The overall goal of the following experiment is to analyze lacrimal gland development using an ex vivo approach for the manipulation and analysis of signaling events during gland development. This is achieved by first dissecting the lacrimal glands of day 16 embryos. As a second step, the glands are placed on filters, floating in culture, medium containing small molecule inhibitors or growth factors that alter lacrimal gland development.
After 24 hours, the glands are fixed and immuno stain for the developmental proteins of interest. Ultimately, the changes in protein expression during ex vivo lacrimal gland development can be analyzed by immunofluorescent microscopy. This method can help answer key questions in the field of lacrimal gland development.
For instance, what are the signaling mechanisms that define lacrimal gland? Morphogenesis Visual demonstration of this method is critical as the lacrimal gland is difficult to recognize during early embryonic development without some visual guidance and practice in locating it. To harvest the lacrimal gland from an embryonic mouse, use 70%ethanol to sterilize the ventral side of an embryonic day, 14 to 16 pregnant mouse.
Then pinch the skin and cut through the skin along the midline of the animal to expose the peritoneum, cut through the peritoneum to expose the abdominal cavity and myometrium and remove both horns of the myometrium and place into cold PBS. Next, use number five forceps to transfer the embryos from the amniotic sacs into a second sterile Petri dish containing cold PBS taking care not to touch the eyes or head area. After severing the head from the torso, just below the lower mandible, rotate the head so that the mandible is now on the dissecting plate.
Then use forceps to stabilize the head and a scalpel to remove about a quarter of the dorsal side of the brain, orient the head so that the scalpel can pierce the nose between the eyes, and then make a cut through the center of the head so that the eyes are now on separate halves. Next, using the above and below illumination, grab the skin from the posterior bottom corner of the eye and carefully pull the skin open with the forceps to expose the lacrimal gland, which should appear as a bud on a duct within a darker condensed mesenchyme. Carefully dissect the lacrimal gland and associated mesenchyme away from the surrounding tissue, taking care not to grab the lacrimal gland or its associated duct.
Then once the lacrimal gland is free from the surrounding tissue, gently grip the epithelial surrounding the eye at the base of the lacrimal gland duct, and remove the entire gland. Take care to preserve the Meen chi surrounding the epithelium, which can be observed as a condensed tissue into which the epithelium invaginates. Then culture four to five lacrimal glands per a filter in a Petri dish containing culture medium and the small molecule inhibitors or growth factors of interest for 24 hours to harvest the lacrimal gland from a postnatal or adult mouse, first spray the eye area with 70%ethanol.
Then lay the animal lateral side up under a dissecting microscope with illumination from above. Next, use small dissection scissors to make an incision in the epidermis laterally from where the lacrimal gland should be located. Then pull open the epidermis toward the ear to expose the lacrimal gland, which in the postnatal and adult mouse is bordered by the carotid artery, the maced muscle and the dermis Gently loosen the lacrimal gland from the surrounding tissue with the forceps, and then once the lacrimal gland is released from the surrounding tissue grip, where the duct and the epithelial surrounding the eye connect and carefully remove the gland.
Finally, fix the lacrimal glands with 4%para formaldehyde for 20 minutes before immuno staining. For the developmental proteins of interest, the lacrimal gland develops through the process of epithelial branching morphogenesis. These brightfield images of embryonic lacrimal glands, dissected at embryonic days 14, 15, 16, 17, and postnatal day two illustrate this process.
Note that as the epithelium increases in size, the amount of mesenchyme decreases as illustrated in these images. Lacrimal glands develop under vivo culture conditions in a similar manner as to within the in vivo gland cultures, and freshly dissected lacrimal glands can be fixed for immunofluorescent analysis. For example, in this image, the staining of the epithelium nerves and blood vessels.
In an embryonic, a 16 lacrimal gland from a wild type CD one embryo can be observed expression of GFP in the lacrimal gland by postnatal or adult PAC six CRE mice facilitates a rapid identification and dissection of the lacrimal gland from these transgenic animals. Though this method can provide insight into lacrimal gland development, it can also be applied to other systems such as lacrimal gland dysfunction due to autoimmune disease or irradiation treatment. After watching this video, you should have a good understanding of how to locate and isolate the murine lacrimal gland for ex vivo culture analysis.
