Ce travail a été soutenu PAR LE American Pediatric Surgical Association Foundation Award (AG) et les National Institutes of Health K08DK098271 (AG).

L&#39;Université du Wisconsin protection des animaux et l&#39;utilisation Comité a approuvé toutes les procédures. 1. Préparation des solutions <ol><li> Utilisez phosphate 1x saline tamponnée (PBS) comme tampon de dissection et la solution de rinçage. </li><li> Préparer 30% de saccharose en pesant 30 g de saccharose et le lieu dans une bouteille. Ajouter 99 ml de 1 x PBS et ajouter de l&#39;azide de sodium à 1 ml de 10%. Mélanger soigneusement jusqu&#39;à ce que le saccharose est dissous. Conserver à 4 ° C jusqu&#39;à ce que nécessaire. </li><li> Préparer la solution de blocage par mélange PBS 1x, 3% d&#39;albumine de sérum bovin (BSA) et 0,1% de Triton-X-100. Mélanger soigneusement et stocker dans le réfrigérateur jusqu&#39;à ce que nécessaire. </li><li> Préparer 8% de paraformaldéhyde (PFA) solution dans PBS 1x par pesée de la quantité appropriée de PFA en 1x PBS puis incuber à 65 ° C jusqu&#39;à ce qu&#39;il soit complètement dissous. Conserver dans 25 ml aliquotes à -20 ° C congélateur jusqu&#39;à ce que nécessaire. Diluer à 4% de PFA dans du PBS 1x, le jour de l&#39;utilisation. </li></ol> 2. embryon et Gut Dissection <ol><li> Conformément aux institutionnels de protection des animaux et l&#39;utilisation Comité protocoles approuvés, euthanasier la souris enceinte chronométré et transférer l&#39;utérus dans un 60 mm boîte de Petri contenant de la glace froide 1x PBS. </li><li> Sous un microscope de dissection à l&#39;aide des ciseaux pointus, couper la paroi utérine ouvert pour exposer les embryons. Retirez les embryons de l&#39;utérus et le lieu dans une boîte de 60 mm propre Petri contenant de la glace froide 1x PBS. </li><li> Euthanasier chaque embryon par décapitation dans glacée 1x PBS. Si vous utilisez des souris transgéniques contenant des protéines fluorescentes, sous l&#39;éclairage de fluorescence, d&#39;identifier les embryons transgéniques positives. </li><li> On dissèque le tractus gastro-intestinal de l&#39;embryon en utilisant un microscope de dissection. En utilisant des pinces fines, orienter l&#39;embryon de telle sorte que le côté gauche est orientée vers le haut et le côté droit est contre le fond de la boîte de Pétri. Retirer la paroi supérieure du corps de l&#39;embryon pour exposer les organes internes. Insérez une pince fine entre la paroi dorsale du corps et les organes internes. Cross forceps contre l&#39;autre dans une action de coupe ciseaux pour enlever les organes internes de l&#39;embryon. </li><li> En outre sous-disséquer le tractus GI loin des organes environnants, puis placer chaque tube digestif dans un tube de 1,5 ml contenant glacée 1x PBS. </li></ol> 3. Fixation des GI Tracts <ol><li> Rincer chaque tractus GI 3 fois avec de la glace froide 1x PBS puis remplacer par 4% de PFA. Fixer les secteurs de GI dans les 1,5 ml microtubes sur une plate-forme à bascule à la température ambiante pendant 1,5 heures. Rincer les voies gastro-intestinaux 3 fois pendant 5 min à température ambiante puis pendant 1 heure sur la plate-forme à bascule. A ce stade, stocker les voies gastro-intestinales à 4 ° C dans 30% de saccharose dans 1 x PBS contenant de l&#39;azide de sodium à 0,1% jusqu&#39;à ce que nécessaire. NOTE: Vous pouvez également stocker les embryons dans 30% de saccharose jusqu&#39;à un an sans aucun effet sur l&#39;intégrité des tissus. Stockage des échantillons