Este trabajo fue apoyado byThe Premio American Pediatric Surgical Association Foundation (AG) y los Institutos Nacionales de Salud K08DK098271 (AG).

La Universidad de Wisconsin Cuidado de Animales y el empleo Comisión aprobó todos los procedimientos. 1. Preparación de soluciones <ol><li> Utilice fosfato 1x solución salina tamponada (PBS) como tampón de disección y solución de lavado. </li><li> Preparar 30% de sacarosa pesando 30 g de sacarosa y el lugar en una botella. Añadir 99 ml de 1x PBS y añadir 1 ml 10% de azida de sodio. Mezclar bien hasta que toda la sacarosa se disuelve. Almacenar a 4 ° C hasta que sea necesario. </li><li> Preparar la solución de bloqueo mediante la mezcla de 1x PBS, 3% de albúmina de suero bovino (BSA) y 0,1% de Triton-X-100. Mezclar bien y guardar en la nevera hasta que se necesite. </li><li> Preparar 8% de paraformaldehído solución (PFA) en PBS 1x pesando la cantidad apropiada de PFA en PBS 1x y luego se incuba a 65 ° C hasta que se disuelva completamente. Almacenar en alícuotas de 25 ml en el congelador a -20ºC hasta que se necesite. Diluir hasta 4% PFA en PBS 1x en el día de uso. </li></ol> 2. Embriones y Gut Dissection <ol><li> De conformidad con el animal de Atención Institucional y el empleo Comisión protocolos aprobados, la eutanasia del ratón embarazada cronometrado y transferir al útero en un 60 mm placa de Petri que contiene hielo frío PBS 1x. </li><li> Bajo un microscopio de disección con tijeras afiladas, corte la pared uterina abierta para exponer los embriones. Retire los embriones del útero y el lugar en una limpia de 60 mm placa de Petri que contiene hielo frío PBS 1x. </li><li> La eutanasia a cada embrión por decapitación en 1x PBS helado. Si está utilizando ratones transgénicos que contienen proteínas fluorescentes, con iluminación de fluorescencia, identificar los embriones transgénicos positivos. </li><li> Diseccionar el tracto GI de cada embrión utilizando un microscopio de disección. Usando unas pinzas finas, orientar el embrión de tal manera que el lado izquierdo está mirando hacia arriba y el lado derecho está en contra de la parte inferior de la placa de Petri. Retire la pared superior del cuerpo del embrión para exponer los órganos internos. Inserte pinzas finas entre la pared dorsal del cuerpo y los órganos internos. CrOSS los fórceps uno contra el otro en una acción de corte de tijera para eliminar los órganos internos del embrión. </li><li> Además sub-diseccionar el tracto GI lejos de los órganos circundantes y luego colocar cada tracto gastrointestinal en un tubo de microcentrífuga de 1,5 ml contiene helada 1x PBS. </li></ol> 3. Fijación de GI Tracts <ol><li> Enjuague cada tracto GI 3 veces con 1x PBS helado y luego vuelva a colocar con 4% PFA. Fijar los tractos gastrointestinales en los tubos de microcentrífuga de 1,5 ml en una plataforma oscilante a temperatura ambiente durante 1,5 hr. Enjuagar los tractos GI 3 veces durante 5 min a RT y luego durante 1 hr en la plataforma oscilante. En esta etapa, almacenar los tractos GI a 4 ° C en 30% de sacarosa en 1x PBS que contiene 0,1% de azida de sodio hasta que se necesite. NOTA: Como alternativa, almacenar los embriones en el 30% de sacarosa hasta por un año sin ningún efecto