Molecular and Cellular Basis of Embryonic Development, Max-Delbrück-Center for Molecular Medicine
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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).
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.
The protocol described here has been adapted from the technique originally described in Percival and Slack9 and optimized for confocal microscopy.
1. Coating of Glass Bottom Culture Dishes
The following steps should be carried out under sterile conditions in a laminar flow hood.
If not all fibronectin-coated dishes are used, they can be stored for up to 1 week at 4 °C. Do not let the dishes dry, fill them with Dissection Medium and place them in the refrigerator.
In addition to fibronectin, pancreatic explants have been successfully cultured on various substrates, such as laminin9, matrigel10,11 or microporous membranes12. Because of its effects on branching, we use fibronectin9 as substrate.
2. Dissection of Dorsal Pancreatic Bud from E11.5 - E12.5 Mouse Embryos
3. Plating and Culture of Pancreatic Explants
Final plating of the explants is carried out in tissue culture room and/or in close proximity to the tissue culture incubator to minimize physical movement of the cultures.
4. Whole-mount Immunofluorescence Staining Protocol for Pancreatic Explants
All steps of the immunofluorescence protocol (from fixation to imaging) are carried out in the same MatTek dish, which yields better images than a plastic-bottomed dish. After fixation, pancreatic explant cultures do not need to be kept under sterile conditions.
5. Live-cell Imaging Protocol of Pancreatic Explants
6. Representative Results
Dorsal pancreatic buds (together with the surrounding mesenchyme) were dissected from mouse embryos between E11.5 and E12.5 and plated directly on glass bottom culture dishes (Figure 1). Blood vessels are present in the mesenchyme surrounding the epithelium and can be visualized by immunostaining for the endothelial marker Pecam12 (data not shown). After two days of culture, pancreatic explants underwent significant growth and started to organize themselves into branched epithelial structures, whose nuclei were positive for the pancreatic transcription factor, Pdx1 (Figure 2A-C). The average z-thickness of pancreatic explants on day 3 of culture is 60-80 μm. The surface and volume of the explants can be measured using appropriate softwares (e.g. Huygens). Morphology and immunofluorescence analyses showed that pancreatic explants cultured using this protocol recapitulated in vivo early pancreatic differentiation and morphogenetic events5,10,11,14 (Figure 2). Pancreatic explants on day 4 of culture underwent typical "tip and trunk" segregation of the epithelium8, displaying cpa1+ progenitor cells at the tips of the epithelial branches (Figure 2C) and differentiated insulin+ and glucagon+ cells organized as clusters inside the trunk (Figure 2D). In addition, the pancreatic epithelium in the ex vivo cultures showed proper cytoskeleton organization and cell polarity, with apical localization of F-actin (Figure 2E, in green) and b1-integrin at the basal membranes (Figure 2E, in red).
To study pancreatic branching in realtime,embryonic pancreatic explants were isolated from the dual-color fluorescent reporter mouse strain membrane-Tomato/membrane-Green15 (mT/mG; mouse strain is available at the Jackson Laboratory) (Figure 3). Still frames from a time-lapse movie of mT/mG pancreatic explants grown in culture are shown in Figure 3. The localization of the mT fluorescent protein to membrane structures outlines cell morphology and allows resolution of fine cellular processes, enabling to track cell remodeling and migration over time.
After a 12-hr time-lapse experiment the mT fluorescence signal remains viable and detectable, even though its intensity is reduced, most likely due to photodamage (Figure 3). Technical solutions to avoid or reduce photodamage and phototoxicity are discussed in the Discussion section.
Figure 1. Schematic representation of the dorsal pancreas bud dissection from E12.5 mouse embryo. (A) Brightfield image of E12.5 mouse embryo. The upper body of the embryo (above the liver) and the tail region are removed to isolate the mid-body (B). Incisions are shown by red dotted lines. (C) Further dissection results in exposure of the stomach and duodenum region (indicated by the dotted box). The dorsal pancreatic bud (see red dotted circle) is at the base of the stomach and next to the spleen primordium. The dissected dorsal pancreatic bud (D) is transferred with a Pasteur pipette into the microwell of a glass bottom dish containing BME culture medium (E, F). Abbreviations: sto (stomach), sp (spleen), dp (dorsal pancreatic bud), duo (duodenum). Scale bars, 1000 μm (A, C).
