Name: dissection and culture of mouse embryonic kidney
Uploaded: 2017-05-17T15:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

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. P...

<ol>
	<li>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=.">.</a> Organogenesis of the kidney. Developmental and Cell Biology Series. 19, (1987).</li><li>Lindstr&#246;m, N. 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>

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 ...

The overall goals of this procedure are to isolate and culture metanephric rudiments from mouse embryos and to observe the effects of specific treatments on ureteric bud branching morphology. This method can help answer key questions in kidney organogenesis about the control of tubular branching morphogenesis, about epithelial mesenchyme interactions, and about the origin of congenital malformations. The main advantages with this technique are that it facilitates direct access to the organ;that it is simple, inexpensive, and easily modified.Before beginning the dissection, use sterile forceps to carefully place one polycarbonate membrane filter with a five-micrometer pore size into each well of a four-well plate containing 500 microliters of fresh culture medium. Transfer the plate into a cell culture incubator. Then use a razor blade to cut approximately 30 100-microliter micropipette tips approximately one millimeter from the tip to widen the tip openings and return the tips to the pipette tip rack.When the tips are ready, place an embryonic day 11 1/2 embryo in PBS under a dissecting microscope. Use a fresh dish for each embryo. And with number five watchmaker forceps, cut the embryo directly under the forelegs.Next, hold the legs with one pair of forceps using a second forceps to remove the leg tissue. Remove the tail in the same manner, and use the tips of the forceps to laterally and superficially open the body wall of the embryo in a rostral and caudal direction parallel to the dorsal aorta. Cutting superficially, make an incision along the dorsal aorta and use the tips of the closed forceps to carefully separate the ventral part of the body wall from the dorsal body wall.Flip the embryo over and cut the body wall laterally in a rostral-to-caudal direction using the dorsal aorta for orientation as just demonstrated. Once the ventral body wall has been separated from the dorsal body, grasp the ventral body wall with the forceps, and carefully pull to remove the wall and the ventral organ mass. Holding the dorsal body wall with one forceps with the ventral side facing up, use the other forceps to grasp the dorsal aorta at its rostral end and carefully peel the dorsal aorta from the dorsal body wall.Using one forceps, cut rostrally to the kidney anlagen to remove the aorta. And use the tips of the forceps to carefully separate the two anlagen. Use the tips of the forceps to carefully peel the unwanted tissue from the kidney anlagen.And use a microliter pipette and a modified pipette tip to transfer one anlagen and 10 microliters of PBS into the center of one half of a single polycarbonate membrane filter in the four-well plate. Then return the plate to the incubator until the metanephric rudiments from the next embryo are ready to be placed on the filter. After three days at 37 degrees Celsius and 5%carbon dioxide, use a micropipette to carefully replace the culture medium in each well with 500 microliters of 4%paraformaldehyde and PBS dropwise to submerge the filters.After 30 minutes at room temperature, remove the fixative and rinse the filters two times with 500 microliters of PBS submerging the filters with each wash. Then add 500 microliters of permeabilization solution to each well for a one-hour incubation at room temperature. At the end of the permeabilization incubation, rinse the filters two times as just demonstrated, and add 500 microliters of blocking solution to each well, After one hour at room temperature, replace the blocking solution with 250 microliters of biotinylated Dolichos biflorus agglutinin and blocking solution, and place the tissues at four degrees Celsius for 24 hours.The next day, wash the filters two times with 500 microliters of PBS per well. And label the tissue with 250 microliters of Alexa 488 conjugated streptavidid and blocking solution for 24 hours at four-degree Celsius. The next day, wash the tissues two times with 500 microliters of PBS per well and use forceps to transfer the filters to prepared glass slides with the explants facing up.Mount the explants with 50 to 100 microliters of embedding medium and cover the tissues with 60-millimeter long number 1.5 coverslips. Then allow the embedding medium to solidify for two hours at room temperature in the dark, and image the metanephric kidney samples. After three days of culture, the ureteric bud of the metanephric kidney anlagen branches up to five times, resulting in a ramification of the initially T-shaped ureteric bud.In the presence of clustered ephrin-B2, the branching complexity has decreased, as compared to control explants incubated with clustered human Fc.Indeed, ureteric bud branching in the majority of the ephrin-B2 treated explants, does not exceed third generation. And the fourth generation is reached in only 8%of the treated explants. Accordingly, the number of endpoints per branch and endpoints per millimeter squared is reduced in the explants treated with clustered ephrin-B2.Further, a third of the explants demonstrate an unusual morphology in the ureteric bud tips, suggesting that ephrin-B2 might have a restricting effect on ureteric bud branching process, most likely by activating FA4 and FB2 forward signaling. Any injury of the mesenchyme decreases the inductive potential leading to a reduced or absent ureteric bud branching. For example, in this explant with an almost-missing mesenchyme, the ureteric bud did not branch beyond the T stage.In this experiment, the metanephric mesenchyme was damaged, leading to poor growth and branching. After it's development in the 1960s, this technique paved the way for researchers in the field of developmental biology to explore kidney organogenesis. After watching this video, you should have a good understanding of how to isolate and culture metanephric rudiments from E11.5 mouse embryo.Once mastered, the dissection can be completed in less than five minutes if performed properly.

Research

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