Name: mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development
Uploaded: 2014-09-04T14:00:00
Description: Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOTE: All mice were handled humanely using protocols approved by the University of Michigan Medical School Unit for Laboratory Animal Medicine and according to the guidelines of the University Committee on Use and Care of Animals.
1. Whole Organ Culture System
<ol>
	<li>Preparation of media and culture plates
	<ol>
		<li>In a tissue culture hood, remove 5 ml of BGJb media from the stock bottle and add 5 ml of Pen/Strep. Prepare a working stock of media in a 50 ml conical tube, by adding 1 ml...

<ol>
	<li>Grosse, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dynamics+in+fetal+intestinal+epithelium:+implications+for+intestinal+growth+and+morphogenesis.">Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis.</a> Development. 138, 4423-4432 (2011).</li><li>Walton, K. 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=Hedgehog-responsive+mesenchymal+clusters+direct+patterning+and+emergence+of+intestinal+villi.">Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.</a> Proc Natl Acad Sci U S A. 109, 15817-15822 (2012).</li><li>Wells, J. M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+endoderm+development.">Vertebrate endoderm development.</a> Annu Rev Cell Dev Biol. 15, 393-410 (1999).</li><li>Hashimoto, H., Ishikawa, H., Kusakabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+vascular+networks+during+the+morphogenesis+of+intestinal+villi+in+the+fetal+mouse.">Development of vascular networks during the morphogenesis of intestinal villi in the fetal mouse.</a> Kaibogaku zasshi Journal of anatomy. 74, 567-576 (1999).</li><li>Mathan, M., Moxey, P. C., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+fetal+rat+duodenal+villi.">Morphogenesis of fetal rat duodenal villi.</a> Am J Anat. 146, 73-92 (1976).</li><li>Burns, R. 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=Requirement+for+fibroblast+growth+factor+10+or+fibroblast+growth+factor+receptor+2-IIIb+signaling+for+cecal+development+in+mouse.">Requirement for fibroblast growth factor 10 or fibroblast growth factor receptor 2-IIIb signaling for cecal development in mouse.</a> Dev Biol. 265, 61-74 (2004).</li><li>Pabst, O., Schneider, A., Brand, T., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Nkx2-3+homeodomain+gene+is+expressed+in+gut+mesenchyme+during+pre-+and+postnatal+mouse+development.">The mouse Nkx2-3 homeodomain gene is expressed in gut mesenchyme during pre- and postnatal mouse development.</a> Dev Dyn. 209, 29-35 (1997).</li><li>Pabst, O., Zweigerdt, R., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+homeobox+transcription+factor+Nkx2-3+in+mice+results+in+postnatal+lethality+and+abnormal+development+of+small+intestine.">Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine.</a> Development. 126, 2215-2225 (1999).</li><li>Kaestner, K. H., Silberg, D. G., Traber, P. G., Sch&#252;tz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mesenchymal+winged+helix+transcription+factor+Fkh6+is+required+for+the+control+of+gastrointestinal+proliferation+and+differentiation.">The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.</a> Genes &amp; Development. 11, 1583-1595 (1997).</li><li>Madison, B. B., McKenna, L. B., Dolson, D., Epstein, D. J., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxF1+and+FoxL1+link+hedgehog+signaling+and+the+control+of+epithelial+proliferation+in+the+developing+stomach+and+intestine.">FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine.</a> J Biol Chem. 284, 5936-5944 (2009).</li><li>Karlsson, L., Lindahl, P., Heath, J. K., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+gastrointestinal+development+in+PDGF-A+and+PDGFR-(alpha)+deficient+mice+implicates+a+novel+mesenchymal+structure+with+putative+instructive+properties+in+villus+morphogenesis.">Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis.</a> Development. 127, 3457-3466 (2000).</li><li>Mao, J., Kim, B., Rajurkar, M., Shivdasani, R., Mcmahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+controls+mesenchymal+growth+in+the+developing+mammalian+digestive+tract.">Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.</a> Development. 137, 1721-1729 (2010).</li><li>Theiler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The house mouse. Development and normal stages from fertilization to 4 weeks of age. , (1989).</li><li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+platelet-derived+growth+factor+alpha+receptor+signaling+mechanisms.">Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.</a> Mol Cell Biol. 23, 4013-4025 (2003).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, 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+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Fu, Y. Y., 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=Microtome-free+3-dimensional+confocal+imaging+method+for+visualization+of+mouse+intestine+with+subcellular-level+resolution.">Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution.</a> Gastroenterology. 137, 453-465 (2009).</li></ol>

The dynamic nature and complex tissue interactions of the developing intestine requires 3D visualization to have a full appreciation of these morphogenetic events. With evolving imaging technology, the ability to examine villus morphogenesis in detail is developing/improving and with it, the understanding of the spatial communication and interaction during organogenesis is greatly enhanced.
Alternative methods for culturing whole intestines have also been tested, but the transwell system remai...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1308">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
			<td style="width:223px;">Fine Science Tools</td>
			<td style="width:133px;">&#160;11254-20</td>
			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Culture of whole explants of fetal intestines allows for the analyses of the location, distribution, and duration of the signaling molecules that coordinate intestinal development, as this system enables the manipulation of the signaling with pharmacological reagents or recombinant proteins. The transwell culture system (Figure 1A, reproduced from Walton et al., 2012)2 provides an air-liquid interface, which makes it possible to place drug- or protein soaked agarose beads onto the tis...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Research

JoVE Journal

Biology

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...