Figure 2. Whole-mount confocal immunofluorescence analysis of pancreatic explant cultures. (A) Brightfield images of pancreatic explants on day 1 and day 2 of ex vivo culture. Red dotted line indicates initiation of branching of the epithelium. (B) 3D reconstruction of whole-mount immunostaining for Pdx1 on day 3 pancreatic culture. Pancreatic explants showed tubules of Pdx1+ cells undergoing extensive branching by 48 hr of cultures. (C) Whole-mount immunostaining of day 3 pancreatic explant with antibodies against the basolateral membrane marker E-cadherin (Ecad), tip progenitor marker, Carboxypeptidase 1 (Cpa1) and phospho-Histone H3 (pHH3). Most of the mitotic active pHH3+ cells colocalize at the peripheral tips with Cpa1+ cells. Dashed lines indicate tip of the branches. (D) Whole-mount immunostaining for endocrine lineage markers, insulin (Ins) and glucagon (Gluca) at day 3. Yellow arrows indicate clusters of Ins+ and Gluca+ cells. Inset (D'), higher-magnification of one of the endocrine cell cluster. (E) Whole-mount immunostaining of day 3 pancreatic explant for Pdx1, β1 integrin (β1int) and F-actin (Fact). Mesenchymal cells (asterisks) interspersed among Pdx1-positive epithelial cells. Images shown in C, D and E are single confocal optical sections of Z-series. Scale bars, 100 μm (A, B); 50 μm (C-E).
Figure 3. Representative live-cell imaging of ex vivo cultured pancreatic explant. (A-D) Frames from a time-lapse movie of a pancreatic explant from a mT/mG mouse transgenic embryo. Time of imaging is shown in hours:minutes. The explant was imaged starting at Day 1. Z-stack images were acquired with a 40X oil immersion objective every 12 min for a total of 15 hr. Dashed lines indicate tip of the branches and border between epithelium and mesenchyme. Scale bar, 50 μm.
Once pancreatic fate is specified, pancreatic progenitor cells undergo extensive proliferation, differentiation and morphogenesis to eventually form a mature and functional organ2,4. At present, how branching takes place in the pancreas and how it is connected to progenitor proliferation and differentiation is largely unknown. Pancreatic explant cultures represent an ideal system to elucidate these processes ex vivo5,9,11. By combining live-cell imaging with ex vivo explants of early pancreatic buds one can obtain a clear picture of the events that take place during pancreas morphogenesis in the embryo. This video article presents a simple approach to establish ex vivo cultures of embryonic pancreas on a glass support, which makes them ideal for whole-mount immunofluorescence and live imaging.
Numerous examples of the use of ex vivo pancreatic explants for imaging cell morphology, growth and differentiation are presented here. Importantly, these examples show that embryonic pancreases cultured ex vivo using this protocol mimic the early stages of in vivo embryonic pancreas development2,3. For example, branching initiates approximately 24 hr after plating of E11.5 pancreas, as expected in vivo (Figure 2A). While morphogenesis and cell differentiation in the cultures closely resembles that seen in vivo, the overall growth of the explants is not comparable and considerably less than that seen in the developing embryo. Under the culture conditions described here, pancreatic explants stop growing at day 5 and undergo degeneration after one week (data not shown). Therefore, the reported ex vivo culturing system is limited and better suited to the investigation of early aspects of pancreas organogenesis.
We present here examples of ex vivo cultures that we established from wild-type as well as mT/mG reporter mouse embryos. The use of a fluorescent reporter strain is indispensable for real-time imaging, enabling the visualization of cellular and subcellular structures and the study of their dynamics in a 3D environment. For instance, the localization of the mT fluorescent protein to membrane structures enables us to precisely visualize cell morphology and track cell remodeling and migration over time in the pancreatic explants. One of the most common problem and limitation in live-cell imaging experiments is photodamage that may occur as a result of repeated exposure to fluorescence excitation illumination. In general, one has to balance image quality with the phototoxicity in the context of its own experimental settings. For instance, to minimize photodamage one possibility is to reduce the frequency of imaging and increase the time lapse between consecutive frames or, alternatively, to optimize the Z stack acquisition by reducing the number of optical slices.
Finally, ex vivo culturing of embryonic pancreas is a valuable system for screening chemical compounds and their effects on pancreatic development. The compound or factors can be added directly to the culture medium and the developmental effects can be easily assessed under a microscope. Using data analysis software, one could even obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. It is also possible to use transgenic or knockout tissues to establish organ cultures or carry out faster gain- and loss-of-function experiments in explants, for instance through lentivirus transduction or morpholino oligonucleotides, respectively (data not shown). All these approaches would allow real-time studies of mutant phenotypes and further understanding of gene regulatory networks in the developing pancreas.
No conflicts of interest declared.
Research in the Spagnoli lab. is funded by the Helmholtz Association, FP7-IRG-2008-ENDOPANC grant and ERC-2009-Starting HEPATOPANCREATIC Grant.
|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|
Inverted Confocal Microscope (LSM 700)
Stereomicroscope (Discovery V12)
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|
|Watchmaker’s foreceps Dumont #5||Roth||K342.1|