Les auteurs remercient Leif Oxburgh et Derek Adams de partager généreusement leurs connaissances, Leif Oxburgh pour les commentaires utiles sur le manuscrit, et Stefan Wölfl et Ulrike Müller pour leur soutien technique et Saskia Schmitteckert, Julia Gobbert, Sascha Weyer et Viola Mayer pour l&#39;aide dans le laboratoire. Ce travail a été soutenu par Development, The Company of Biologists (to CP).

Les souris ont été maintenues selon la réglementation suédoise et la législation de l&#39;Union européenne (2010/63 / UE). Toutes les procédures ont été effectuées selon les directives du Comité suédois d&#39;éthique (permis C79 / 9, C248 / 11 et C135 / 14). Les procédures à l&#39;Université de Heidelberg portant sur des matières animales ont été approuvées par le Regierungspräsidium Karlsruhe et les agents de protection des animaux à l&#39;Université de Heidelberg. 1. Préparation des réactifs et des matériaux pour la culture REMARQUE: Utilisez un capot à flux laminaire pour minimiser la contamination. <ol><li> Le jour de la dissection, la protéine d&#39;éphrine chimère recombinante Fc seule ou recombinante est fusionnée à Fc humaine en la mélangeant avec des anticorps contre la chèvre Fc anti-humains à un rapport molaire de 1: 5 dans une solution saline stérile tamponnée au phosphate (PBS). Incuber pendant 1 h à 37 ° C 5 . </li><li> Préparer le milieu de culture en complétant l&#39;Eagle Eagle modifié par Dulbecco: NutrientMélange F-12 (DMEM / F12) avec 1% de pénicilline / streptomycine (v / v), 1% de glutamine (v / v), 1% d&#39;insuline-transferrine-sélénium (v / v), 50 μg / mL d&#39;holo- La transferrine et 1,5 μg / ml d&#39;amphotéricine. REMARQUE: 10 ml de milieu sont suffisants pour 16 embryons. </li><li> Ajouter une solution d&#39;éphrine recombinante en grappe à une concentration finale de 20 μg / ml sur le milieu de culture. Utilisez le Fc humain en cluster comme contrôle. </li><li> Préparer des plaques de culture en ajoutant 500 μl de milieu de culture à chaque puits d&#39;une plaque stérile, non revêtue, à base de polystyrène à fond plat et à 4 puits. </li><li> En utilisant des pinces stériles, placez soigneusement un filtre à membrane en polycarbonate avec une taille de pore de 5 μm par puits sur le dessus du milieu. Transférer la plaque préparée à l&#39;incubateur (37 ° C / 5% de CO 2 ). Assurez-vous que le filtre reste sec sur la surface supérieure et les flotteurs. REMARQUE: Un filtre suffit pour deux anilènes rénaux. </li><li> En utilisant une lame de rasoir, couper environ 30 pointes de pipette (volume: 100 μL) approximatelY 1 mm de la pointe pour aboutir à une ouverture plus large. Placez les embouts dans une crémaillère à pipette. </li></ol> 2. Dissection de Rudiments Metanephriques à E11.5 <ol><li> Sacrifiez une souris enceinte chronométrée à E11.5 par dislocation cervicale (ou utilisez une méthode approuvée par le comité local d&#39;éthique). </li><li> Supprimer l&#39;utérus et les embryons, comme décrit par Brown et al 6 . Sur ce point, travailler sous un microscope à dissection. </li><li> En utilisant la pince à horlogerie n ° 5, couper l&#39;embryon directement sous les pattes antérieures. Utilisez un plat de Petri frais pour chaque embryon. Retirer les jambes et la queue de l&#39;embryon. Pour ce faire, prenez le tissu à enlever avec une paire de pince à horlogerie n ° 5 et utilisez les pointes d&#39;une deuxième paire pour couper. </li><li> Pour enlever la masse d&#39;organe ventrale, utilisez les pointes d&#39;une pince pour ouvrir latéralement la paroi du corps de l&#39;embryon dans une direction rostrale à caudale. <ol><li> Couper superficiellement, parallèlement à tL&#39;aorte dorsale. Utilisez l&#39;aorte dorsale remplie de sang et donc visible pour l&#39;orientation. Séparez soigneusement la partie ventrale de la paroi du corps de la partie dorsale en utilisant les pointes de la pince fermée et retournez l&#39;embryon. Coupez la paroi du corps latéralement dans une direction rostral à caudale et utilisez l&#39;aorte dorsale pour l&#39;orientation, comme pour le côté précédent. </li></ol></li><li> Une fois que la paroi du ventral a été séparée du corps dorsal, saisissez-la avec une paire de pince et tirez soigneusement pour l&#39;enlever, avec la masse de l&#39;organe. REMARQUE: l&#39;anilène métanéphrique reste habituellement attaché à la paroi du corps dorsal. Ce sont des structures ovales, de 200 μm de long situées au niveau des membres postérieurs. </li><li> Tenez la paroi du corps dorsal avec une paire de pince, avec le côté ventral vers le haut. À l&#39;aide de l&#39;autre pince, saisissez l&#39;aorte dorsale à son extrémité rostrale et épluchez soigneusement l&#39;aorte dorsale de la paroi du corps dorsal. NOTE: Les deux reins métasphériques restent habituellement attachésÀ l&#39;aorte dorsale. </li><li> À l&#39;aide d&#39;une paire de pinces, coupez vers le riz anlagen pour éliminer l&#39;aorte. Ensuite, séparez soigneusement les deux anols du rein en utilisant les pointes de la pince. </li><li> Éloignez soigneusement les tissus indésirables du rein anlagen en utilisant les pointes de la pince. Veillez à ne pas endommager le mésenchyme, car les dommages peuvent entraîner une mauvaise croissance et l&#39;exclusion de l&#39;explant à partir d&#39;analyses ultérieures. </li><li> Transférer le rein anlagen séparément à l&#39;aide d&#39;une pipette à microlitres et d&#39;une pointe de pipette large-ouverte dans 10 μL de PBS au filtre à membrane en polycarbonate dans une plaque à 4 puits. REMARQUE: En raison de la tension de surface, l&#39;explant sera recouvert d&#39;une mince couche de milieu. Un filtre suffit pour deux anilènes rénaux. <ol><li> Placez chacun des deux anilènes rénaux au centre de chaque moitié respective du filtre. Transférer la plaque à l&#39;incubateur à 37 ° C / 5% de CO 2 . Laisser la plaque dans l&#39;incubateur jusqu&#39;à ce que la rud metanephricLes imites du prochain embryon sont prêtes