에 대한 마우스 태아의 전체 소장 문화 시스템<em> 전의 VIVO</em> 신호 전달 경로와 융모 개발의 3 차원 라이브 영상의 조작
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
태아 소장에서 대부분의 형태 형성 과정은 발달 단계를 통해 변화의 스냅 샷을 제공하는 고정 된 조직의 얇은 섹션에서 유추되고있다. 얇은 시리얼 섹션에서 세 가지 차원 정보로 인해 완벽하게 시리얼 섹션을 재구성 시리얼 섹션을 통해 조직의 올바른 방향을 유지의 어려움의 해석에 도전 할 수 있습니다. 그로 의해 외. 최근 연구 결과, 2011은 한 부위의 현상 융모의 형태 형성을 이해 삼차원 정보의 중요성을 강조. 단독 표지 장 세포의 3 차원 재구성은 장 상피 세포의 대부분은 모두 혀끝과 기저 표면에 문의하는 것이 보여 주었다. 더욱이, 상피의 선 단면에서 액틴 세포 골격의 삼차원 재구성 장내 루멘 연속이며 그 보조 루멘 (S)의 이슈임을 입증ectioning. 그 두 점은, 장 상피 세포에서 interkinetic 핵 마이그레이션 시연과 함께 pseudostratified 상피 세포로 개발하는 장 상피 세포를 정의하고 이전에 한 생각으로 계층화 없습니다. 상피 세 차원을 관찰 할 수있는 능력은이 점을 증명하고 태아 소장에서 상피 형태 형성을 재정에 정액이었다. 다중 광자 이미징 기술 및 3 차원 재구성 소프트웨어의 발전으로, 능력은 급속히 향상되고 장기 개발, 그대로 시각화. 이광자 여기는 고해상도 조직에 덜 파괴적인 침투를 깊게한다. 두 광자 이미징 및 월튼 등.에 전체 태아 마우스 내장의 3 차원 복원은 2012 융모 파생물이의 패턴을 정의하는 데 도움을 주었다. 여기에서는 허용하도록 그 배양 시스템의 융모와 확장 생체 개발을 허용 전체 기관 배양 시스템을 설명창자는 세 가지 차원으로 자신의 개발 기간 동안 이미지를 만들 수 있습니다.
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 Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen.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
동적 특성 및 개발 소장의 복잡한 조직 상호 작용은 이러한 형태 발생 이벤트의 전체 감사의 마음을 가지고 3D 시각화가 필요합니다. 이미징 기술 진화로, 능력 개발 / 개선과 함께, 기관 형성기 동안 공간 의사 소통과 상호 작용의 이해가 크게 향상되어 자세히 융모 형태 형성을 검사합니다.
전체 창자 배양 방법에 대해서도 테스트되었지만, 트랜스 웰 시스템은 긴 배양 시간?…
Divulgazioni
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 |
Riferimenti
- Grosse, A. S., et al. Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis. Development. 138, 4423-4432 (2011).
- Walton, K. D., et al. Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi. Proc Natl Acad Sci U S A. 109, 15817-15822 (2012).
- Wells, J. M., Melton, D. A. Vertebrate endoderm development. Annu Rev Cell Dev Biol. 15, 393-410 (1999).
- Hashimoto, H., Ishikawa, H., Kusakabe, M. Development of vascular networks during the morphogenesis of intestinal villi in the fetal mouse. Kaibogaku zasshi Journal of anatomy. 74, 567-576 (1999).
- Mathan, M., Moxey, P. C., Trier, J. S. Morphogenesis of fetal rat duodenal villi. Am J Anat. 146, 73-92 (1976).
- Burns, R. C., et al. Requirement for fibroblast growth factor 10 or fibroblast growth factor receptor 2-IIIb signaling for cecal development in mouse. Dev Biol. 265, 61-74 (2004).
- Pabst, O., Schneider, A., Brand, T., Arnold, H. H. The mouse Nkx2-3 homeodomain gene is expressed in gut mesenchyme during pre- and postnatal mouse development. Dev Dyn. 209, 29-35 (1997).
- Pabst, O., Zweigerdt, R., Arnold, H. H. Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine. Development. 126, 2215-2225 (1999).
- Kaestner, K. H., Silberg, D. G., Traber, P. G., Schütz, G. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes & Development. 11, 1583-1595 (1997).
- Madison, B. B., McKenna, L. B., Dolson, D., Epstein, D. J., Kaestner, K. H. FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine. J Biol Chem. 284, 5936-5944 (2009).
- Karlsson, L., Lindahl, P., Heath, J. K., Betsholtz, C. Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis. Development. 127, 3457-3466 (2000).
- Mao, J., Kim, B., Rajurkar, M., Shivdasani, R., Mcmahon, A. Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract. Development. 137, 1721-1729 (2010).
- Theiler, K. . The house mouse. Development and normal stages from fertilization to 4 weeks of age. , (1989).
- Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol. 23, 4013-4025 (2003).
- Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. A global double-fluorescent Cre reporter mouse. Genesis. 45, 593-605 (2007).
- Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science. 277, 1109-1113 (1997).
- Fu, Y. Y., et al. Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution. Gastroenterology. 137, 453-465 (2009).
