Mus Fetal Hele tarmen Kultur System for<em> Ex Vivo</em> Manipulering av signalveier og tredimensjonale Levende Imaging av Villus Development
Summary
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.
Abstract
De fleste morphogenetic prosesser i foster tarmen har blitt utledes fra tynne snitt av fast vev, og gir øyeblikksbilder av endringer over utviklingsstadier. Tredimensjonal informasjon fra tynne seriesnitt kan være utfordrende å tolke på grunn av vanskeligheter med å rekonstruere seriesnitt perfekt og opprettholde riktig orientering av vevet enn seriesnitt. Nyere funn av Grosse et al., 2011 markere betydningen av tredimensjonalt informasjon for å forstå morphogenesis av utviklings villi av tarmen en. Tredimensjonal rekonstruksjon av enkeltvis merkede tarmceller viste at de fleste av de intestinale epitelceller kontakt med både de apikale og basaloverflater. Videre tredimensjonal rekonstruksjon av aktin cytoskjelettet på den apikale overflate av epitelet viste at tarmlumen er kontinuerlig, og at sekundære lumen er en gjenstand av sectioning. Disse to punkter, sammen med en påvisning av interkinetic atom migrasjon i intestinal epitelium, defineres det fremkallende intestinal epitelium som pseudostratified epitel og ikke som tidligere antatt stratifisert 1. Evnen til å observere Epitel tredimensjonalt var banebrytende for å demonstrere dette punktet, og redefinerte epitelial morphogenesis i fosterets tarmen. Med utviklingen av multi-foton avbildningsteknologi og tredimensjonal rekonstruksjon programvare, til evnen til å visualisere intakt, utvikle organer er i sterk forbedring. To-foton eksitasjon tillater mindre skadelig inntrengning dypere inn i vev med høy oppløsning. To-foton avbildning og 3D rekonstruksjon av hele føtale mus tarmen i Walton et al. 2012 bidratt til å definere mønsteret av villus utvekst 2. Her beskriver vi et helt organ kultur system som lar ex vivo utvikling av villi og utvidelser av at kultur system for å tillatetarmen til å være tredimensjonalt fotografert under utvikling.
Introduction
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.
Protocol
Representative Results
Discussion
Den dynamiske natur og komplekse vev interaksjoner av utviklings tarmen krever 3D-visualisering for å ha en full forståelse av disse morphogenetic hendelser. Med utvikling bildeteknologi, muligheten til å undersøke villus morphogenesis i detalj er utvikling / forbedring og med det, er forståelsen av den romlige kommunikasjon og samhandling under organogenesen sterkt forbedret.
Alternative metoder for dyrking av hele tarmen er også testet, men Transwell systemet forblir den mest pålite…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Fine dissecting forceps | Fine Science Tools | 11254-20 | 2 pairs |
| 70% Ethanol | |||
| 1x sterile Dulbecco's Phosphate-Buffered Saline (DPBS) | Gibco | 14040-133 | 500 ml |
| 6 well plates | Costar | 3516 | |
| 24 well plates | Costar | 3524 | |
| 60 x 15 mm petri dishes | Falcon | 451007 | |
| Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes | Corning Costar | 3428 | 6 inserts per plate |
| BGJb media | Invitrogen | 12591-038 | 500 ml |
| PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin) | Gibco | 15140 | |
| Ascorbic Acid | Sigma | A0278 | make 5 mg/ml stock, filter, aliquot and store at -20 °C |
| Mouth pipet (Drummond 1-15 inch aspirator tube assembly) | Fisher | 21-180-10 | remove the aspirator assembly and replace it with a 1000 µl pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet. |
| 6 inch glass pasteur pipets | |||
| Agarose beads | BioRad | 153-7301 | |
| Capillary Tubes | World Precision Instruments | TW100F-4 | pull to needles |
| 4% Paraformaldehyde | made in 1 x PBS, pH to 7.3 | ||
| Live Imaging Materials | |||
| Name of Material/Equipment | Company | Catalog Number | Comments/Description |
| Culture plates | Falcon | 353037 | |
| Fine mesh stainless steel screen | purchase at hardware store | ||
| Polycarbonate membranes | Thomas scientific | 4663H25 | alternatively, cut Corning Costar 3428 membranes off of transwell supports |
| Instant glue | purchase at hardware store | gel based preferrably | |
| 35 x 10 mm plates | Falcon | 351008 | |
| 7% agarose | Sigma | A9414 | prepare w/v in 1x DPBS, heating to dissolve in a waterbath |
| minutien pins | Fine Science Tools | 26002-20 | |
| Phenol red free media (DMEM) | Gibco | 21063-029 | |
| Xylazine (100 mg/ml) | AnaSed | 139-236 | |
| Matrigel | BD | 356231 | basement membrane matrix, growth factor reduced, phenol red-free |
| 3-4% agarose | Sigma | A9414 | prepare w/v in 1x DPBS, heating to dissolve in a waterbath |
| Imaging of fixed intestines | |||
| Name of Material/Equipment | Company | Catalog Number | Comments/Description |
| vaseline | purchase at pharmacy | used to make VALAP: equal parts vaseline, lanolin, paraffin | |
| lanolin | Sigma | L7387 | used to make VALAP: equal parts vaseline, lanolin, paraffin |
| paraffin | Surgipath | 39601006 | used to make VALAP: equal parts vaseline, lanolin, paraffin |
| 70% glycerol in 1 x PBS | |||
| Focus clear and Mount Clear | CelExplorer Labs Co. | F101-KIT | |
| Modeling clay | purchase at art supply store | ||
| double stick tape | |||
| cotton applicator swabs | |||
| plastic molds, 10mm x 10mm x 5 mm) | Tissue Tek | 4565 | |
| slides | |||
| coverslips | |||
| lab wipe | Kimberly Clark | 34155 | lint free delicate task wipe |
| Theiler staging chart | http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf | ||
| Leica SP5X confocal microscope | Leica | Used to conduct the live imaging | |
| Leica DMI 6000 stand | Leica | Used to conduct the live imaging | |
| Aqueous mounting medium (Prolong Gold) | Molecular Probes | P36930 | |
| Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines | |||
| Name of Material/Equipment | Company | Catalog Number | Comments/Description |
| 24 well plate | Costar | 3524 | |
| Triton X-100 | Sigma | T-8787 | used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS |
| Goat serum | used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS | ||
| Tween20 | Sigma | P9416 |
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