorsal-specifying RNA between the 8- and 16-cell stages of Xenopus.</a> Dev. Genetics. 19, 210-221 (1996).</li><li>Pandur, P. D., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+maternal+influences+on+dorsal-ventral+fate+of+Xenopus+animal+blastomeres.">Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.</a> Dev. Dyn. 225, 581-587 (2002).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Development+of+Xenopus+laevis.">Early Development of Xenopus laevis.</a> A Laboratory Manual. , (2000).</li><li>Sullivan, S. A., Akers, L., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=foxD5a,+a+Xenopus+winged+helix+gene,+maintains+an+immature+neural+ectoderm+via+transcriptional+repression+that+is+dependent+upon+the+C-terminal+domain.">foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent upon the C-terminal domain.</a> Dev. Biol. 232, 439-457 (2001).</li><li>Yan, B., Neilson, K. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxD5+plays+a+critical+upstream+role+in+regulating+neural+fate+and+onset+of+differentiation.">FoxD5 plays a critical upstream role in regulating neural fate and onset of differentiation.</a> Dev. Biol. 329, 80-95 (2009).</li><li>Vincent, J. -. P., Gerhart, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcortical+rotation+in+Xenopus+eggs:+an+early+step+in+embryonic+axis+specification.">Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification.</a> Dev. Biol. 123, 526-539 (1987).</li><li>Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+A:+Solutions+and+Protocols.">Appendix A: Solutions and Protocols.</a> Methods in Cell Biology. 36, 657-662 (1991).</li><li>Klein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+cleavage+furrow+demarcates+the+dorsal-ventral+axis+in+Xenopus+embryos.">The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos.</a> Dev. Biol. 120, 299-304 (1987).</li><li>Masho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+the+first+cleavage+plane+and+the+body+axis+in+early+Xenopus+embryos.">Close correlation between the first cleavage plane and the body axis in early Xenopus embryos.</a> Dev. Growth Diff. 32, 57-64 (1990).</li></ol>

No hay conflictos de interés declarado.

Los pasos más críticos para el cultivo con éxito de blastómeros individuales son: 1) identificar correctamente el blastómero de interés, 2) el mantenimiento de un cultivo estéril, saludable y 3) células de disección en la parte correcta del ciclo celular, y 4) la elaboración del manual necesario destreza para evitar daños durante la disección y transferir a la cultura también. Para manipular blastómeras específicas, es esencial para ser capaz de identificar los ejes cardinales...