à être placées sur le filtre. </li></ol></li><li> Répétez les étapes 2.3-2.9.1 pour chaque embryon. REMARQUE: avec une certaine pratique, la procédure de dissection prend environ 5 min par embryon. </li><li> Incuber le rein anlagen pendant 3 jours à 37 ° C / 5% de CO 2 . </li></ol> 3. Préparation des réactifs pour la fixation et la coloration <ol><li> Pour préparer 4% de paraformaldéhyde (PFA) dans du PBS sans calcium et magnésium (p / v), dissoudre le PFA à 60 ° C dans du PBS, en agitant continuellement. Refroidissez à température ambiante et utilisez un filtre en papier pour enlever les particules restantes. Aliquote et conservez à 4 ° C pour une utilisation immédiate ou un gel pour un stockage à long terme. ATTENTION: le PFA est toxique. Portez l&#39;équipement de protection individuelle spécifié dans les normes de sécurité locales et travaillez dans une hotte aspirante. Jeter les déchets en suivant les directives institutionnelles et en utilisant les conteneurs de déchets désignés. </li><li> Pour préparer une solution de perméabilisation, diluer Triton X-100Dans du PBS sans calcium et magnésium à 0,3% (v / v). </li><li> Pour préparer une solution de blocage, ajouter 5% (v / v) de sérum de chèvre et 0,1% de Triton X-100 à PBS sans calcium et magnésium. Ajouter de l&#39;azoture de sodium à une concentration finale de 0,02% (p / v). Conserver à 4 ° C. ATTENTION: L&#39;azide de sodium est toxique. Manipuler l&#39;azide de sodium conformément à la réglementation locale sur la sécurité et la protection de l&#39;environnement. NOTE: Lorsque vous utilisez des anticorps pour la coloration, utilisez un sérum de 5% parmi les espèces dont l&#39;anticorps secondaire a été dérivé pour obtenir une solution de blocage. </li><li> Diluer l&#39;agglutinine biotinylée de Dolichorus biflorus 1: 200 dans la solution bloquant. Diluer la streptavidine conjuguée Alexa488 1: 200 dans la solution de blocage. </li><li> Pour préparer un milieu d&#39;incorporation prêt à l&#39;emploi (voir la table des matériaux), ajouter 0,1 g de mucoadhésif à base d&#39;alcool polyvinylique à l&#39;eau / ml à 25% (p / v) de glycerol dans du Tris-HCl 0,1 M (pH 8,5) . Chauffer à 50 ° C sous agitation continue pendant 1 h, refroidir et régler le pH à 8,0-8,5 en utilisant 1M NaOH. Évitez les concentrations de NaOH plus élevées. Ajouter 100 μg / mL de N-propylgallate et thimerosal à une concentration finale de 0,02% (p / v). Mélanger pendant 30 minutes à température ambiante. ATTENTION: Le thimerosal est toxique. Manipulez-le conformément à la réglementation locale sur la sécurité et la protection de l&#39;environnement. <ol><li> Remplissez le milieu d&#39;incorporation dans des tubes de 50 ml, équilibrez le rotor et centrifugez à 3,200 xg et la température ambiante pendant 10 min. Décrivez la solution transparente dans des tubes de 15 mL et jeter la pastille. Aliquotez et conservez à -20 ° C pendant plusieurs semaines. Une fois décongelée, maintenir la solution à 4 ° C. Chaud à ~ 30 ° C immédiatement avant utilisation. </li></ol></li><li> Préparez autant de diapositives en verre que les filtres avec des explants doivent être montés. Pour cela, collez deux petits lamelles, 18 x 18 mm, de chaque côté de la glissière en verre à l&#39;aide d&#39;un adhésif instantané, laissez un espace de ~ 15 mm pour le filtre entre les deux. </li></ol> 4. Fixation et coloration <ol><li> Après une période de culture de 3Jours in vitro (div), retirer les plaques de l&#39;incubateur et aspirer soigneusement le milieu de culture de chaque puits à l&#39;aide d&#39;une micropipette. Assurez-vous de ne pas toucher le filtre et de garder la pointe en direction de la paroi du puits. </li><li> Ajouter 500 μL de PFA à 4% / PBS à chaque puits. Les filtres flottent sur le fixateur. À l&#39;aide d&#39;une micropipette, ajoutez soigneusement le fixateur PFA pour immerger les filtres. Incuber à température ambiante pendant 30 min. REMARQUE: Pour toutes les étapes suivantes, il faut prendre soin de ne pas laver les explants du filtre. Gardez l&#39;ouverture de la pointe de la pipette vers le mur du puits. </li><li> Retirez le PFA et rincez deux fois avec 500 μL de PBS, en immergeant le filtre. Ajouter 500 μL de solution de perméabilisation à chaque puits et incuber à température ambiante pendant 1 h. </li><li> Rincer deux fois avec 500 μL de PBS et ajouter 500 μl de solution bloquant à chaque puits. Incuber à température ambiante pendant 1 heure. </li><li> Remplacez la solution de blocage par250 μL d&#39;agglutinine biotinylée de Dolichorus biflorus diluée à 1: 200 dans une solution bloquant et incubée à 4 ° C pendant 24 h. </li><li> Enlever la solution d&#39;agglutinine Dolichorus biflorus et rincer deux fois avec 500 μL de PBS par puits. </li><li> Ajouter 250 μL de streptavidine conjuguée avec Alexa488 dilué 1: 200 dans la solution de blocage et incuber à 4 ° C pendant 24 h. Alternativement, incuber à température ambiante pendant 2 h. REMARQUE: Un meilleur rapport signal / bruit est obtenu lorsque l&#39;on incube à 4 ° C. </li><li> Rincer deux fois avec 500 μL de PBS par puits. En utilisant une pince, transférez les filtres sur les lames de verre préparées, avec les explants vers le haut. Monter avec ~ 50-100 μL de support d&#39;encastrement. Couvrir avec des lamelles de 60 mm de long n ° 1.5. Laisser la solution du milieu d&#39;incorporation se solidifier pendant 2 h à température ambiante dans l&#39;obscurité. Procédez à l&#39;imagerie ou rangez les diapositives, emballées dans du papier d&#39;aluminium, à 4 ° C jusqu&#39;à ce qu&#39;elles soient prêtes à l&#39;image. </li><li> Image avec un microscope épifluorescent large champ 7 &lt;/ Sup&gt; couplé à une lampe Hg et utiliser une unité miroir pour l&#39;excitation bleue (passe-bande d&#39;excitation, 460 - 495 nm, miroir dichromatique, 505 nm et bande passante d&#39;émission, 510 à 550 nm) et 20X lentilles Plan-Apochromat, 0,75 numériques ouverture. </li></ol>