sobre la integridad de los tejidos. Almacenamiento de las muestras en 30% de sacarosa permite el procesamiento posterior de las muestras o bien para inmunotinción o en PTU para su crio-SECCIng. Como alternativa, proceda con el protocolo de inmunotinción se detalla a continuación. </li></ol> 4. Protocolo Inmunoticción <ol><li> Si las muestras se han almacenado en el 30% de sacarosa, enjuagarlos 3 veces durante 20 minutos en 1x PBS en una plataforma oscilante. </li><li> Coloque los tractos GI en solución de bloqueo en una plataforma oscilante durante 1 hora a RT. </li><li> Eliminar la solución de bloqueo e incubar los tractos GI con la cantidad apropiada de anticuerpos primarios diluidos en solución de bloqueo, ya sea para 4 horas a RT o O / N a 4 ° C en una plataforma oscilante. Utilice 1: 1000 dilución de anticuerpo humano anti-Hu (suero obtenido de paciente), 1: 1000 dilución de pollo anti-proteína verde fluorescente (GFP) de anticuerpos y dilución 1: 100 de anticuerpo de cabra anti-chat. NOTA: Utilizamos un anticuerpo anti-Hu humano que fue obtenida localmente a partir de un paciente, sin embargo, los anticuerpos anti-Hu están disponibles comercialmente, por ejemplo, de ratón anti-Hu, (uso en dilución 1: 500). </li><li> Enjuague los tractos gastrointestinales en 1x PBS3 veces durante 5 min y luego durante 1 hora a RT en una plataforma oscilante. </li><li> Vuelva a colocar la PBS 1x con anticuerpos secundarios diluidos 1: 500 en solución de bloqueo en una plataforma oscilante, ya sea para 4 horas a RT o O / N a 4 ° C. Utilice burro tinte amina reactiva antihumano como DyLight, Cy2 anti-pollo burro y burro anti-cabra Cy5. Si se utiliza el ratón anti-Hu luego use una dilución 1: 500 de burro anti-ratón-amina reactiva de colorante 405. NOTA: La intensidad de la GFP y la expresión endógena tdTomato y la claridad de la inmunotinción aumento con la edad de desarrollo. Por lo general inmunotinción tripas embrionarias individualmente en tubos de 0,2 ml con 150 l de solución de tinción con el fin de reducir el volumen de anticuerpo utilizado y también para asegurar la tinción eficiente del tejido. </li><li> Enjuagar los tractos GI en 1X PBS 3 veces durante 5 min y luego durante 1 hora a RT en una plataforma oscilante. </li><li> Coloque unas pocas gotas de medio de montaje de fluorescencia con DAPI en un portaobjetos de vidrio. Sumerja los tra GIct en el medio de montaje de fluorescencia y añadir una cubierta de vidrio directamente en la parte superior del tejido. El medio de montaje -G fluorescencia es un compuesto soluble en agua que proporciona un sello semi-permanente. NOTA: El uso medio de montaje de fluorescencia como Vectashield que es un glicerol basado medio que previene la decoloración y fotoblanqueo de anticuerpos de montaje. Ambos productos permiten a los tejidos para ser almacenados por largos períodos de tiempo a 4 ° C. </li><li> Capturar imágenes de cada uno de fluoróforo utilizando un microscopio confocal. Utilice longitudes de onda de excitación de los fluoróforos y filtros empleados se presentan en la Tabla 3. </li><li> Realizar análisis de imagen asistido por ordenador usando el software apropiado dependiendo del tipo de confocal utilizado para capturar imágenes. </li></ol>

