Summary

के लिए एक प्रणाली<em> पूर्व vivo</emभ्रूण अग्न्याशय> संवर्धन

Published: August 27, 2012
doi:

Summary

यहाँ, हम अलगाव, संस्कृति और माउस भ्रूणीय अग्न्याशय के हेरफेर के लिए एक विधि का वर्णन है. यह एक उत्कृष्ट प्रतिनिधित्व करता है<em> पूर्व vivo</em> Morphogenesis भेदभाव, और अग्नाशय के विकास के विकास सहित विभिन्न पहलुओं, अध्ययन के लिए प्रणाली. अग्नाशय के कली explants कई दिनों के लिए संवर्धित किया जा सकता है और पूरे माउंट immunofluorescence और रहते इमेजिंग सहित विभिन्न अनुप्रयोगों, की एक श्रृंखला में प्रयोग किया जाता है.

Abstract

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.

During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing β-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.

Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

Protocol

यहाँ वर्णित प्रोटोकॉल मूल पेर्सिवल और सुस्त 9 में वर्णित और confocal माइक्रोस्कोपी के लिए अनुकूलित तकनीक से अनुकूलित किया गया है. 1. गिलास नीचे संस्कृति व्यंजन की कोटिंग निम्नलिखित…

Discussion

एक बार अग्नाशय भाग्य निर्दिष्ट किया जाता है, अग्नाशय पूर्वपुस्र्ष कोशिकाओं व्यापक प्रसार, भेदभाव और morphogenesis अंततः एक परिपक्व और कार्यात्मक 2,4 अंग फार्म से गुजरना. वर्तमान में, कैसे शाखाओं में बंटी अग…

Disclosures

The authors have nothing to disclose.

Acknowledgements

में अनुसंधान Spagnoli प्रयोगशाला. Helmholtz एसोसिएशन, FP7-IRG-2008-ENDOPANC अनुदान और ईआरसी-2009-शुरू HEPATOPANCREATIC अनुदान द्वारा वित्त पोषित है.

Materials

Name of the reagent Company Catalogue number Comments
Antibodies:
Carboxypeptidase
E-cadherin
F-actin
Glucagon
Insulin
β1-integrin
Pdx1
Pdx1
Phospho-Histone H3
AbD Serotec
Invitrogen
Molecular Probes
ImmunoStar
Millipore
Millipore
Abcam
Hybridoma bank
Cell Signalling
1810-0006
13-1900
A-12373
20076
4011-01
MAB1997
ab47267
F109-D12
9706
 
Basal Medium Eagle (BME) Sigma B1522-500ML Kept in sterile conditions
Cell culture grade water PAA S15-012 Kept in sterile conditions
Culture dishes (glass-bottomed), 35-mm MatTek Corporation P35G-0-20-C  
Donkey Serum Chemicon S30-100 ml  
Fetal calf serum Gold PAA A15-151 Kept in sterile conditions
Fibronectin Invitrogen 330100-8 Stock sol. 1 mg/ml in cell culture grade water
Gentamicin Invitrogen 15750-037 Kept in sterile conditions
Glutamine Invitrogen 25030-024 Kept in sterile conditions
4-well Multidishes Nunc 176740  
Microscopes:
Inverted Confocal Microscope (LSM 700)
Stereomicroscope (Discovery V12)
Zeiss

Zeiss
  Objectives:
C-Apochromat 10X / 0.45 W M27 (work. dist. 1.8 mm; imaging depth ~100 mm); C-Apochromat 40X / 1.2 W Corr M27 (work. dist. 0.28 mm; ~imaging depth 50 μm)

Transillumination from below and fiber-optic illumination from above
Paraformaldehyde Roth 0335.3 Stock solution 20%
Pasteur Pipet (Glass), 150 mm VWR HECH567/1  
Penicillin/Streptomycin PAA P11-010 Kept in sterile conditions
Petri dishes, 60 mm Greiner Bio-One 628102  
Petri dishes, 35 mm Greiner Bio-One 627161  
1X PBS, pH7.4 PAA H15-002 Kept in sterile conditions
Spring Scissors 8 mm blade curved Fine Science Tools 15023-10  
Triton-X100 Roth 3051.3  
Watchmaker’s foreceps Dumont #5 Roth K342.1  

References

  1. Slack, J. Developmental biology of the pancreas. Development. 121, 1569-1580 (1995).
  2. Pan, F., Wright, C. Pancreas organogenesis: from bud to plexus to gland. Dev. Dyn. 240, 530-565 (2011).
  3. Puri, S., Hebrok, M. Cellular Plasticity within the Pancreas- Lessons Learned from Development. Developmental Cell. 18, 342-356 (2010).
  4. Spagnoli, F. M. From endoderm to pancreas: a multistep journey. Cell. Mol. Life Sci. 64, 2378-2390 (2007).
  5. Hick, A. -. C. Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1. BMC Dev. Biol. 9, 1-17 (2009).
  6. Villasenor, A., Chong, D., Henkemeyer, M., Cleaver, O. Epithelial dynamics of pancreatic branching morphogenesis. Development. 137, 4295-4305 (2010).
  7. Kesavan, G. Cdc42-Mediated Tubulogenesis Controls Cell Specification. Cell. 139, 791-801 (2009).
  8. Zhou, Q. A Multipotent Progenitor Domain Guides Pancreatic Organogenesis. Developmental Cell. 13, 103-114 (2007).
  9. Percival, A., Slack, J. Analysis of pancreatic development using a cell lineage label. Exp. Cell Res. 247, 123-132 (1999).
  10. Miralles, F., Czernichow, P., Ozaki, K., Itoh, N., Scharfmann, R. Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas. Proc. Natl. Acad. Sci. U.S.A. 96, 6267-6272 (1999).
  11. Puri, S., Hebrok, M. Dynamics of embryonic pancreas development using real-time imaging. Dev. Biol. 306, 82-93 (2007).
  12. Magenheim, J. Blood vessels restrain pancreas branching, differentiation and growth. Development. 138, 4743-4752 (2011).
  13. Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. . Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).
  14. Horb, L. D., Slack, J. M. Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas. Int. J. Dev. Biol. 44, 791-796 (2000).
  15. Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. A global double-fluorescent Cre reporter mouse. Genesis. 45, 593-605 (2007).

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Cite This Article
Petzold, K. M., Spagnoli, F. M. A System for ex vivo Culturing of Embryonic Pancreas. J. Vis. Exp. (66), e3979, doi:10.3791/3979 (2012).

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