The overall goal of the following experiment is to understand the behavior of the Melano blasts population as it migrates s doso laterally through the developing embryonic epidermis. This is achieved by dissecting skin from E 14.5 mouse embryos to allow ex vivo organotypic culture. As a second step, the dissected skin is mounted in a custom built culture apparatus, which allows culture at an eliquid interface.
Next, the chamber is mounted onto the stage of a confocal microscope to image the migrating melano blasts population. Imaging the six embryonic skin cultures in parallel over an 18 hour period demonstrates the migration of Mel Oblasts as well as the development of primary hair follicles. The main advantage of this technique over older methods to visualize Mel Oblasts like immunohistochemistry in seizure hybridization, or staining lactase, is that we can do live imaging and so view individual cells to assay migration and proliferation over time.
This method can help answer key questions in a pigment cell biology field, such as how melon blasts behavior is disrupted in pigmentation mutants, and how melano blasts localize to developing hair follicles For this procedure at embryonic day 14.5 In cold PBS harvest embryos that express a melano blasts, specific Cree recombinase and a fluorescent Cree activat reporter construct if needed, screen the embryos for expression of fluorescent reporters After selecting the required embryos, decapitate them and remove their limbs with a razor blade. Retain these tissues for genotyping if required to collect embryonic skin. First, make a Ros coddle incision in the venum close to the midline.
The next step requires tools described by Kashiwa and company. Make a small slit in the flat end of a 15 centimeter bamboo skewer length using a razor blade, and then insert a soft toothbrush bristle into the slit. Using these hair stick tools, carefully tease away the skin from the underlying connective tissue while rotating the embryo.
Leave the skin attached along the ventral edge of the first incision, but nowhere else to mount the skin. Some preparations must be made. Have the clips, inserts and O-rings sterilized in an overnight 70%ethanol bath.
Prepare the clips by first mixing culture media warmed to 60 degrees Celsius with an equal volume of molten 60 degrees Celsius, 2%aros. Then fill the imaging clips with the resultant 1%Aros solution dropwise, and allow them to cool and solidify. Once solid, prevent the clips from drying out by adding a milliliter of PBS to each well.
The clips will be good for several hours before use. Now prepare the inserts by cutting alum's membrane from alum's dish, and securing the membrane to each sterile insert using an O-ring. The base in which the clip insert assemblies are housed is cleaned by wiping down with 70%ethanol.
The six inserts are then placed in the base. Use a sterile lid from a six well plate to enclose the inserts in the base. Now, after having nearly peeled the skin off the first embryo, transfer it to a dry petri dish.
Using the hair sticks, flatten the skin onto the dish dermal side up without any folds. Then cut the skin free with a razor. It should be about half a square centimeter.
Take an imaging clip from the PBS, dry the surface of the aeros and set it on the skin so the aeros is touching the dermal side of the tissue. Wait 60 seconds while the dermis adheres to the aros, and then carefully tease the edges of the tissue up the sides of the clip. Using the hair sticks.
Turn the clip over and with minimal touching of the area to be imaged, remove bubbles and flatten the skin onto the aros. Using the hairs sticks, secure the skin to both sides of the clip with silk suture thread and a few simple thumb knots when tightened, the thread should pull the skin into the groove on top of the clip. Now push the clip into the imaging insert that was previously mounted in the base.
Then fill the insert with about five milliliters of culture, media, and proceed with imaging. The procedure is repeated with an additional five embryos for each of the remaining insert and clip assemblies. The confocal microscope requires an environmental chamber or stage top chamber providing 5%carbon dioxide in air and an automated stage.
Prewarm the environmental chamber to 37 degrees Celsius for at least one hour before using it. Using the control software, prepare a multi-position template to quickly execute imaging experiments. Define the six positions to image.
Typically collect a small Zack at each position every two minutes over 18 to 24 hours. Selecting a wider than optimal pinhole setting and taking small three to five image, Zacks helps prevent the laser from damaging the tissue over the long imaging period. When mounting to the stage, be sure to use the appropriate plate insert, then proceed with the imaging program.
Embryonic skin cultures were prepared and time-lapse images were made. According to the protocol viewing the YFP channel, Mel Oblasts moved almost constantly only pausing to undergo mitosis under transmitted light. The developing skin and the formation of the primary hair follicle pattern were observed in the composite sequence.
Melano blasts, mitosis, and developing hair follicles could be identified together using the WR MAC plugin. For Image J, it was possible to track the migrating melano blasts population in each of the six time lapse sequences captured. In parallel, a representation of the Melano blasts.
Tracking results shows the degree of movement within the developing skin culture. A summary of the tracking data using zero to tracking highlights the random nature of the Mel Oblasts movements and shows how they spread out. Once mastered the dissection and setting up of the culture, operators can be completed in around 90 minutes.
This technique has paved the way for researchers in the fields of developmental biology and cancer to explore melano blasts behavior during embryonic development on a number of mutant mouse backgrounds.