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+determinants+of+the+independence+and+complexity+of+the+enteric+nervous+system.">Developmental determinants of the independence and complexity of the enteric nervous system.</a> Trends Neurosci. 33 (10), 446-456 (2010).</li><li>Amiel, J., Sproat-Emison, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J Med Genet. 45 (1), 1-14 (2008).</li><li>Erickson, C. S., Barlow, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+enteric+nervous+system+analysis+during+parenteral+nutrition.">Colonic enteric nervous system analysis during parenteral nutrition.</a> J Surg Res. 184 (1), 132-137 (2013).</li><li>Erickson, C. S., Zaitoun, I., Haberman, K. M., Gosain, A., Druckenbrod, N. R., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sacral+neural+crest-derived+cells+enter+the+aganglionic+colon+of+Ednrb(-/-)+mice+along+extrinsic+nerve+fibers.">Sacral neural crest-derived cells enter the aganglionic colon of Ednrb(-/-) mice along extrinsic nerve fibers.</a> J Comp Neurol. 20 (3), 620-632 (2011).</li><li>Zaitoun, I., Erickson, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neuronal+density+and+neurotransmitter+expression+in+the+ganglionated+region+of+Ednrb+null+mice:+implications+for+Hirschsprung's+disease.">Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.</a> Neurogastroenterol Motil. , (2013).</li><li>Margolis, K. G., Stevanovic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neuronal+density+contributes+to+the+severity+of+intestinal+inflammation.">Enteric neuronal density contributes to the severity of intestinal inflammation.</a> Gastroenterology. 141 (2), 588-598 (2011).</li><li>Qu, Z. -. D., Thacker, M., Castelucci, P., Bagy&#225;nszki, M., Epstein, M. L., Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis+of+neuron+types+in+the+mouse+small+intestine.">Immunohistochemical analysis of neuron types in the mouse small intestine.</a> Cell Tissue Res. 334 (2), 147-161 (2008).</li><li>Bian, X. -. C., Bornstein, J. C., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+transmission+at+functionally+distinct+synapses+in+descending+reflex+pathways+of+the+rat+colon.">Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.</a> Neurogastroenterol Motil. 15 (2), 161-171 (2003).</li><li>Johnson, C. D., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+polyvalent+antiserum+to+chicken+choline+acetyltransferase.">Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase.</a> J Neurochem. 46 (3), 968-976 (1986).</li><li>Tooyama, I., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+encoded+by+an+alternative+splice+variant+of+choline+acetyltransferase+mRNA+is+localized+preferentially+in+peripheral+nerve+cells+and+fibers.">A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.</a> J Chem Neuroanat. 17 (4), 217-226 (2000).</li><li>Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+Choline+Acetyltransferase+of+Peripheral-Type+(pChAT)+in+the+Trigeminal+Ganglion+Neurons+of+the+Non-Human+Primate+Macaca+fascicularis.">Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis.</a> Acta histochemica et cytochemica. 46 (2), 59-64 (2013).</li><li>Sang, Q., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+chemical+coding+of+cholinergic+neurons+in+the+small+and+large+intestine+of+the+mouse.">The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.</a> Anat Rec. 251 (2), 185-199 (1998).</li><li>Lei, J., Howard, 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=Targeted+deletion+of+Hand2+in+enteric+neural+precursor+cells+affects+its+functions+in+neurogenesis,+neurotransmitter+specification+and+gangliogenesis,+causing+functional+aganglionosis.">Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.</a> Development (Cambridge, England). 138 (21), 4789-4800 (2011).</li><li>Erickson, C. S., Lee, S. J., Barlow-Anacker, A. J., Druckenbrod, N. R., Epstein, M. L., Gosain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+cholinergic+myenteric+neurons+during+enteric+nervous+system+development:+comparison+of+different+ChAT+fluorescent+mouse+reporter+lines.">Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.</a> Neurogastroenterol Motil. 26 (6), 874-884 (2014).</li><li>Teitelbaum, D. H., Caniano, D. A., Qualman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+Hirschsprung's-associated+enterocolitis:+importance+of+histologic+correlates.">The pathophysiology of Hirschsprung's-associated enterocolitis: importance of histologic correlates.</a> J Pediatr Surg. 24 (12), 1271-1277 (1989).</li><li>Aslam, A., Spicer, R. D., Corfield, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Children+with+Hirschsprung's+disease+have+an+abnormal+colonic+mucus+defensive+barrier+independent+of+the+bowel+innervation+status.">Children with Hirschsprung's disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status.</a> J Pediatr Surg. 32 (8), 1206-1210 (1997).</li><li>Puig, I., Champeval, D., De Santa Barbara, P., Jaubert, F., Lyonnet, S., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Pten+in+the+mouse+enteric+nervous+system+induces+ganglioneuromatosis+and+mimics+intestinal+pseudoobstruction.">Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction.</a> J Clin Invest. 119 (12), 3586-3596 (2009).</li></ol>