<ol>
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O., 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=Integrated+&#946;-catenin,+BMP,+PTEN,+and+Notch+signalling+patterns+the+nephron.">Integrated &#946;-catenin, BMP, PTEN, and Notch signalling patterns the nephron.</a> eLife. 4, e04000 (2015).</li><li>Peuckert, C., 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=Multimodal+Eph/Ephrin+signaling+controls+several+phases+of+urogenital+development.">Multimodal Eph/Ephrin signaling controls several phases of urogenital development.</a> Kidney Int. 90 (2), 373-388 (2016).</li><li>Pasquale, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph+receptor+signalling+casts+a+wide+net+on+cell+behaviour.">Eph receptor signalling casts a wide net on cell behaviour.</a> Nat Rev Mol Cell Biol. 6 (6), 462-475 (2005).</li><li>Bonanomi, D., 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=Ret+Is+a+Multifunctional+Coreceptor+that+Integrates+Diffusible-+and+Contact-Axon+Guidance+Signals.">Ret Is a Multifunctional Coreceptor that Integrates Diffusible- and Contact-Axon Guidance Signals.</a> Cell. 148 (2), 568-582 (2012).</li><li>Brown, A. C., 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=Isolation+and+Culture+of+Cells+from+the+Nephrogenic+Zone+of+the+Embryonic+Mouse+Kidney.">Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney.</a> J Vis Exp. (50), e2555 (2011).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+interaction+in+the+development+of+the+mouse+metanephros.">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li><li>Sax&#233;n, L., Toivonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary Embryonic Induction. , (1962).</li><li>Sax&#233;n, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system.">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li><li>Dudley, A. T., Godin, R. E., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme.">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li><li>Per&#228;l&#228;, N., 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=Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney.">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li><li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+branching+morphogenesis,+growth+and+differentiation+of+the+embryonic+kidney.">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li><li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+ureteric+bud+branching+morphogenesis.">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li><li>Sebinger, D. D. R., Unbekandt, M., Ganeva, V. V., Ofenbauer, A., Werner, C., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel,+Low-Volume+Method+for+Organ+Culture+of+Embryonic+Kidneys+That+Allows+Development+of+Cortico-Medullary+Anatomical+Organization.">A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization.</a> PLoS One. 5 (5), e10550 (2010).</li><li>Ekblom, P., Miettinen, A., Virtanen, I., Wahlstr&#246;m, T., Dawnay, A., Sax&#233;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+segregation+of+the+metanephric+nephron.">In vitro segregation of the metanephric nephron.</a> Dev Biol. 84 (1), 88-95 (1981).</li><li>Davies, J. A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture.">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li><li>Sax&#233;n, L., Lehtonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+kidney+in+organ+culture.">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li><li>Bard, J. B. 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+development+of+the+mouse+kidney+embryogenesis+writ+small.">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li><li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments.">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li><li>Batourina, 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=Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret.">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li></ol>

