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

De auteurs bedanken Leif Oxburgh en Derek Adams om hun kennis, Leif Oxburgh, voor de handige opmerkingen over het manuscript te delen en Stefan Wölfl en Ulrike Müller voor hun technische ondersteuning en Saskia Schmitteckert, Julia Gobbert, Sascha Weyer en Viola Mayer voor hulp in de laboratorium. Dit werk werd ondersteund door Development, The Company of Biologists (aan CP).

Muizen werden gehandhaafd volgens de Zweedse regelgeving en de wetgeving van de Europese Unie (2010/63 / EU). Alle procedures werden uitgevoerd volgens de richtlijnen van het Zweedse Etiekkomitee (vergunningen C79 / 9, C248 / 11 en C135 / 14). Procedures bij de Universiteit van Heidelberg met betrekking tot diergeneeskunde zijn goedgekeurd door het Regierungspräsidium Karlsruhe en de dierenwelzijnsambtenaren aan de Universiteit van Heidelberg. 1. Bereiding van reagentia en materialen voor cul...

<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>

Dit manuscript beschrijft een methode om de ontwikkelende metanephrische anlagen van het muisembryo te isoleren en de orgaansrudimenten te cultiveren. Deze methode is een standaard techniek, zoals ontwikkeld door Grobstein 8 en Saxén 9 , 10 , en werd aangepast en aangepast door vele anderen 11 , 12 . Het succes van de methode hangt voornamelijk af van de duur van de dissectie, aa...

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

Het doel van dit protocol is om een ​​methode te beschrijven voor de dissectie, isolatie en cultuur van muismetanefrische rudimenten. Tijdens de ontwikkeling van zoogdierontwikkeling, communiceren de twee voorvaderweefsels, de ureterische knop en het metanefrische mesenchym, cellulaire mechanismen en vormen zij uiteindelijk het verzamelstelsel en de nefronen van de nier. Aangezien de embryo&#39;s van de zoogdier intracuterine groeien en derhalve ontoegankelijk zijn voor de waarnemer, is er een orgelcultuur ontwikkeld. Met deze methode is het mogelijk om epitheliale mesenchymale interacties en cellulaire gedragingen tijdens nierorganogenese te bestuderen. Bovendien kan de oorsprong van aangeboren nier- en urogenitale tract misvormingen worden onderzocht. Na zorgvuldige dissectie worden de metanefrische rudimenten overgebracht op een filter dat op cultuurmedium drijft en gedurende enkele dagen in een celcultuur-incubator kan worden gehouden. Men moet echter bewust zijn dat de voorwaarden zijnKunstmatig en kan het metabolisme in het weefsel beïnvloeden. Ook kan de penetratie van teststoffen beperkt worden door de extracellulaire matrix en het basale membraan aanwezig in de explant. Een belangrijk voordeel van orgelcultuur is dat de experimentator direct toegang tot het orgaan kan krijgen. Deze technologie is goedkoop, eenvoudig en maakt een groot aantal wijzigingen mogelijk, zoals de toevoeging van biologisch actieve stoffen, de studie van genetische varianten en de toepassing van geavanceerde beeldvormingstechnieken.

Metanephrische nier anlagen werden afgeleid van zwangere Black-6 inbloed muizen bij E11.5 en werden gekweekt. Na 3 dagen had de ureterische knop tot 5 maal vertakt, waardoor de initiële T-vormige ureterische knop werd verdubbeld. Elke explant werd gefotografeerd en de aantallen segmenten en eindpunten werden gekwantificeerd om de vertakkende generaties te bepalen en het aantal eindpunten per tak te berekenen ( Figuur 1 ). ImageJ ( rd generatie; de 4e</su...

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

Dissection and Culture of Mouse Embryonic Kidney

Dit protocol beschrijft een methode voor het isoleren en cultiveren van metanefrische rudimenten uit muisembryo&#39;s.

Dissectie en Cultuur van Muis Embryonale Nier

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

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