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1Department of Biological Sciences, University at Albany, SUNY
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A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.
Keywords: Genetics, Issue 71, Molecular Biology, Cellular Biology, Developmental Biology, Virology, Medicine, Adenovirus, Embryonic, Epithelial rudiments, Extracellular matrix, Mesenchyme, Organ culture, Submandibular gland, ex vivo, cell culture, tissue engineering, embryo, mouse, animal model
Sequeira, S. J., Gervais, E. M., Ray, S., Larsen, M. Genetic Modification and Recombination of Salivary Gland Organ Cultures. J. Vis. Exp. (71), e50060, doi:10.3791/50060 (2013).
Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.
The protocol contains four major steps, as depicted in Figure 1. All steps are described in full detail. Adenovirus construction and viral purification should be performed in advance of the organ harvesting for use in the genetic transduction of dissected epithelial rudiments. All standard BSL-2 safety precautions should be followed when working with adenoviruses.
1. Mouse Embryonic Submandibular Gland (SMG) Harvesting and Microdissection
2. SMG Epithelial Rudiment Separation
3. Adenoviral Infection of Epithelial Rudiments
4. Ex vivo Culture of Recombined SMGs
The flow of the major experimental steps is outlined in Figure 1. An example of an intact SMG, an isolated epithelial rudiment, and its corresponding mesenchyme are shown in Figure 2. Brightfield images of recombined SMGs, which continue to undergo branching morphogenesis when cultured ex vivo for the indicated times, are shown in Figure 3. Recombined glands grown for 48 hr expressing epithelial GFP are shown in Figure 4. Confocal images of recombined SMGs fixed and imaged after 72 hr in culture followed by immunocytochemistry, are shown in Figure 5. The epithelial marker, E-cadherin, demarcates the GFP-expressing epithelial cell population from the surrounding mesenchyme. Detection of the basement membrane protein, perlecan, localized at the periphery of the epithelium demonstrates at least partial reconstitution of the basement membrane in recombined glands. Figure 6. Parasympathetic submandibular ganglia undergo neurite outgrowth and innervate the glands in recombination cultures as demonstrated previously.19
Figure 1. (A) Flowchart and (B) Schematic depicting the major experimental steps.
Figure 2. Brightfield images of (A) a whole embryonic day 13 (E13) salivary gland showing the sublingual gland epithelium (SL Epi) and the submandibular gland epithelium (SMG Epi) surrounded by mesenchyme (mes). (B) Isolated epithelial rudiment, and (C) Separated Mesenchyme. Scale bars = 100 μm.
Figure 3. Brightfield images of epithelial rudiments (outlined by white dashed lines) surrounded by mesenchyme pieces grown in ex vivo culture for the indicated times. Epithelial cells undergo branching morphogenesis and mesenchymal condensation is evident as early as 24 hr in culture. Scale bars = 100 μm
Figure 4. Brightfield (A), fluorescent (B) and overlaid (C) images of recombined SMGs in which only the epithelial cells (outlined in white dashed lines) were infected with GFP-expressing adenovirus and cultured ex vivo for 48 hr. Scale bars = 100 μm.
Figure 5. Confocal images or brightfield images (BF) of recombined SMGs labeled with (A) nuclei (DAPI, blue), adenoviral GFP (green), the epithelial marker. E-cadherin (red), scale bars = 250 μm , or, (B) nuclei (DAPI, blue), adenoviral GFP (green), the epithelial marker, E-cadherin (red), and the basement membrane marker, Perlecan (cyan), scale bars = 50 μm. Dashed white lines outline epithelium. Click here to view larger figure.
Figure 6. Parasympathetic submandibular ganglia undergo neurite outgrowth and innervate the glands in recombination cultures, scale bars = 250 μm.
The ex vivo epithelial-mesenchymal recombination technique was first published for submandibular salivary glands in 198116. In this protocol, we expand upon the original method, using adenoviral infection to manipulate epithelial cell gene expression within the context of a recombined gland. We show that a percentage of the epithelial cells are infected with the adenovirus, whereas the percentage of cells that are infected depends upon the properties of the viral promoter, viral titer, and viral purity. We have found that it is necessary to use CsCl gradient-purified viruses for efficient epithelial transduction, the purification of which is not described here. Since we have also been able to infect the mesenchyme cells prior to recombination (data not shown), independent genetic manipulation of the mesenchymal population is also possible. We recently used this adenoviral transfection/tissue recombination method to identify a role for the polarity protein, PAR-1b, in the control of the placement of basement membrane by the epithelium in developing salivary glands. Kinase-dead PAR-1b adenovirus and wild-type PAR-1b adenovirus were both used in this study13 to infect epithelial rudiments and recombined with E13 mesenchymes. The ability to use mutant viruses to interrogate functions of specific molecules greatly enhances the utility of this method.
There is currently a lack of effective tools to target specific molecules within the epithelial cell population in intact embryonic salivary glands without affecting the mesenchymal compartment. Although multiple studies have used pharmacological inhibitors to manipulate signal transduction in intact organ cultures, these inhibitors affect both the epithelium and mesenchyme. To directly assess effects of inhibitors on the epithelium, epithelial rudiments must be cultured in the absence of mesenchyme in either Matrigel or laminin-111 gels6,7. Small inhibitory RNAs (siRNAs) are an effective method to down-regulate epithelial gene expression since siRNAs are preferentially taken up by the epithelial cells in the presence of most lipid carriers13,14,17,18. However, neither of these methods for interfering with protein levels or function allow overexpression of a wild-type or mutant gene. Attempts at traditional (non-viral) transfection of embryonic whole SMG cultures have met with limited success in that only a small number of epithelial cells can be targeted (M.L., unpublished data). In comparison, adenoviral transduction represents an effective method for specifically transfecting salivary epithelial cells.