Les auteurs n&#39;ont rien à dévoiler.

Ce manuscrit décrit une méthode pour isoler l&#39;anlagen méthanephrique en développement de l&#39;embryon de souris et pour cultiver les rudiments d&#39;organes. Cette méthode est une technique standard, développée par Grobstein 8 et Saxén 9 , 10 , et a été adaptée et modifiée par beaucoup d&#39;autres 11 , 12 . Le succès de la méthode dépend principalement de la d...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040117</td>
			<td>use for dissection</td>
		</tr>
		<tr>
			<td>holo-Transferrin human</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>Sigma-Aldrich</td>
			<td>T5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Dolichorus Biflorus Agglutinin</td>
			<td>Vector Laboratories</td>
			<td>B-1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 conjugated Streptavidin</td>
			<td>Jackson Immuno Research</td>
			<td>016-540-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>496-EB</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IgG1 Fc, CF</td>
			<td>R&#38;D Systems</td>
			<td>110-HG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Human IgG Fc Antibody</td>
			<td>R&#38;D Systems</td>
			<td>G-102-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>use for fixation and immunostaining</td>
		</tr>
		<tr>
			<td>Dumont #5, biologie 
			tips, INOX, 11cm</td>
			<td>agnthos.se</td>
			<td>0208-5-PS</td>
			<td>2 pairs of forceps are needed</td>
		</tr>
		<tr>
			<td>Iris scissors, straight, 12cm</td>
			<td>agnthos.se</td>
			<td>03-320-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps, 
			straight, delicate, 13cm</td>
			<td>agnthos.se</td>
			<td>08-032-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes Nunclo Delta treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>150679</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &#181;m, 13 mm, white, plain</td>
			<td>MerckMillipore</td>
			<td>TMTP01300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon Multidishes 
			4 wells, flat bottom</td>
			<td>Sigma-Aldrich</td>
			<td>D6789-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm</td>
			<td>nordicbiolabs</td>
			<td>107222</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses No.1.5, 18x18mm</td>
			<td>nordicbiolabs</td>
			<td>102032</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides ~76x26x1, 1/2-w. ground plain</td>
			<td>nordicbiolabs</td>
			<td>1030418</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430055</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1213125</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed stage research mircoscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice, male, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice,female, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Greenough Stereo Microscope</td>
			<td>Leica</td>
			<td>Leica S6 E</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and culture of mouse embryonic kidney