The authors have nothing to disclose.

Nuestro laboratorio y otros han demostrado que los defectos intestinales en HSCR no se limitan al colon aganglionar pero extenderse proximalmente, incluso en el intestino delgado ganglionar 5,15,16. Estas alteraciones incluyen cambios en la densidad y ENS fenotipo neuronal neurotransmisor y puede dar cuenta de dismotilidad que se ha observado en pacientes con HSCR. Hemos utilizado las técnicas anteriores en nuestros esfuerzos para comprender los determinantes de la formación de la ENS. En concreto, estas t?...

Un funcionamiento del sistema nervioso entérico (ENS), que controla la motilidad, la absorción de nutrientes, y el flujo sanguíneo local, es esencial para la vida 1. El ENS está formado por células de la cresta neural (NCC) que proliferan, migran y colonizar el intestino, donde se diferencian en neuronas de los ganglios que contiene y células gliales. Enfermedad de Hirschsprung (herencia mendeliana HSCR, en línea en el hombre), un trastorno congénito multigeneic con una incidencia de 1 en 4.000 nacidos vivos, se puede considerar la enfermedad prototípico para el estudio de la formación de ENS interrumpido. En HSCR, NCC dejan de migrar y colonizar a longitudes variables del intestino posterior distal 2. Además, otra gastrointestinal común (GI) defectos en el desarrollo de la población pediátrica, como malformaciones anorrectales, atresias intestinales y trastornos de la motilidad se asocian con alteraciones en las funciones básicas de la ENS, y es probable que, asociados con cambios anatómicos subestimados sutiles y cambios funcionales enel 3-6 ENS. Por lo tanto, las técnicas que nos permitan comprender los determinantes del desarrollo de la formación ENS pueden arrojar luz sobre la patogénesis y el tratamiento potencial de trastornos del tracto GI pediátricos. A raíz de la migración y colonización, NCC se diferencia en neuronas con marcadores específicos para su fenotipo neurotransmisor. Las neuronas colinérgicas representan aproximadamente el 60% de las neuronas entéricas 7, y pueden ser detectados por tinción para la colina acetiltransferasa (ChAT), la enzima sintetizadora de la acetilcolina del neurotransmisor excitatorio. Históricamente, los intentos de visualizar las neuronas colinérgicas fueron confundidos por los diferentes antígenos especificidad de anticuerpos dirigidos contra el sistema nervioso central (SNC) Chat contra el sistema nervioso periférico (SNP) Chat 8-10. Sin embargo, los anticuerpos dirigidos contra ChAT placentaria reconocer tanto ChAT central y periférico 11-13, y tenemos técnicas recientemente descritos que permiten visualización de las neuronas colinérgicas ENS con alta sensibilidad anteriormente en el desarrollo que se ha logrado con líneas reportero ChAT 14. A continuación, presentamos una técnica de disección, la fijación y la inmunotinción de la murino tubo digestivo embrionario visualizar ENS expresión neurotransmisor en las neuronas. Para estos estudios, hemos utilizado ratones chat-Cre se aparearon con R26R: animales tdTomato para producir chat-Cre; R26R:: floxSTOP ratones tdTomato (definidos como Chat-Cre tdTomato todo el manuscrito): floxSTOP. Estos animales fueron entonces aparearon con ratones reportero GFP-ChAT homocigotos, para obtener ratones que expresan ambos reporteros fluorescentes que detectan la expresión de ChAT 14. Estos dos animales reportero están en un fondo C57BL / 6J y están disponibles comercialmente (Jackson Laboratories, Bar Harbor, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining to visualize murine enteric nervous system development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Hemos descrito previamente la generación de ratones que expresan tanto las buenas prácticas agrarias y tdTomato reporteros fluorescentes que detectan la expresión de ChAT 14. En pocas palabras, los ratones chat-Cre se aparearon con R26R: floxSTOP: animales tdTomato para producir chat-Cre; R26R: ratones tdTomato (llamados chat-Cre tdTomato): floxSTOP. Estos animales fueron entonces aparearon con ratones reportero GFP-ChAT homocigotos. Se aislaro...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

Immunostaining to Visualize Murine Enteric Nervous System Development | Developmental Biology | Video

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

La inmunotinción para visualizar murino entérica Desarrollo del Sistema Nervioso

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

immunostaining-to-visualize-murine-enteric-nervous-system-development

Research

Education

JoVE Journal

JoVE Core

Biology

Developmental Biology

Human Biology

Nervous System

SCIENTISTS IN ACTION

11.3K vistas.  University of Wisconsin School of Medicine and Public Health. The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Video: Immunostaining to Visualize Murine Enteric Nervous System Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimizado<em&gt; Ex-ovo</em&gt; El cultivo de embriones del polluelo a etapas avanzadas de desarrollo

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Investigación

Biología del Desarrollo

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Utilización de hojas de luz de microscopía de fluorescencia a la imagen de ojo de pez cebra Desarrollo

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Una nueva técnica de trasplante de grasa mamaria para visualizar la generación de vasos de células madre endoteliales vasculares

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

La proyección de imagen de lapso tiempo multi-Photon para visualizar el desarrollo en tiempo real: visualización de la migración de las células de cresta Neural en embriones de pez cebra

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Cultura Organotypic método para estudiar el desarrollo de tejidos de embrión de pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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

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

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

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

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

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

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Cultivo de tejido ovárico para visualizar fenómenos en ovario de ratón

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

Visualizar Drosophila pierna axones de la neurona de Motor a través de la cutícula del adulto

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Sistema de ensayo de angiogénesis tridimensional utilizando esferoides de cocultura formados por células formadoras de colonias endoteliales y células madre mesenquimales

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Hibridación en situ de montaje completo en embriones de pez cebra y ensayo de formación de tubos en iPSC-CE para estudiar el papel de la endoglina en el desarrollo vascular

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...