![]() The filter paper attaches to the vitelline membrane, thereby maintaining its natural tension, and surrounds the embryo like a picture frame 1. The EC (Early Chick) culture, a more recent technique developed by Chapman and Collignon 1, circumvents the stretching of the vitelline membrane by using a filter paper carrier with a central aperture. Moreover, the embryo can only be cultured with its ventral side up. The blastoderm often detaches when the vitelline membrane, carrying the blastoderm, is stretched around a glass ring to achieve the proper tension 1. Despite its great success, New's technique is relatively demanding and time consuming. For many years, a technique pioneered by Denis New in 1955 and its variations were the golden standards of explant culture methods for chick embryos, thereby making the embryo accessible for time-lapse microscopy and microsurgical interventions 20,21. The explants are easily accessible for experimental manipulations and require less space for culturing. These early embryos can be imaged best if they are excised from the yolk and cultured as in vitro explants. These techniques are especially useful for studying the development of embryos after the initiation of blood circulation (which increases the contrast), while younger embryos remain hard to visualize. Embryos in whole yolk cultures can ideally be cultured until hatching and are accessible to micromanipulations. This container can be a surrogate egg shell 17, a Petri dish 19, or a hammock made of plastic cling wrap 13,18. In whole yolk culture systems, the embryo remains on top of the intact yolk, and the whole content of the egg is transferred to another container for incubation. Therefore, several techniques have been proposed to culture chick embryos ex ovo, either in whole yolk culture systems 13,17 - 19 or as explant cultures 1,20 - 23. The chick embryo can be visualized in ovo 16 but with poor accessibility for experimental manipulations and limited visibility for transmission light microscopy imaging. Morphogenetic processes can be understood best if they are studied dynamically, such as through the acquisition of time-lapse images. ![]() Moreover, it has an almost flat geometry during early morphogenetic events, such as heart and brain formation, gastrulation, neurulation, and somitogenesis 15. How do the structure and morphology of an embryo develop in such a well-defined temporal and spatial manner? The chick embryo has become one of the classical vertebrate animal models for embryogenesis for several reasons: It is easily available, inexpensive, transparent in its early stages, and accessible for experimental manipulations, as it develops outside the mother. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).Įmbryogenesis has fascinated man since the beginning of history. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. Representative time-lapse frames and movies are shown. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5) 15 up to at least the 28-somite stage (HH16) 15, embryos can be cultured with either their ventral or dorsal side up. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture) 1 and allows for the culturing of chick embryos without the need for a climate chamber. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo 14. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation 2, neurulation 3 - 5, somitogenesis 6, heart bending 7,8, and brain formation 9 - 13, during early embryogenesis.
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