Le but de ce protocole est de décrire une méthode de dissection, d&#39;isolement et de culture des rudiments métanéphriques de souris. Au cours du développement du rein des mammifères, les deux tissus progéniteurs, le bourgeon urétéral et le mésenchyme métasphorique, communiquent et induisent réciproquement des mécanismes cellulaires pour former éventuellement le système collecteur et les néphrons du rein. Au fur et à mesure que les embryons de mammifères se développent par voie intra-utérine et qui sont inaccessibles à l&#39;observateur, une culture d&#39;organe a été développée. Avec cette méthode, il est possible d&#39;étudier les interactions épithéliales-mésenchymateuses et le comportement cellulaire pendant l&#39;organogenèse rénale. En outre, l&#39;origine des malformations rénales congénitales et des voies urogénitales peut être étudiée. Après une dissection minutieuse, les rudiments métanéphriques sont transférés sur un filtre qui flotte sur un milieu de culture et peuvent être conservés dans un incubateur de culture cellulaire pendant plusieurs jours. Cependant, il faut savoir que les conditions sontArtificielle et pourrait influencer le métabolisme dans le tissu. En outre, la pénétration des substances d&#39;essai pourrait être limitée en raison de la matrice extracellulaire et de la membrane basale présents dans l&#39;explant. Un des principaux avantages de la culture des organes est que l&#39;expérimentateur peut avoir un accès direct à l&#39;organe. Cette technologie est peu coûteuse, simple et permet un grand nombre de modifications, telles que l&#39;ajout de substances biologiquement actives, l&#39;étude des variantes génétiques et l&#39;application de techniques avancées d&#39;imagerie.

L&#39;anlagen rénale métasphorique a été dérivé de souris enceintes enceintes Black-6 chez E11.5 et ont été cultivées. Après 3 jours, le bourgeon urétéral s&#39;est ramifié jusqu&#39;à 5 fois, entraînant une ramification du bourrelet urétéral initialement en forme de T. Chaque explant a été photographié et les nombres de segments et de points d&#39;extrémité ont été quantifiés pour déterminer les générations de ramifications et pour calculer le nombre de point...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

Ce protocole décrit une méthode pour isoler et cultiver des rudiments métasphoriques à partir d&#39;embryons de souris.

Dissection et culture du rein embryonnaire de la souris

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

11.8K vues.  Uppsala University.  Ce protocole décrit une méthode pour isoler et cultiver des rudiments métasphoriques à partir d'embryons de souris. 

Vidéo: Dissection and Culture of Mouse Embryonic Kidney

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

À grande échelle de poisson zèbre embryonnaire Coeur Dissection d&#39;analyse transcriptionnelle

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Recherche

Biologie du développement

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Culture de souris embryonnaires cochléaires explants et Gene Transfer par électroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Culture et co-culture de souris ovaires et follicules ovariens

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Différenciation neurale des souris de cellules souches embryonnaires dans le sérum sans culture monocouche

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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

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

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

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

Transcriptome profilage des<em&gt; In-Vivo</em&gt; embryons de bovins pré-implantation à l&#39;aide de deux couleurs Microarray Plate-forme

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

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Culture de murines embryonnaires Métatarsiens: un modèle physiologique de endochondrale ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Dissection et préparation de tranches coronales de développer la glande pituitaire souris

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Dissection et la coloration des Drosophila ovaires Pupal

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

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 ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Dissection, culture et analyse des cellules neurales primaires de crête neurale de la souris pour l'étude de la délamination et de la migration neurales de cellules de crête

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...