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Jun 1, 2007 (Vol. 27, No. 11)

Long-Term Time-Lapse Studies Focus on Cells

Small-scale Environmental Chamber Allows Real-time Monitoring of Feather Morphogenesis

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    In our lab, we study the development of chicken skin appendages (feathers, scales) as a model for organogenesis. We are interested in characterizing the cellular and molecular activities underlying the developmental process. For this purpose, we commonly grow the skin as explants on culture inserts (Falcon) where time-lapse video microscopy is easily performed. This is a valuable tool that aids the understanding of such events.

    Skin appendages start to develop along the midline of dorsal skin about embryonic day 6.5 (E6.5) resulting from a series of epithelial-mesenchymal interactions. Subsequent rows of feathers develop bilaterally from the midline. The feather buds first become evident as epithelial placodes overlying dermal condensations. At this stage, each feather bud is surrounded by a hexagon of adjacent buds. Hence, experimental perturbations can be readily assessed. The buds then become slightly raised into symmetric short feather buds. As they elongate they develop anterior–posterior asymmetries. They then invaginate to form feather follicles. Apoptosis causes the previously ensheathed feather follicles to open, allowing them to unfurl into diverse feather shapes.

    To study this process, explants are grown in an environmental chamber to maintain proper conditions while filming. E7 skin was grown for three days in either a humidified incubator at 37ºC or the WaferGen (www.wafergen.com) Smart Slide. This small environmental chamber is based on a 6-well dish. It uses heated glass to maintain tissue temperature even at the spot being observed. It also regulates CO2 flow and provides for rapid media exchange at selectable intervals. Everything is controlled via a computer interface.

    For the purposes of this study, HEPES buffer (10 µM) to control the pH was used. Explants were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10 % fetal bovine serum plus gentamycin (diluted 1:1000). The bottom plate of the WaferGen Smart Slide was set to 37ºC. The cover plate was heated to 37.5ºC to avoid condensation, which would obscure the view. Time-lapse video pictures of the explants at 15-minute intervals were taken using Leica DFC300 FX camera mounted on a Leica Z16 APO A macroscope to assess their development and overall health.

    Over the three-day interval feather buds can be seen to develop and elongate. Formation and elongation of feather buds at 24, 48, and 72 hours can be seen (Figure). Here the midline (older feather buds) runs horizontally through the middle of the skin explant. Feather buds are elongating toward the right side of the figure.

    Movies capturing these processes, particularly when comparing control and experimental skin explants, provide tremendous insight into skin morphogenesis. At 24 hours, six rows of feathers have developed, and the lateral regions have not yet formed feather buds. By 48 hours, 10 rows of feathers are apparent, and buds toward the center have begun to elongate. After 72 hours, the number of buds remains unchanged, but most of the buds have begun to elongate. With movies at higher power, movements over time can be tracked to document cell trafficking.

    The developmental progression of feather bud growth from explants grown in the WaferGen Smart Slide versus those grown in the incubator for the same time interval was then compared. Survival and growth appeared to be similar in both explant. Additionally, morphological characteristics were also alike.

    My team concluded that this small-scale environmental chamber enables the monitoring of cell and molecular interactions in developing skin explant cultures in real time. This will facilitate investigations into the effects of chemical and molecular manipulations in developmental system.

    The Smart Slide platform can have a myriad of other applications. Biological specimens grown in a traditional incubator have to be shuttled in and out of the incubator to be photographed over time. These still images have provided a wealth of information to biologists over the years, but the ability to observe the movement of cells and tissues in real time or by time-lapse videomicroscopy provides a much clearer picture of cell behavior.

    Those interested in exploring the effects of therapeutic drugs on cells or tissues over time will appreciate the ability to exchange media within the chamber, controlled by user adjustable parameters. This also can be helpful in environmental or toxicology studies. We have found that this system is more amenable to high-throughput research than the older, bulkier environmental chambers that enclose whole microscopes.



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