Xenopus laevis embriones se han utilizado ampliamente para identificar los mecanismos por los cuales las células embrionarias adquieren sus destinos específicos debido a que sus huevos son lo suficientemente grandes para permitir que los enfoques de microcirugía. Además, se desarrollan de forma externa sin la necesidad de suplementación nutricional del medio de cultivo debido a que cada célula contiene un rico suministro intracelular de las plaquetas de yema de que proporcionan una reserva de energía intrínseca. Un activo importante para el estudio de los mecanismos por los cuales se determina el destino celular es el conjunto completo de mapas de destino de los blastómeros estadio de división (de 2 - a través de las etapas de 32 células) 1, 2, 3, 4, 5, 6. Estos mapas fueron construidos por microinyección de una molécula detectable en un blastómeras, identificables y seguimiento posterior en el desarrollo de los tejidos que están pobladas por los descendientes etiquetados. Mapas consistentes destino son posibles porque los ejes cardinales de los embriones pueden ser identificadas con toda fiabilidad en muchos embryos. En primer lugar, en todos los embriones de tipo salvaje del hemisferio animal es pigmentada, mientras que el hemisferio vegetal no lo es. En segundo lugar, la entrada del esperma en la fertilización produce una contracción de la pigmentación hemisferio animal hacia el lado ventral futuro; en muchos embriones una diferencia pigmentación por lo tanto se puede utilizar para discriminar entre los lados dorsal y ventral. En tercer lugar, el surco de división primera aproxima el plano sagital medio en la mayoría de los embriones, y por lo tanto se puede utilizar para identificar los lados derecho e izquierdo del embrión. Los mapas de destino también se basan en el hecho de que, naturalmente, los huevos fertilizados con frecuencia se unirá en patrones regulares que hacen que cada blastómero identificable a través de una gran población de embriones. Si bien existe variabilidad dentro y entre las puestas de huevos respecto a los patrones de pigmentación y de escisión, utilizando procedimientos de selección descritos en este documento permite que las células con destino prescritos a ser identificado con una precisión del 90% aproximadamente. Destinos celulares pueden disuadirextraído durante la embriogénesis por varios mecanismos. Los factores intrínsecos, tales como diferencialmente heredados mRNAs citoplasmáticas o proteínas, contribuyen a varios aspectos del patrón temprano. Por ejemplo, específicos de mRNAs materna determinar qué células contribuirán a la línea germinal, se convierten en el endodermo, o contribuir al eje del cuerpo dorsal (revisado en 7). Los factores extrínsecos proporcionados localmente por células vecinas o más alejadas de un centro embrionario de señalización, son responsables de la inducción de tipos específicos de tejidos y patrones casi todos los sistemas de órganos. Ejemplos de centros de señalización incluyen la organizador / nodo en la gástrula que induce el ectodermo neural y los patrones del mesodermo, y la zona de actividad polarizante que los patrones del eje anterior-posterior del primordio de la extremidad. Aunque los mapas destino identificar los precursores de los diferentes órganos y revelar la vía de desarrollo adoptadas por sus descendientes en el embrión normal, que no pueden distinguir entre intrínsecay influencias extrínsecas en esas células. También no revelan el potencial de desarrollo de una célula, cuyos descendientes diferenciar en el complejo entorno de señalización del embrión. Dos enfoques experimentales pueden probar si el destino de una célula está determinada por factores intrínsecos o sea posteriormente influida por factores externos: 1) el trasplante de la célula a una ubicación nueva en el embrión, o 2) la eliminación de la célula del embrión seguido por cultivo en la ausencia de señales exógenas. Ambos enfoques experimentales han sido factible en Xenopus porque las células son suficientemente grandes para separar manualmente. Por ejemplo, numerosos estudios han eliminado las células individuales a partir de embriones (para cambiar interacciones célula-célula) o células trasplantadas a lugares nuevos en los embriones de acogida para probar los cambios de destino (revisado en 7, 8, 9). Además, el segundo enfoque de la explantación pequeño número de células de diferentes regiones del embrión icultura ONT para dilucidar las interacciones inductivas de tejido en ausencia de factores exógenos es posible debido a que las células del embrión de Xenopus se llenan con un almacén intracelular de nutrientes, las plaquetas de yema. Por lo tanto, se pueden cultivar durante unos días en un medio definido sin sal suplementación nutricional o factor de crecimiento del medio de cultivo. Hemos usado este enfoque para demostrar que los blastómeros animales dorsales tienen una capacidad autónoma para producir tejidos mesodérmicos neuronales y dorsal debido a la herencia materna mRNAs 10, 11, y otros mostraron que mesodermo especificación de 32-celulares blastómeros se basa tanto en la información intrínseca y extrínseca 12 . Una ventaja de cultivar células como explantes es que el medio también se puede complementar con factores de señalización definidos para determinar que la célula-a-célula vía de comunicación podrían influir en el destino de la célula explantado 12, 13, 14. Además, se puede inyectar el pr blastómeroior a la cultura con ARNm para sobre-expresar un gen, o con oligonucleótidos anti-sentido para evitar la traducción de los ARNm endógenos. Estos, ganancia y pérdida de función de análisis puede identificar moléculas que se requieren para un destino autónoma expresado. Para analizar el destino del explante, la identificación de tipos específicos de células puede llevarse a cabo por la expresión de genes estándar (por ejemplo, hibridación in situ, RT-PCR) y ensayos inmunocitoquímicos. Este protocolo proporciona un simple, pero poderoso, forma de distinguir entre los mecanismos intrínsecos y extrínsecos que regulan el funcionamiento de una célula embrionaria se desarrolla en tejidos específicos.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Película abrasivo de alúmina </td><td> Thomas Scientific </td><td> # 6775E-38 </td><td> Campo (12 micras), para grandes reparaciones de puntas de las pinzas </td></tr><tr><td> Película abrasivo de alúmina </td><td> Thomas Scientific </td><td> # 6775E-46 </td><td> Medio (3 m), para bien afilado de puntas de las pinzas </td></tr><tr><td> Película abrasivo de alúmina </td><td> Thomas Scientific </td><td> # 6775E-54 </td><td> Fine (0,3 m), para el pulido de puntas de las pinzas </td></tr><tr><td> Pinzas: Dumont, Dumoxel Biologie # 5 </td><td> Herramientas Artes Ciencias </td><td> # 11252-30 </td><td> Estos tienen las puntas finas que no necesitan ser afilados cuando se adquirió. Resistente a la corrosión, para que puedanse esteriliza en autoclave. </td></tr><tr><td> La gentamicina solución </td><td> Sigma </td><td> G1397 </td><td> Añadir al medio en el mismo día de uso </td></tr></tbody></table>