dans 30% de saccharose permet un traitement ultérieur des échantillons soit pour un marquage ou dans octobre pour cryo-SECTIONIng. Sinon, continuez avec le protocole d&#39;immunocoloration détaillé ci-dessous. </li></ol> 4. Protocole Immunocoloration <ol><li> Si les échantillons ont été stockés dans 30% de saccharose, les rincer trois fois pendant 20 minutes dans 1 x PBS sur une plate-forme à bascule. </li><li> Placez les voies gastro-intestinales dans la solution de blocage sur une plate-forme à bascule pendant 1 h à température ambiante. </li><li> Retirer la solution de blocage et incuber les voies gastro-intestinaux avec la quantité appropriée d&#39;anticorps primaires dilués dans une solution de blocage soit pour 4 heures à température ambiante ou O / N à 4 ° C sur une plateforme à bascule. Utilisez 1: 1000 dilution de l&#39;anticorps anti-Hu humaine (sérum obtenu à partir du patient), 1: 1000 dilution de poulet anti-anticorps protéine fluorescente verte (GFP) et 1: 100 dilution des anticorps de chèvre anti-ChAT. REMARQUE: Nous utilisons un anticorps anti-Hu humain qui a été obtenue localement à partir d&#39;un patient, cependant, les anticorps anti-Hu sont disponibles dans le commerce, par exemple, souris anti-Hu, (utilisation à dilution 1: 500). </li><li> Rincer les voies gastro-intestinales en 1x PBSTrois fois pendant 5 min, puis pendant une heure à température ambiante sur un agitateur oscillant. </li><li> Remplacez le 1x PBS avec des anticorps secondaires dilué 1: 500 dans une solution de blocage sur une plate-forme à bascule pour soit 4 heures à température ambiante ou O / N à 4 ° C. Utilisez âne colorant amine réactif anti-humain comme DyLight, Cy2 anti-poulet âne et d&#39;âne anti-chèvre Cy5. Si l&#39;on utilise l&#39;anticorps anti-souris Hu alors utiliser une dilution 1: 500 d&#39;âne anti-souris-amine réactif colorant 405. NOTE: L&#39;intensité de la GFP endogène et l&#39;expression tdTomato et la clarté de immunocoloration augmentation avec l&#39;âge de développement. Nous typiquement immunocoloration cran embryonnaires individuellement dans des tubes de 0,2 ml avec 150 ul de solution de coloration afin de réduire le volume de l&#39;anticorps utilisé et également efficace pour assurer la coloration du tissu. </li><li> Rincer les voies gastro-intestinales dans du PBS 1X 3 fois pendant 5 min, puis pendant 1 heure à température ambiante sur une plate-forme à bascule. </li><li> Placez quelques gouttes de milieu de montage à fluorescence DAPI sur une lame de verre. Plonger les tra IGct dans le moyen de montage de fluorescence et ajouter un verre de protection directement au-dessus du tissu. La fluorescence moyenne de montage -G est un composé soluble dans l&#39;eau qui fournit un joint d&#39;étanchéité semi-permanent. REMARQUE: Utilisez fluorescence milieu de montage tels que Vectashield qui est un milieu à base de glycérol qui empêche la décoloration et photoblanchiment d&#39;anticorps de montage. Ces deux produits permettent tissus à être stockés pendant de longues périodes de temps à 4 ° C. </li><li> Capturer des images de chaque fluorophore de l&#39;aide d&#39;un microscope confocal. Utilisez des longueurs d&#39;ondes d&#39;excitation pour les fluorophores et les filtres utilisés présentés dans le Tableau 3. </li><li> Effectuer l&#39;analyse d&#39;images assistée par ordinateur en utilisant le logiciel approprié selon le type de confocal utilisé pour capturer des images. </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.

