4D Fluorescent Imaging of Embryonic Quail Development

Traditionally, our understanding of developmental biology has been based on the fixation and study of embryonic samples. Detailed microscopic scrutiny of static specimens at varying ages allowed for anatomical assessment of tissue development. The advent of confocal and two-photon excitation (2PE) microscopy enables researchers to acquire volumetric images in three dimensions (x, y, and z) plus time (t). Here, we present techniques for acquisition and analysis of three-dimensional (3D) timelapse data. Both confocal microscopy and 2PE microscopy techniques are used. Data processing for tiled image stitching and time-lapse analysis is also discussed. The development of a transgenic Japanese quail system, as discussed here, has provided an embryonic model that is more easily accessible than mammalian models and more efficient to breed than the classic avian model, the chicken. BACKGROUND The fieldofdevelopmentalbiologyisevolvingfromthestaticstudyofembryoanatomytothedynamic analysis of the roles of genetics and mechanics during cell differentiation, tissue morphogenesis, and organogenesis. Theabilityto visualizeand comprehend howmillionsofcellsinteract withone another to form tissues and organs in three dimensions (x, y, z) over time (t) is just beginning to be realized. The advancement of fluorescent protein (FP) fusions permits researchers to discriminate individual cell movement and development in the context of gene expression within the embryo (Tsien 1998) with unprecedented detail. Combined with the advanced four-dimensional (4D [x, y, z, t]) light microscopy techniques outlined here, scientists can now study these developmental events in real time in living embryos. An essential requirement of time-lapse imaging is that the specimen continues to live and to function normally throughout the course of image acquisition. Successfully culturing a specimen is made much more difficult by the requirements of imaging: The specimen must be incubated under conditions that permit normal development, within the working distance of the objective, and be capable of resisting phototoxicity from the constant bombardment of the photons used for imaging. Few animal models are amenable to the rigors imposed by vital imaging; fewer still have the optical transparency to permit visual insight and the molecular tools to offer genetic approaches for the study of normal and pathologic embryogenesis. The present mismatch between the best species for molecular and imaging experiments motivated us to develop the tools for transgenesis in the quail embryo. We have developed several lines of transgenic, FP-expressing Japanese quail (Coturnix coturnix japonica) as an experimental system that permits vital imaging of embryogenesis. Coturnix offers advantages with respect to the accessibility of the developing embryo, the small size of its egg, the moderate size of breeding adults, and its short generation time. The Tg(tie1::H2B:eYFP) transgenic line fluorescently labels nuclei in the endothelial cells that line the lumina of all blood vessels. To better