blastomere explants to test for cell fate commitment during embryonic development

Mapas de Destino, construidas a partir de linaje rastreo de todas las células de un embrión, revelan que los tejidos descienden de cada célula del embrión. Aunque los mapas destino son muy útiles para la identificación de los precursores de un órgano y para elucidar la vía de desarrollo por el que las células descendientes rellenar ese órgano en el embrión normal, no ilustran el potencial de desarrollo de una célula precursora o identificar los mecanismos por los cuales su destino está determinado. Para poner a prueba el compromiso del destino celular, se compara repertorio normal de una célula de descendientes en el embrión intacto (el mapa de destino) con los que se expresan después de una manipulación experimental. Es el destino de la célula fija (comprometido), independientemente del entorno celular, o es influenciado por factores externos facilitados por sus vecinos? Usando los mapas completos destino del embrión de Xenopus, se describe cómo identificar, aislar y única cultura precursores estadio de división, llamado blastomeres. Este enfoque permite evaluar si estas células tempranas están comprometidos con el destino que adquieren en su entorno normal en el embrión intacto, requieren interacciones con sus células vecinas, o puede ser influenciada para expresar destinos alternativos, si expuesto a otros tipos de señales.

La capacidad de este ensayo para evaluar con precisión el potencial de desarrollo de la célula se basa en la disección de la blastómero correcto basado en el destino mapas 1, 2, 3, 4, 5, 6. Por lo tanto, es fundamental para elegir embriones con el patrón de pigmentación correcta en la etapa 2-células, como se ilustra en la Figura 1, que posteriormente se ajustan a los patrones de escisión regulares, como se ilustra en la Figura 2. Si se observa según los embriones a ...

Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

El destino de una célula embrionaria individual puede ser influenciada por las moléculas hereditarias y / o por las señales de las células vecinas. Utilizando los mapas de destino de la etapa de escisión de embriones de Xenopus, blastómeras individuales pueden ser identificados por la cultura de forma aislada para evaluar las contribuciones de moléculas hereditarias frente a las interacciones célula-célula.

Los explantes blastómeras para la Prueba de Compromiso destino celular durante el desarrollo embrionario

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

blastomere-explants-to-test-for-cell-fate-commitment-during-embryonic

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

15.2K vistas.  The George Washington University. El destino de una célula embrionaria individual puede ser influenciada por las moléculas hereditarias y / o por las señales de las células vecinas. Utilizando los mapas de destino de la etapa de escisión de embriones de Xenopus, blastómeras individuales pueden ser identificados por la cultura de forma aislada para evaluar las contribuciones de moléculas hereditarias frente a las interacciones célula-célula.

Video: Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Investigación

Biología

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Un enfoque práctico de elaboración de mapas genéticos inducible destino: una guía visual para marcar y rastrear las células En Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Un enfoque de genética inversa para probar la redundancia funcional durante la embriogénesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

El destino de la asignación de células estaminales embrionarias humanas mediante la formación de teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

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, ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

El uso de LysoTracker para detectar la muerte celular programada en embriones y la diferenciación de células madre embrionarias

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...