老鼠胎儿整肠文化系统<em>前体内</em>信号通路和绒毛开发的三维实时成像的操作
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
在胎儿小肠大部分形态过程已经推断出从固定的组织薄片，提供了发展阶段变化的快照。由薄的连续切片的三维信息可以是具有挑战性的，因为完美重构连续切片和保持组织在连续切片的适当定向的难度来解释。由格罗斯等人最近的研究结果，2011年重点在了解肠1的开发绒毛形态三维信息的重要性。单独使用标记的肠细胞的三维重建表明大多数肠上皮细胞的接触既心尖和基底表面。此外，肌动蛋白细胞骨架在上皮的顶端表面的三维重建表明肠腔是连续的，并且次要流明是第一个工件ectioning。这两点，伴随着interkinetic核移植的肠上皮示范，明确了发展中肠上皮为假复层上皮，不分层，如以前认为1。观察到上皮三维的能力是开创性到证明了这一点，重新定义上皮形态在胎儿肠。用多光子成像技术和三维重建软件的演化，能够可视化完好，显影机构正在迅速提高。双光子激发可损害较小穿透更深的组织具有较高的分辨率。双光子成像和三维重建整个胎鼠肠中的沃顿等人 ，2012帮助定义绒毛生长2的格局。在这里，我们描述了整个器官培养系统，使离体的发展是文化系统的绒毛和扩展，使肠道是三维的开发过程中进行成像。
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 Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen.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
动态性和发展肠道复杂的组织相互作用的，需要三维可视化有这些形态发生事件的全面理解。随着不断变化的影像技术，能够检查绒毛形态进行了详细的发展/改善有了它，空间通信和交互的器官形成期的理解大大提高。
另一种方法培养整个肠子也进行了测试，但Transwell小系统仍是最可靠的更长的培养时间，并维持正常的集群和绒毛图案。三维培养系统（基质胶或胶原蛋白）?…
Disclosures
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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