Notre laboratoire et d&#39;autres ont montré que les défauts intestinales dans HSCR ne sont pas limitées au côlon aganglionnaire mais étendues proximale, même dans l&#39;intestin grêle ganglionated 5,15,16. Ces modifications incluent des changements dans la densité et ENS neurotransmetteur neuronale phénotype et peuvent expliquer dysmotility qui a été observé chez les patients avec HSCR. Nous avons utilisé les techniques ci-dessus dans nos efforts pour comprendre les déterminants de la formation...

Un fonctionnement du système nerveux entérique (SNE), qui contrôle la motilité, l&#39;absorption des nutriments, et la circulation sanguine locale, est essentiel à la vie 1. L&#39;ENS est formé par les cellules de la crête neurale (CCN) qui prolifèrent, migrent et colonisent l&#39;intestin, où ils se différencient en neurones des noyaux contenant et les cellules gliales. Maladie de Hirschsprung (Héritage de mendélienne HSCR, en ligne chez l&#39;homme), un trouble congénital multigeneic avec une incidence de 1 sur 4000 naissances vivantes, peut être considéré comme la maladie prototypique pour l&#39;étude de la formation ENS perturbé. Dans HSCR, CCN ne parviennent pas à migrer vers et de coloniser des longueurs variables de l&#39;intestin distal 2. En outre, d&#39;autres gastro-intestinales Fréquent (GI) les défauts de développement dans la population pédiatrique, tels que des malformations ano-rectales, atrésie intestinale et des troubles de la motilité sont associés à des perturbations dans les fonctions de base de l&#39;ENS, et sont susceptibles d&#39;être associées avec subtils, sous-estimés, les changements anatomiques et des changements fonctionnels dansl&#39;ENS 3-6. Par conséquent, les techniques qui nous permettent de comprendre les déterminants du développement de la formation ENS peuvent faire la lumière sur la pathogenèse et le traitement potentiel des troubles du tractus GI pédiatriques. Après la migration et de la colonisation, la CCN se différencie en neurones avec des marqueurs spécifiques de leur phénotype de neurotransmetteur. Les neurones cholinergiques comprennent environ 60% des neurones entériques 7, et peuvent être détectés par coloration de choline acétyltransférase (ChAT), l&#39;enzyme de synthèse de l&#39;acétylcholine, un neurotransmetteur excitateur. Historiquement, les tentatives de visualiser les neurones cholinergiques ont été confondus par les différentes spécificité antigénique des anticorps dirigés contre le système nerveux central (SNC) Chat contre le système nerveux périphérique (SNP) Tchat 8-10. Cependant, les anticorps dirigés contre placentaire ChAT reconnaître à la fois central et périphérique ChAT 11-13, et nous avons récemment techniques décrites qui permettent de visualisation des neurones cholinergiques ENS avec une grande sensibilité plus tôt dans le développement que ce qui a été réalisé avec des lignes ChAT rapporteurs 14. Ici, nous présentons une technique de dissection, fixation et immunologique de la GI embryonnaire murin voies de visualiser ENS neurotransmetteur expression dans les neurones. Pour ces études, nous avons utilisé des souris Chat-Cre accouplées avec R26R: animaux tdTomato pour produire Chat-Cre; R26R: floxSTOP souris tdTomato (définis comme CHAT-Cre tdTomato au long du manuscrit): floxSTOP. Ces animaux ont ensuite été accouplées avec des souris rapporteurs de Chat-GFP homozygotes, pour obtenir des souris exprimant les deux reporters fluorescents qui détectent expression ChAT 14. Ces deux animaux rapporteurs sont sur un fond C57BL / 6J et sont disponibles dans le commerce (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.

Nous avons déjà décrit la génération de souris exprimant les deux GFP et tdTomato journalistes fluorescentes qui détectent expression ChAT 14. Bref, des souris Chat-Cre ont été accouplés avec R26R: floxSTOP: animaux tdTomato pour produire Chat-Cre; R26R: souris tdTomato (appelés CHAT-Cre tdTomato): floxSTOP. Ces animaux ont ensuite été accouplées avec des souris rapporteurs de Chat-GFP homozygotes. Les embryons ont été isolés et les tissus ...

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

Immunocoloration pour visualiser le développement du système nerveux entérique murin | Biologie du développement | Vidéo

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.

Immunocoloration à Visualisez murin nerveux entérique développement du système

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 vues.  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.

Vidéo: 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).

Optimisé<em&gt; Ex-ovo</em&gt; Culture des embryons de poulet aux stades avancés de développement

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

Recherche

Biologie du développement

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.

Utilisation de feuilles Lumière Fluorescence Microscopy à l&#39;image du développement Zebrafish Eye

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.

Une nouvelle technique de transplantation de graisse mammaire mammaire pour visualiser la génération de vaisseaux de cellules souches endothéliales vasculaires

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.

Multiphotonique Time Lapse imagerie de visualiser le développement en temps réel : visualisation de la migration des cellules neurales de crête dans des embryons de poisson-zèbre

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.

Méthode de Culture organotypique pour étudier le développement des tissus embryonnaires de poulet

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.

Analyse de calcul de la lignée germinale de Caenorhabditis elegans pour étudier la Distribution des noyaux, les protéines et le cytosquelette

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.

Culture de tissus ovariens pour visualiser les phénomènes dans les ovaires de souris

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.

Visualiser les Drosophila jambe axones des motoneurones à travers la cuticule de l’adulte

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.

Système d'analyse d'angiogenèse tridimensionnel utilisant des sphéroïdes de co-culture formés par des cellules de formation de colonies endothéliales et des cellules souches mésenchymales

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.

Hybridation in situ à monture entière chez les embryons de poissons zèbres et l’essai de formation de tubes dans iPSC-ECs pour étudier le rôle de l’Endoglin dans le développement vasculaire

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