Parasympathetic nerves have recently been shown to be critical for normal salivary gland branching by maintaining a keratin 5-positive progenitor cell population19. An advantage of this tissue recombination method over the mesenchyme-free epithelial rudiment culture method is that the parasympathetic ganglion can be maintained in the recombination culture system. By keeping careful track of the approximate location of the parasympathetic ganglion, which is located in the mesenchyme adjacent to the primary epithelial duct19, it is possible to ensure that each rudiment ends up being associated with a ganglion. However, we find that by combining 3-4 glands worth of mesenchymes with every epithelial rudiment, each rudiment is associated with a ganglion sufficient to innervate the buds to some extent (Figure 6), although not always to the same as in the intact unmodified glands.
There are alternate methods for infecting the epithelium in the context of the intact gland. We previously reported that microinjection can be used to introduce adenovirus into epithelial cells in intact glands20,21. However, microinjection is difficult to control, may physically damage the glands, and typically leads to infection of only a localized subset of cells20. Adeno-associated viruses (AAV) have been shown to be useful for transfection of distinct cell populations within the context of intact SMG organ explants22. The serotype scAAV2 was shown to be substantially more efficient at specific transduction of the epithelium of intact SMGs over other recombinant AAV vectors23. Since AAVs are not widely commercially available, nor does the expertise to prepare AAV exist in most laboratories, the adenoviral transduction and tissue recombination protocol provided here is more generally accessible to most laboratories than is the use of AAV vectors.
While this tissue recombination method recapitulates epithelial-mesenchymal interactions to some extent, it is also possible to infect the epithelium with adenovirus and then grow the epithelium outside of its native context. As previously mentioned, we infected intact salivary epithelial rudiments with adenovirus and then grew the cultures in Matrigel with exogenously added growth factors17,20,21. Salivary gland organ explants can also be cultured on nanofiber scaffolds, although these scaffolds do not recapitulate the full function of the mesenchyme in their current form15. While neither of these methods recapitulates the native mesenchyme, such methods allow for imitation of specific properties of the mesenchyme in ex vivo culture.
Each ex vivo culture method has its own advantages and disadvantages but is applicable for addressing specific scientific questions. While the recombined glands do not undergo as much branching morphogenesis as do intact salivary gland organ explants, organ explants, in general, only recapitulate in vivo branching morphogenesis for a few days. A solution to this problem is of course to create transgenic animals containing targeted gene expression within the epithelial compartment. Since there is a lack of known promoters that are activated specifically in early developing SMG epithelium, and transgenic technology remains significantly more expensive than the described methods, the tissue recombination experiments described here constitute a viable model system to study both epithelial and mesenchymal cell signaling in early developing salivary gland cells within their tissue context.
No conflicts of interest declared.
The authors would like to thank Dr. Deirdre Nelson for helpful comments and for critical reading of the manuscript. This work was funded by NIH grants DE019244, DE019197, and DE021841 to M.L., F32DE02098001 to S.J.S, and C06 RR015464 to the University at Albany, SUNY.
|DMEM/Ham's F12 Medium without phenol red||Life Technologies||21041-025|
|Penicillin and Streptomycin||Life Technologies||15070-163||10X stock|
|Dispase||Life Technologies||17105-041||Freeze single use aliquots at -20C|
|BSA||Sigma||A2934-100G||Fraction V, low endotoxin|
|Adeno-X-GFP||BD Biosciences||8138-1||Should be high titer (1x1010 pfu/ml). CsCl purified viruses are more effective than column-purified viruses in this assay.|
|16% Paraformaldehyde||Electron Microscopy Sciences||15710||Diluted to 2% in PBS with 5% sucrose (w/v)|
|1X Phosphate-buffered saline (PBS)||Life Technologies||70011-044||Prepared from 10X stock|
|Hank's Balanced Salt Solution||Life Technologies||14175095||no Calcium, no Magnesium, no Phenol Red|
|Transferrin||Sigma||T8158||25 mg/ml stock solution in DMEM/F12 media. Freeze single-use aliquots at -20C|
|L- Ascorbic acid (Vitamin C)||Sigma||A4403||75 mg/ml stock solution in DMEM/F12 media.Freeze single-use aliquots at -20C|
|Table 1. List of reagents required for SMG recombination protocol.|
|10 cm sterile plastic dishes||Corning||430167||Non-tissue culture-treated plates can also be used.|
|Stereo dissecting microscope with transmitted light base||Nikon||SMZ645||Any stereo dissecting microscope can be used that has a transmitted light base.|
|35 mm tissue culture dishes||Falcon||353001||Non-tissue culture-treated plates can also be used.|
|50 mm diameter microwell dishes||MatTek Corporation||P50-G-1.5-14F|
|Nuclepore Track-Etch membrane filters||Whatman||110405||13 mm diameter, 0.1 mm pore size|
|Widefield fluorescence microscope||Carl Zeiss, USA||Axio Observer Z1||Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera.|
|Confocal microscope||Leica Microsystems||TCS SP5||Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.|
|Timed-pregnant female mice, strain CD-1 or ICR||Charles River Labs||Embryos are harvested on day 13 (with day of plug discovery designated as day 0).|
|Scalpel blade #11||Fine Science Tools||10011-00|
|Scalpel handle #3||Fine Science Tools||10003-12|
|Dumont #5 forceps inox alloy, 0.05mm X 0.02mm||Fine Science Tools||11252-20||Ideal for harvesting glands from embryos|
|Dumont #5 forceps dumostar alloy, 0.05mm X 0.01mm||Fine Science Tools||11295-20||Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.|
|Table 2. Equipment used in SMG recombination protocol.|
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